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Recent Advances in Marine Biotechnology Vol 8
Bioremediation 1st Edition Milton Fingerman (Editor)
Digital Instant Download
Author(s): Milton Fingerman (Editor)
ISBN(s): 9781578082452, 1578082455
Edition: 1
File Details: PDF, 31.49 MB
Year: 2003
Language: english
Recent Advances in
MARINE BIOTECHNOLOGY
VOLUME 8
BIOREMEDIATION
Editors
M ilton Fingerm an
Rachakonda N agabhushanam
Department of Ecology and Evolutionary Biology
Tulane University
New Orleans, Louisiana 70118
USA
CRC Press
Taylor & Francis Group
Boca Raton London New York
CRC Press is an imprint of the

Taylor & Francis Group, an inform a business
A SC IEN C E PUBLISHERS BO OK
SCIENCE PUBLISHERS, INC.
Post Office Box 699
Enfield, New Hampshire 03748
United States of America
Internet site: http://www.scipub.net
[email protected] (marketing department)

[email protected] (editorial department)
[email protected] (for all other enquiries)
Library of Congress Cataloging-in-Publication Data
Bioremediation/ editors, Milton Fingerman, Rachakonda Nagabhushanam.

p. cm. -- {Recent advances in marine biotechnology ; v. 8)
Includes bibliographical references and index.
ISBN 1-57808-245-5
1. Bioremediation. 2. Marine organisms. 3. Marine bioremediation.
I. Fingerman, Milton, 1928-11. Nagabhushanam, Rachakonda. III. Series.
TD1 9 2 .5.B55683 2003

628.5 -- dc21
2002030451
ISBN Set 1-57808-010-X

ISBN Vol. 8 1-57808-245-5
© 2003, Copyright Reserved
All rights reserved. No part of this publication may be reproduced, stored

in a retrieval system, or transmitted in any form or by any means, electronic,
mechanical, photocopying or otherwise, without the prior permission from
the publisher. The request to reproduce certain material should include a
statement of the purpose and extent of the reproduction.
Published by Science Publishers, Inc., Enfield, NH, USA

Preface
T h is v o lu m e is th e e ig h th in th e o n g o in g s e rie s w e h a v e e n title d Recent

Advances in Marine Biotechnology. F o r th e firs t v o lu m e th e to p ic E n d o c r in e
and R e p ro d u c tiv e B io lo g y w a s s e le c te d . V o lu m e 2 w a s s u b title d
E n v ir o n m e n ta l M a rin e B io te c h n o lo g y , a n d V o lu m e 3 w a s s u b title d B io film s,
B io a d h e s io n , C o r r o s io n , a n d B io fo u lin g . V o lu m e 4 is c o n c e r n e d w ith
A q u a c u ltu r e . T h e s u b title o f V o lu m e 5 is Im m u n o b io lo g y a n d P a th o lo g y .
V o lu m e 6 d e a ls w i th th e im p o r t a n t s u b je c t B io -o r g a n ic C o m p o u n d s :
C h e m is tr y a n d B io m e d ic a l A p p lic a tio n s . V o lu m e 7 is d e v o te d to S e a fo o d
S a f e ty a n d H u m a n H e a lth . N o w , f o r V o lu m e 8 w e tu r n o u r a tte n tio n to
a n o th e r c u tti n g - e d g e s u b je c t, n a m e ly B io r e m e d ia tio n .
T h e u s e o f m a r in e o rg a n is m s to d e g r a d e a v a r ie t y o f n a tu ra l a n d s y n th e tic
s u b s ta n c e s in th e m a r in e e n v ir o n m e n t, th e r e b y r e d u c in g th e le v e ls o f
h a z a r d o u s c o m p o u n d s , is in c r e a s in g ly d r a w i n g a tte n tio n b e c a u s e o f th e
p o te n tia l s u c h b io r e m e d ia tio n h a s fo r e n v ir o n m e n ta l r e s to ra tio n . A m o n g
th e c u r r e n t r e s e a r c h e ffo rts in b io r e m e d ia tio n a r e s o m e d ir e c te d to w a r d
id e n tif y in g o r g a n is m s th a t p o s s e s s th e a b ility to d e g r a d e s p e c ific p o llu ta n ts .
W ith s u c h o r g a n is m s , w h ic h h a v e a l r e a d y b e e n id e n tifie d , b io c h e m ic a l
s tu d ie s a r e g o in g o n w ith th e a im o f e lu c id a ti n g th e p a th w a y s o f th e s e
d e g r a d a t i v e p r o c e s s e s a n d th e e n z y m e s in v o lv e d . H e r e in a r e c h a p te r s ,
a m o n g o th e r s , th a t a r e d e v o te d to p e tr o l e u m s p ill b io r e m e d ia tio n , u s e o f
s p e c tr o s c o p y to id e n tify m ic r o b ia l m e ta b o lic p a th w a y s , d e to x if ic a tio n o f
m e r c u r y b y u s in g r e c o m b in a n t m e r c u r y -r e s is t a n t b a c te r ia , a n d th e u s e o f
m a n g a n e s e -o x id iz in g b a c te r ia f o r b io r e m e d ia tio n . A b r o a d -b a s e d a p p r o a c h
to b io r e m e d ia tio n o f m a r in e h a b ita ts , a s e x e m p lifie d b y th e c h a p te r s h e re in ,
is r e q u ir e d b e c a u s e o f th e w id e v a r ie t y o f c o n ta m in a n ts w e h a v e in th e
w o r ld 's o c e a n s .
T h is v o lu m e , w h ich p re s e n ts th e m o s t r e c e n t in fo r m a tio n on
b io r e m e d ia tio n , w a s w r itte n b y a h ig h ly ta le n te d g r o u p o f s c ie n tis ts w h o
a r e n o t o n ly a b le to c o m m u n ic a t e v e r y e f f e c tiv e ly th r o u g h th e ir w r itin g ,
b u t a r e a ls o re s p o n s ib le fo r m a n y o f th e a d v a n c e s th a t a r e d e s c rib e d h e re in .
A s w ith th e firs t s e v e n v o lu m e s o f th is s e r ie s , w e , th e e d ito r s , h a v e b e e n
m o s t f o r tu n a te in a ttr a c tin g a h ig h ly ta le n te d , in te r n a tio n a lly r e s p e c te d
g r o u p o f in v e s tig a to r s to s e r v e a s a u th o r s . W e in te n tio n a lly s e t o u t to
p r e s e n t a tr u ly in te rn a tio n a l s c o p e to th is v o lu m e . C o n se q u e n tly , a p p r o p r ia te
a u th o r s fr o m s e v e r a l c o u n tr ie s w e r e s o u g h t , a n d to e v e r y o n e 's b e n e f it, o u r
in v ita tio n s to c o n tr ib u te w e r e a c c e p te d .
IV MARINE BIOTECHNOLOGY
W e ta k e p le a s u r e in th a n k in g th e a u th o r s fo r th e ir c o o p e r a tio n a n d
e x c e lle n t c o n trib u tio n s , a n d f o r k e e p in g to th e p u b lic a tio n s c h e d u le . T h e
e ffo rts o f th e se in d iv id u a ls m a d e o u r ta s k m u c h less d iffic u lt th a n it m ig h t
h a v e b e e n . A ls o , w e e s p e c ia lly w is h to th a n k o u r w iv e s , M a r ia E s p e r a n z a
F in g e r m a n a n d R a c h a k o n d a S a ro jin i, fo r th e ir c o n s ta n t a n d u n d im in is h in g
e n c o u r a g e m e n t a n d s u p p o r t d u r in g th e p r o d u c tio n o f th is s e rie s . W e tr u s t
th a t y o u , th e r e a d e r s , w ill a g r e e w ith u s th a t th e e ffo rts o f th e a u th o r s o f th e
c h a p t e r s in th is v o lu m e w ill s e r v e c o lle c tiv e ly to p r o v id e a m a jo r th r u s t
t o w a r d s a b e tte r u n d e r s t a n d i n g o f e n v ir o n m e n ta l b io r e m e d ia tio n a n d
w h a t m u s t b e d o n e to i m p r o v e th e h e a lth o f o u r o c e a n s .
M il to n F i n g e r m a n
R ach ak on d a N agab h u sh an am
Contents
Preface iii
The Contributors v ii
C o ld -A c tiv e E n z y m e s fr o m C o l d -A d a p te d M ic r o o r g a n is m s : 1
T h e ir P o s s ib le A p p lic a tio n s a n d S tr u c tu r a l R e la tio n s h ip s w ith
M e s o p h ilic a n d T h e r m o p h ilic C o u n te r p a r ts
Andrey Galkin, Ljudmila Kulakova, Toru Nakayama,
Tokuzo Nishino and Nobuyoshi Esaki
E n g in e e rin g o f M ic r o b ia l D io x y g e n a s e s fo r th e D e g r a d a tio n 29
o f X e n o b io tic C o m p o u n d s
Jun Hirose and Kensuke Furukawa
P ie z o p h ilic B a c te ria in th e D e e p -S e a : Is o la tio n , T a x o n o m y , 47
D iv e rs ity , M o le c u la r A d a p ta ti o n a n d B io te c h n o lo g ic a l P o te n tia l
Jiasong Fang and Chiaki Kato
M ic r o b ia l C o n tr o l o f H e a v y M e ta l P o llu tio n : A n O v e r v i e w 81
Tomas Ruml and Pavel Kotrba
M o le c u la r C h a r a c te r iz a t io n o f P h e n o l-D e g r a d in g B a c te ria 155
in A c tiv a te d S lu d g e
Kazuya Watanabe
C a d m iu m a n d M a n g a n e s e T r a n s p o r t G e n e s in B a c te r ia 171
Zhiqi Hao, Shaolin Chen and David B. Wilson
M e r c u r y R e m e d ia tio n U s in g N a t u r a l a n d R e c o m b in a n t M ic r o b e s 189
Irene Wagner-Dobler
A p p lic a tio n s o f M o le c u la r T e c h n o lo g ie s in E n v ir o n m e n ta l 203
B io te c h n o lo g y fo r M o n ito r in g B a c te ria
Jacques Theron and Thomas E. Cloete
B io te c h n o lo g ic a l T r e a tm e n t o f S u lfu r-C o n ta in in g W a s te w a te r s 233
Marcus V.G. Vallero, Jan Sipma, Ajit Annachhatre, Piet N.L. Lens
and Look W. Hulshoff Pol
P h y to r e m e d ia tio n o f H e a v y M e ta ls : A n O v e r v ie w 269
Alexander A. Kamnev
VI MARINE BIOTECHNOLOGY
C h e m ic a l a n d B io lo g ic a l T ra n s fo rm a tio n o f P a r a ly ti c S h ellfish 319

P o is o n in g T o xin s
Shigeru Sato and Masaaki Kodama
In d e x 33 7
The Contributors
A jit A n n a c h h a tre
S u b -d e p a r tm e n t o f E n v ir o n m e n ta l T e c h n o lo g y
W a g e n in g e n U n iv e r s ity
" B io te c h n io n " -B o m e n w e g / 2
P.O . B o x 8 1 2 9 ,
6 7 0 0 E V W a g e n in g e n
T h e N e th e r la n d s
S h a o l in C h e n
D e p a r tm e n t o f M o le c u la r B io lo g y a n d G e n e tic s
C o rn e ll U n iv e r s ity
I th a c a , N e w Y o rk 1 4 8 5 3
USA
T h o m a s E . C lo e te
D e p a r tm e n t o f M ic r o b io lo g y a n d P la n t P a th o lo g y
U n iv e r s ity o f P r e to r ia
0 0 0 2 P r e to r ia
S o u th A f r ic a
N o b u y o sh i E sak i
In s titu te f o r C h e m ic a l R e s e a r c h
K y o to U n iv e r s ity
U ji, K y o to -F u 6 1 1 , J a p a n
Jia so n g F an g
D e p a r tm e n t o f G e o lo g ic a l a n d A tm o s p h e r ic S c ie n c e s
Io w a S ta te U n iv e r s ity
A m e s, Io w a 5 0 0 1 1 -3 2 1 2
USA
K en su k e F u ru k aw a
D e p a r tm e n t o f B io s c ie n c e a n d B io te c h n o lo g y
F a c u lty o f A g r ic u ltu r e
K y u s h u U n iv e r s ity
F u k u o k a 8 1 2 -8 5 8 1
Ja p a n
viii MARINE BIOTECHNOLOGY
A n d re y G a lk in
In s titu te fo r C h e m ic a l R e s e a r c h
U ji, K y o to -F u 611
Jap an
Z h iq i H ao
C o r n e ll U n iv e r s ity
I th a c a , N e w Y o rk 1 4 8 5 3
U .S .A .
J u n H ir o s e
D e p a r tm e n t o f A p p lie d C h e m is tr y
F a c u lt y o f E n g in e e rin g
M iy a z a k i U n iv e r s ity
M iy a z a k i 8 8 9 - 2 1 9 2
Jap an
L o o k W . H u ls h o ff P o l
S u b -d e p a r t m e n t o f E n v ir o n m e n ta l T e c h n o lo g y
" B io te c h n io n ' -B o m e n w e g , 2
P.O . B o x 8 1 2 9
A le x a n d e r A . K am n ev
L a b o r a to r y o f B io c h e m is try o f P la n t-B a c te r ia l S y m b io s e s
I n s titu te o f B io c h e m is try a n d P h y s io lo g y
o f P la n ts a n d M ic r o o r g a n is m s
R u s s ia n A c a d e m y o f S c ie n c e s
1 3 P r o s p . E n tu z ia s to v
4 1 0 0 1 5 S a r a to v
R u s s ia
C h i a k i K a to
T h e D E E P S T A R G ro u p
J a p a n M a rin e S c ie n c e a n d T e c h n o lo g y C e n t e r
2 - 1 5 N a ts u s h im a -c h o
Y o k o s u k a 2 3 7 -0 0 6 1
Jap an
THE CONTRIBUTORS IX
M asaak i K od am a
S c h o o l o f F is h e r ie s S cie n ce s
K ita s a to U n iv e r s ity
O f u n a to
I w a te 0 2 2 -0 1 0 1
Jap an
P a v e l K o tr b a
R e s e a r c h In s titu te o f In n o v a tiv e T e c h n o lo g y
fo r th e E a r t h
9 - 2 K iz u g a w a d a i
K iz u -c h o , S o r a k u -g u n
K y o to 6 1 9 - 0 2 9 2
Jap an
L ju d m ila K u la k o v a
In s titu te fo r C h e m ic a l R e s e a r c h
U ji, K y o to -F u 611
Jap an
P ie t N .L . L e n s
S u b -d e p a r t m e n t o f E n v ir o n m e n ta l T e c h n o lo g y
" B io te c h n io n " -B o m e n w e g / 2
P.O . B o x 8 1 2 9
T o ru N a k a y a m a
D e p a r tm e n t o f B io m o le c u la r E n g in e e r in g
G r a d u a te S c h o o l o f E n g in e e rin g
T o h o k u U n iv e r s ity
A o b a -y a m a 0 7
S e n d a i, M iy a g i 9 8 0 - 8 5 7 9
Jap an
T o k u z o N ish in o
D e p a r tm e n t o f B io m o le c u la r E n g in e e r in g
G r a d u a te S c h o o l o f E n g in e e rin g
T o h o k u U n iv e r s ity
A o b a -y a m a 0 7 , S e n d a i, M iy a g i 9 8 0 - 8 5 7 9
Jap an
X MARINE BIOTECHNOLOGY
T om as R um l
D e p a r tm e n t o f B io c h e m is tr y a n d M ic r o b io lo g y
a n d C e n te r f o r I n te g r a te d G e n o m ic s
In s titu te o f C h e m ic a l T e c h n o lo g y
T e c h n ic k a 3 ,
1 6 6 2 8 P rag u e
C z e c h R e p u b lic
S h i g e r u S a to
S c h o o l o f F is h e r ie s S c ie n c e s
K ita s a to U n iv e r s ity
O f u n a to ,
I w a te 0 2 2 -0 1 0 1
Jap an
J a n S ip m a
" B io te c h n io n ”-B o m e n w e g , 2
P.O . B o x 8 1 2 9
Ja cq u e s T h ero n
D e p a r tm e n t o f M ic r o b io lo g y a n d P la n t P a th o lo g y
U n iv e r s ity o f P re to ria
0 0 0 2 P r e to r ia ,
S o u th A f ric a
M a r c u s V .G . V a lle ro
" B io te c h n io n " -B o m e n w e g , 2
P.O . B o x 8 1 2 9
I r e n e W a g n e r - D o b le r
G e s e lls c h a ft fu r B io te c h n o lo g is c h e F o r s c h u n g m b H
M a sch e ro d e r W eg 1
D -3 8 1 2 4 B ra u n s c h w e ig
G erm an y
THE CONTRIBUTORS
K a z u y a W a ta n a b e
M a r in e B io te c h n o lo g y In s titu te
3 -7 5 - 1 H e ita , K a m a is h i
I w a te 0 2 6 -0 0 1
Jap an
D a v id B . W ils o n
C o r n e ll U n iv e r s ity
I th a c a , N e w Y o rk 1 4 8 5 3
U .S .A .
Cold-Active Enzymes from Cold-Adapted
Microorganisms: Their Possible Applications and
Structural Relationships with Mesophilic and
Thermophilic Counterparts
Audrey Galkin', Ljudmila Kulakova1, Toru Nakayama2,

Tokuzo Nishino2 and Nobuyoshi Esaki1*
'Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan
’Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku,
University, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan
Introduction
T h e m ic ro o rg a n is m s in h a b itin g e x tr e m e e n v iro n m e n ts a re c a lle d
" e x tr e m o p h ile s ." T h e y c a n s u r v iv e a n d e v e n g r o w u n d e r h a r s h c o n d itio n s
s u c h a s e x tr e m e te m p e r a tu r e s , e x tr e m e p H s , h ig h s a lt c o n c e n tr a tio n s , a n d
h ig h p r e s s u r e s , in w h ic h th e m a j o r ity o f o r g a n is m s c a n n o t s u r v iv e .
E x tr e m o p h ile s a r e im p o r ta n t s o u r c e s o f in d u s tr ia l e n z y m e s th a t c a n b e
u s e d u n d e r s u c h e x tr e m e r e a c tio n c o n d itio n s in b io te c h n o lo g y . S o far,
th e r m o p h ile s , a c la s s o f e x tr e m o p h ile s th a t c a n g r o w a t h ig h te m p e r a tu r e s
(> 5 5 ° C ), a s w e ll as th e ir e n z y m e s and p ro te in s , h a v e a ttr a c te d
b io te c h n o lo g is ts ' a tte n tio n b e c a u s e th e e n z y m e s fro m th e rm o p h ile s a re
g e n e r a lly m o r e "ro b u s t" th a n th o s e o f m e s o p h ilic o rig in , a llo w in g th e
e f f ic ie n t a p p l ic a ti o n o f e n z y m e s in b io te c h n o lo g y . T h e u s e f u ln e s s o f
th e rm o s ta b le e n z y m e s fro m t h e r m o p h i l i c m i c r o o r g a n i s m s is w e l l
d o c u m e n t e d b y th e a p p lic a tio n o f th e r m o s ta b le D N A p o ly m e r a s e s fr o m
e x tr e m e th e rm o p h ile s in P C R te c h n o lo g y .
D u r in g th e p a s t d e c a d e , in c r e a s in g a tte n tio n h a s a ls o b e e n p a id to th e
a p p lic a tio n o f th e o th e r c la s s o f e x tre m o p h ile s , c o ld -a d a p te d
m ic r o o r g a n is m s w h ic h c a n g r o w a t lo w t e m p e r a tu r e s (b e lo w 1 5 ° C ), a n d
th e ir c o ld -a c t iv e e n z y m e s in b io te c h n o lo g y . T h is r e v ie w s u r v e y s c u r r e n t
k n o w le d g e o n c o ld -a c t iv e e n z y m e s f r o m c o ld -a d a p te d m ic r o o r g a n is m s ,
m a in ly in te r m s o f th e ir a p p lic a tio n s in b io te c h n o lo g y a n d th e m e c h a n is tic
a s p e c ts o f th e ir c o ld a d a p ta tio n .
‘ Corresponding author Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611,
Japan
2 MARINE BIOTECHNOLOGY
1. Cold-adapted Microorganisms as Sources of Cold-active Enzymes

C o ld -a d a p t e d m ic r o o r g a n is m s a r e g o o d s o u r c e s o f c o ld -a c t iv e e n z y m e s
and can be c la s s ifie d in to tw o m a jo r c la s s e s , p s y c h r o p h ile s and
p s y c h r o tr o p h s , on th e b a s is of th e ir w ays of c o ld a d a p ta tio n . A
p s y c h r o p h ile is a m ic r o o r g a n is m w ith o p tim a l g r o w th a t 1 5 ° C o r lo w e r,
w h e r e a s a p s y c h r o tr o p h (o r a ls o c a lle d " p s y c h r o to le r a n t" ) is e s s e n tia lly a
m e s o p h ilic m ic r o o r g a n is m th a t c a n a ls o g r o w u n d e r c o ld c o n d itio n s
b e lo w 1 5 ° C (M o r ita 1 9 7 5 ,1 9 7 6 ) . S u c h lo w te m p e r a tu r e c o n d itio n s m a y n o t
n e c e s s a r ily b e " e x tr e m e " o n e s fo r m o s t m ic r o o r g a n is m s b e c a u s e th e y
r e m a in v ia b le (b u t d o n o t g r o w ) u n d e r s u c h c o n d itio n s . N o n e th e le s s ,
p s y c h r o p h ile s a n d p s y c h r o tr o p h s a re r e g a r d e d a s a c la s s o f " e x tr e m o p h ile s "
d u e to th e ir d is tin c tiv e a b ility to r a p id ly g r o w a t s u c h lo w te m p e r a tu r e s .
T h e r e is p r o b a b ly a c o n tin u ity in th e d is tr ib u tio n o f o r g a n is m s w h o s e
h ig h e s t g r o w th te m p e r a tu r e r a n g e s fr o m n e a r 0 ° C to 1 1 0 ° C , s u c h as
p s y c h r o p h ile s , p s y c h r o tr o p h s , m e s o p h ile s , m o d e r a t e th e r m o p h ile s , a n d
h y p e r th e r m o p h ile s (A g u ila r 1 9 9 6 , G e r d a y et al. 2 0 0 0 ).
C o ld -a d a p t e d m ic r o o r g a n is m s s h o w a b r o a d p h y lo g e n e tic d iv e r s ity
c o v e r in g th e A r c h a e a , P r o k a r y a , a n d E u k a r y a d o m a in s o f life (S h e r id a n et
al. 2 0 0 0 , T h o m a s a n d C a v ic c h io li 1 9 9 8 ). T h u s far, c o ld -a d a p te d p r o k a r y o te s ,
in c lu d in g b o th g r a m - n e g a t i v e a n d g r a m - p o s i tiv e b a c te r i a , h a v e m o s t
fr e q u e n tly b e e n id e n tifie d in d iv e r s e e n v ir o n m e n ts — in fre sh a n d sa lin e
w a t e r s , s o ils , a n d m a n -m a d e e n v ir o n m e n ts s u c h a s r e f r ig e r a to r s , a n d in
a s s o c ia tio n w i th p la n ts a n d a n im a ls (M o r ita 1 9 7 5 ,1 9 7 6 , B a r r o s a n d M o r ita
1 9 7 8 , R u s s e ll 1 9 9 0 , W y n n -W illia m s 1 9 9 0 , G o u n o t 1 9 9 1 ) 1. S o m e o f th e s e
m ic r o o r g a n is m s c a n g r o w e v e n u n d e r in h o s p ita b le c o n d itio n s s u c h a s
a r c t ic , A n ta r c tic , a n d a lp in e r e g io n s a s w e ll a s a b y s s e s w h o s e a v e r a g e
t e m p e r a tu r e s a r e b e lo w 5 ° C .
D u r in g th e c o u r s e o f o u r r e c e n t id e n tif ic a tio n o f A n ta r c tic a n d S ib e ria n
c o l d -a d a p te d b a c te r ia (C h o o 1 9 9 8 , C h o o et al. 1 9 9 8 , G a lk in et al. 1999,
K u la k o v a 2 0 0 0 , K u la k o v a et al. 1 9 9 9 ) (s e e T ab le 1 ), w e o b s e r v e d t h a t, in
g e n e r a l, A n ta r c tic is o la te s h a v e a n a r r o w e r g r o w t h te m p e r a tu r e r a n g e a n d
l o w e r o p t i m u m /m a x i m u m g r o w th te m p e r a tu r e s in c o m p a r is o n w i th th e
s p e c ie s is o la te d fr o m S ib e ria n s o il. T h e o b s e r v a tio n c o r r o b o r a te s a n o th e r
r e p o r t th a t d e s c r ib e s th e d o m in a tio n o f p s y c h r o p h ile s in A n ta r c tic s e a ice
(B o w m a n et al. 1 9 9 7 ), w h ile p s y c h r o tr o p h s , b u t n o t p s y c h r o p h ile s , a re
o fte n th e d o m in a n t ty p e in o th e r lo w te m p e r a tu r e e n v ir o n m e n ts (R u s s e ll
1 9 9 0 ).
'Today the largest publicly accessible source of cold-adapted bacterial strains is the Australian
Collection of Antarctic Microorganisms (ASAM), holding about 300 isolates of heterotrophic
Antarctic bacteria. A private collection that of AMRAD Discovery Technologies Pty Ltd.,
which works with ASAM, consists of several thousand isolates of Antarctic bacteria and
fungi, but the strains collected are not publicly available (Nichols et al. 1999).
COLD-ACTIVE ENZYMES FROM COLD-ADAPTED MICROORGANISMS 3
Table 1. Antarctic and Siberian bacterial isolates and their most similar organisms on the
basis of 16S rDNA sequences
Isolate 16S rDNA Accession Similar organism Gram staining

(% identity) number
Siberian isolates
SI 98 X83408 A rth ro b a cter o xy d a n s positive
S2 99 U81990 N ocardioides sim p lex positive
S3 94 AB02026 S p h in g o b a d e r iu m sp. negative
S4 98 D16277 B acillus p sy ch ro p h ilu s positive
S8 94 AJ131117 Sten o iro p h o m o n a s m altophilia negative
S9 99 X79186 R hod ococcu s fa cien ce positive
S10 98 X77444 A u reo b a cteriu m liq u efa d e n c e positive
Antarctic isolates
AclO 98 U85906 Sheiuanella fr ig id im a rin a negative
St2 96 Z73313 C a m o b a cteriu m a lte rfu n d iu m positive
PI 96 M59053 C y to p h a ga jo h so n a e negative
Ant300 98 U85875 P sy ch ro b a d e r gla cincola negative
2. General Properties of Cold-adapted Enzymes

If m ic r o o r g a n is m s h a v e to r e m a in s u c c e s s f u l c o m p e tito r s f o r th e e c o lo g ic a l
n ic h e o f a c o ld e n v ir o n m e n t, th e y m u s t b e a b le to a d ju s t th e ir m e ta b o lic
flu x e s , w h ic h a r e e n z y m a ti c p r o c e s s e s , to b e e ffe c tiv e a t lo w te m p e r a tu r e s .
T h e r e fo re , th e ir e n z y m e s th e m s e lv e s h a v e to b e c o ld -a d a p te d . T h u s , th e
s p e c ific a c tiv ity o f a c o ld -a d a p te d e n z y m e is c h a r a c te r is tic a lly m u c h h ig h e r
th a n th o s e o f its m e s o p h ilic a n d th e rm o p h ilic c o u n t e r p a r ts a t te m p e r a tu r e s
o f 0 - 3 0 ° C (T ab le 2 ). T h e a c tiv a tio n e n e rg ie s o f r e a c tio n s c a t a l y z e d b y c o ld -
a c tiv e e n z y m e s a r e g e n e r a lly lo w e r th a n th o s e c a ta ly z e d b y th e ir m e s o p h ilic
Table 2. Catalytic properties of hydrolases from cold-adapted and mesophilic organisms
Enzyme Source E. Reference

(s-1)* (kj m o l')*
a-amylase A ltero m o n a s h a lo p la n d i& (Antarctica) 187 73 (Feller et al. 1992)

B acillus a m y lo liq u e fa c ie n c f 34 99
subtilisin Shew anella sp. strain AclOb 10.4 41.6 (Kulakova 2000)
B acillus subtilis (Carlsberg)1 1.27 57.5
lipase P seud om o nas sp. strain B -ll-lb(Alaska) nr 11.2 (Choo et al. 1998)
A c in e to b a d e r sp. strain No. 6b(Siberia) nr 11.2 (Suzuki et al. 2001)
P seu d o m o n a s cepacia1 nr 28.4
■ At 15° C
bPsychrotroph
'Mesophile
nr: not reported
a n d th e r m o p h ilic c o u n t e r p a r ts (G e r d a y et al. 1 9 9 7 ). It s h o u ld b e m e n tio n e d

that the apparent optimum temperatures o f reactions catalyzed b y cold-
a c tiv e e n z y m e s a r e g e n e r a lly lo w e r th a n th o s e c a ta ly z e d b y th e ir m e s o p h ilic
a n d th e r m o p h ilic c o u n t e r p a r ts ; h o w e v e r , th is o b s e r v a tio n s h o u ld b e o f
le s s b io c h e m ic a l s ig n ific a n c e b e c a u s e th e a p p a r e n t o p tim u m te m p e r a tu r e
s h o u ld a ls o d e p e n d o n m a n y p a r a m e t e r s u n r e la te d to th e c o ld a d a p ta tio n
o f e n z y m e s (F e lle r a n d G e r d a y 1 9 9 7 ).
O n e o f th e o th e r r e m a r k a b le p r o p e r tie s th a t h a v e fr e q u e n tly b e e n
o b s e r v e d w i th c o ld -a c t iv e e n z y m e s is th e ir th e rm o la b ility (G e r d a y et al.
1 9 9 7 ) ; c o ld -a c t iv e e n z y m e s a r e in a c tiv a te d a t c o ld e r te m p e r a tu r e s , w h ile
th e ir m e s o p h ilic a n d th e rm o p h ilic c o u n t e r p a r ts a r e n o t in a c tiv a te d (s e e
F ig . 1 , f o r e x a m p l e ) . S u c h a n i n v e r s e r e l a tio n s h ip b e tw e e n p r o te i n
th e r m o s ta b ility a n d " c o ld a c tiv ity " h o ld s fo r th e r m o p h ilic e n z y m e s a s
w e ll, w h ic h d is p la y h ig h s ta b ility a t h ig h te m p e r a tu r e s b u t v e r y lo w
s p e c ific a c tiv ity a t lo w t e m p e r a tu r e s . T h e th e rm o la b ility a n d ’’c o ld a c ti v i ty ”
o f c o ld -a d a p te d e n z y m e s s e e m to b e c lo s e ly r e la te d to e a c h o th e r, a n d b o th
h a v e b e e n p r o p o s e d to a r is e fro m a n e n z y m e 's s tr u c t u r a l fle x ib ility [se e
S e c tio n 5 f o r d e ta ils , (H o c h a c h k a a n d S o m e r o 1 9 8 4 ) ] . H o w e v e r , it h a s
100
80
60
40
20
0
40 50 60 70 80 90
Temperature (°C)
Figure 1. Thermal stabilities of alanine dehydrogenases from psychrotrophic microorganisms

C a m o b a cteriu m sp. strain St2 (recombinant, □ ; wild type, ♦ ) and Sch ew a n ella sp. strain AclO
( O ), the mesophilic microorganisms V. p ro teo ly ticu s ( • ) and B. su b tilis ( ■ ), and the
thermophilic microorganism B . stea ro th em w p h ilu s ( ▲ ). The enzyme activity remaining after
incubation for 30 min at different temperatures and pH 7.2 is shown. Analyses of the structural
characteristics responsible for cold adaptation of the psychrotrophic AlaDHs presented in
this figure are described in Section 5.
recently been claimed that the general thermolability of cold-active enzymes

could be mechanistically unrelated to their cold activities (Sheridan el al.
2000). Namely, although many cold-active enzymes are indeed
thermolabile, it does not necessarily follow that cold adaptation requires
thermolability. It is likely that most cold-active enzymes are thermolabile
simply because the microorganisms producing these enzymes have never
experienced any selective pressures to maintain enzyme stability at higher
temperatures. If this is the case, it may be possible to improve the
thermostability of a cold-active enzyme, through protein engineering
approaches, by maintaining their catalytic efficiency at low temperatures.
Attempts addressing this issue will be described in Section 6.
3. Potential Applications of Cold-active Enzymes in Biotechnology

Enzymes offer an environmentally benign alternative to chemical catalysts
in many commercial and industrial applications, which also include a
wide variety of low-temperature processes to be accelerated by means of
cold-active enzymes. Many attempts have been made to obtain and develop
cold-active enzymes that perform both effectively and economically at
low temperatures under defined, application-specific conditions.
Food industries—There are numerous potential applications of cold-
active enzymes in the food industries; cold-active enzymes allow the
processing of food materials at low temperatures, minimally affecting the
native properties of the materials, and would also be beneficial in reducing
the possibility of contamination by mesophilic microorganisms during the
processing. This is typified by the application of cold-active [i-galactosidase
in the milk and dairy industries for the purpose of alleviating the "lactose
problem" (Nakayama and Amachi 1999). Cow milk generally contains
4.5-5.0% (w/v) lactose with low digestibility, low solubility, and low
sweetness. However, lactose intolerance is widespread, and 70% of the
world’s adult population is unable to metabolize large quantities of lactose
in milk due to lack of intestinal lactase. In addition, the high lactose
content in non-fermented milk products, such as ice-cream and condensed
milk, often causes lactose crystallization during preservation, which would
make the products sandy. All of these problems can be effectively
circumvented by decomposing lactose in the products by the action of
cold-active ^-galactosidase at low temperatures.
The following illustrates other potential applications of cold-active
enzymes in the food industries, (i) Cold-active proteinases can be used for
tenderizing the meat in the meat industry, (ii) Cold-active transglutaminases
catalyzing the intermolecular cross-linking of proteins between lysine and
glutamine residues can be used for hardening the meat as well as controlling
the rheological properties of protein-based food products, such as noodles
and fish-paste products, (iii) In the fruit-juice-making processes, cold-

active pectinases can be used to reduce the viscosity and clarify the juice
products at low temperatures; (iv) In the baking industry, cold-active
amylases and proteinases can be used to reduce the time for dough
fermentation and improve the rheological properties of bakery products.
In this case, the thermolability of these enzymes would especially be
advantageous because the actions of these enzymes are easily stopped by
heat treatment.
Synthesis of useful compounds—The cold-adapted microorganisms and
their enzymes have been used as catalysts for organic syntheses at low
temperatures of unstable compounds (Choo 1998). This is exemplified by
enantioselective production of optically active amino acids from the
corresponding a-keto acids by a coupled multienzyme system consisting
of formate dehydrogenase, alanine dehydrogenase, alanine racemase, and
amino acid aminotransferase (Fig. 2) (Galkin et al. 1997a, b, Hummel and
Kula 1989). Some of the substrate a-keto acids are unstable and are
degraded during prolonged incubation at moderate temperatures, such as
37°C. Cold-adapted enzymes that exhibit high levels of activities at low
temperatures may be useful for converting such unstable a-keto acids [see
also Section 5, (Galkin et al. 1999)].
[AteRj
Formate NAD* • L-Alanine ^ S D-Alantne a-Keto acids
|FDH | jAlaPH | D-ATA
COs - 'NADH ♦ H * — Pyruvate «--------^ D -A m in o acids

NH4*
Figure 2. Enantioselective production of D-amino acids by a multienzyme system consisting

of formate dehydrogenase (FDH), alanine dehydrogenase (AlaDH), alanine racemase (AlaR),
and D-amino add aminotransferase (D-ATA). A similar system, where AlaDH and AlaR are
substituted by glutamate dehydrogenase and glutamate racemase, respectively, can also be
used for amino add production (Nakajima et al. 1988). It should be mentioned that the cold-
active AlaDHs and glutamate racemases mentioned in Section 5 are applicable to this system.
Additives in laundry detergents—Hydrolases such as proteinases, amylases,

and lipases have been used as additives in laundry detergents for efficient
removal of proteinaceous, starchy, and lipid stains on clothes, respectively,
during washing. Importantly, under laundry conditions, enzymes must
be able to withstand highly alkaline environments (pH 8.5-10) containing
detergents, chelators, fluorescence enhancers, and bleaching agents. They
must also withstand the action of proteinases added in the detergents
during washing. The preferred washing temperature depends on
conventions and circumstances, and, in several countries including Japan,
washing is conventionally done with tap water at cold or moderate

temperatures, mainly for the purpose of saving energy costs. Some
hydrolytic enzymes, such as an alkaline proteinase from B. lentus [Savinase,
(Christensen et al. 1986, Betzel et al. 1992)] and a lipase from Humicola
lanuginosa (Jensen 1988), exhibit relatively high activity at low and moderate
temperatures and are currently used for such cold laundry conditions.
However, the source microorganisms of these enzymes are mesophilic.
Despite the high catalytic activity at low and moderate temperatures of
cold-active enzymes from cold-adapted microorganisms, their use as
detergent additives has been precluded by their instability under the
conditions of washing as well as those of long-term storage in detergents.
Enhancing the stability of cold-active enzymes without altering the specific
activity will enhance their usefulness as agents in laundry detergents (see
Section 6).
Bioremediation—The earth's surface, even in cold environments that are
inhospitable to humans, has been found to be contaminated with harmful
compounds such as hydrocarbons, aromatic compounds, lipids, and heavy
metals as a result of human activities. Bioremediation is a recently
established technology that utilizes microorganisms for the restoration of
such contaminated environments (Timmis and Pieper 1999). In some
cases, bioremediation can be enhanced by adding microorganisms with
specific metabolic functions, a procedure that is referred to as
bioaugmentation (Tanaka 1998). However, the effectiveness of
microorganisms in degrading these compounds in the cold districts or
seasons is significantly reduced because of low temperatures. Cold-adapted
microorganisms and their enzymes should be ideal for such processes at
low or moderate temperatures due to the high catalytic efficiency of their
enzymes, and one of such potential applications is as follows.
The fast-food industry and restaurants produce large amounts of fat-
containing wastewater during their daily kitchen activities, which can
give rise to environmental problems (Wakelin and Forster 1997). Until
now, this problem is, in part, circumvented by removing fats from
wastewater through physical and biological approaches, namely, the grease-
trap and the microbial (or enzymatic) degradation methods, respectively
(Bednarski et al. 1994, Wakelin and Forster 1997). Microbial removal of fats
from aqueous systems has been extensively examined, and some microbial
agents (biological/nutrient supplements) for this purpose are now
commercially available. However, these agents are of mesophilic origin
and are not effective during cold seasons or in cold districts because of
their insufficient growth rates and very low lipolytic activities under cold
environments. Recently, we have isolated, from Siberian tundra soil, a
lipolytic psychrotroph Acinetobacter strain No. 6 capable of growing at 4°C
(Suzuki et al. 2001). The strain No. 6 extracellularly produces a lipolytic
enzyme to efficiently hydrolyze triglycerides such as soybean oil during

bacterial growth even at 4°C; it efficiently degraded added oil after
cultivation at 4°C for 7 days. Thus, the bacterium is potentially applicable
to in situ bioremediation or bioaugmentation of the fat-contaminated cold
environments.
4. Structural Characteristics Associated with Cold-active Enzymes

and Mechanistic Consideration of Protein Cold Adaptation
Many cold-active enzymes have been thoroughly characterized, and
structural factors for protein cold adaptation have been deduced from
sequence comparison and/or homology modeling studies in comparison
with their mesophilic and thermophilic counterparts [(Feller et al. 1996,
Gerday et al. 1997) and references therein]. Recently, the crystal structures
of some cold-adapted enzymes have been determined, providing important
information for insights into the mechanism of protein cold adaptation:
e.g., citrate synthase from an Antarctic bacterium (Gerike et al. 1998,
Russell et al. 1998), a-amylase from A ltero m o n a s haloplanctis (Aghajari et al.
1998 a, b), malate dehydrogenase from A q u a sp irilliu m a rcticu m (Kim et al.
1999), and triose-phosphate isomerase from Vibrio m a rin u s (Alvarez et al.
1998). An important conclusion obtained from these studies is that the
enzymes may use different strategies for cold adaptation. The structural
characteristics that have been identified as those which may be responsible
for cold adaptation are summarized as follows:
(i) Increased protein-solvent interactions through increased occurrence
of charged (or polar) residues in the loops or on the surface of a protein
molecule.
(ii) An increase in solvent-exposed hydrophobic residues, which is
thought to destabilize protein structure due to the ordering of water
molecules.
(iii) A loosened or relaxed N-terminal or C-terminal segment of the
protein molecule.
(iv) A decrease in the number of stabilizing interactions within the
protein molecule or between subunits, such as aromatic-aromatic
interactions, hydrogen bonds, and salt bridges. For example, arginine
residues form more stable bonds and provide more favorable interactions
than lysine residues. Thus, the arginine content, or Arg/(Arg + Lys) ratio,
of a protein is considered as an important factor for protein stability.
Lower contents of arginine have frequently been observed with cold-
adapted enzymes.
(v) A decreased occurrence of proline residues in loops and turns,
which provides a higher degree of freedom for structural segments
connected by loops. Proline is believed to restrict the main-chain flexibility
and therefore decrease the backbone entropy of unfolding (Hardy et al.

1993, Matthews et al. 1987).
(vi) Extension of the surface loop, causing higher freedom of motion of
the loop itself.
(vii) Lowered binding constants of metal ions, which may cause protein
stability.
(viii) A more accessible active site with optimized electrostatic potentials.
Some of these structural characteristics have been proved to be, at least
in part, responsible for the thermolability of cold-active enzymes (see
Section 6 for details). In addition, many of these structural characteristics
potentially result in the structural flexibility of proteins, providing
supporting evidence for a theory (Hochachka and Somero 1984) which
proposes that protein cold adaptation is attained by an increase in the
relative flexibility of the enzyme. According to this theory, the high catalytic
activity at low temperatures of cold-active enzymes is explained as follows.
Enzymes generally undergo small conformational changes during catalysis
with an energy barrier between such conformational transitions. At low
temperatures, only insufficient energy could be supplied to an enzyme to
overcome this barrier, and the kinetic motion of the enzyme would hence
be restricted. If an enzyme possesses higher structural flexibility (i.e., a
lower energy barrier for conformational changes), higher activity at low
temperatures of the enzyme should be attained.
The crystallographic temperature factor (B factor) can be a measure of
the structural flexibility of a protein; a higher B factor value represents a
higher degree of flexibility of the protein structure. X-ray crystallographic
studies of some cold-active enzymes showed that the B factors of their
whole protein structures are not necessarily higher than those of their
mesophilic and thermophilic counterparts, but the B factors of their active-
site environments are indeed higher (Kim et al. 1999, Russell et al. 1998,
Lonhienne et al. 2000). Unfortunately, however, there is currently no direct
experimental evidence connecting the molecular flexibility and high
catalytic activities at low temperatures. Furthermore, the mechanistic
relevance of the structural characteristics presented above to the high
activity at low temperatures of cold-active enzymes remains to be
unambiguously demonstrated. Nonetheless, the characteristics that may
cause the structural flexibility of proteins have usually been found with
cold-active enzymes and are indicative of protein cold adaptation. To
illustrate these circumstances, we briefly survey below the crystallographic
analyses of structural characteristics associated with some cold-active
enzymes.
Citrate synthase—The crystal structure of citrate synthase (DsCS) from
an Antarctic bacterium, strain DS2-3R, has been determined in a complex
with substrates at 2 A resolution and extensively compared with those of
hyperthermostable citrate synthase from archaeon Pyrococcusfuriosus (PfCS)

(Gerike et al. 1998, Russell et al. 1998). These enzymes show high amino
acid sequence identity (40%) to each other. However, DsCS has a
temperature optimum for the activity of 31°C, which is lower by 70°C than
that of PfCS; at 6°C, DsCS is 29-fold more active than PfCS. In addition,
DsCS is far more thermolabile than PfCS; DsCS and PfCS lose half of their
catalytic activity within eight minutes at 45°C and 100°C, respectively
(Gerike et al. 1997).
Structural analyses revealed that DsCS has a loop that is three residues
shorter than the equivalent loop in PfCS. Also, Arg353, which is located on
the opposite side of the active site of PfCS, is replaced by alanine in DsCS.
These differences are suggested to allow easier entrance of the substrates
into the active site cleft of DsCS. The increase in the number of positive
charges at and around the substrate-binding site in DsCS could also
enhance the catalytic efficiency toward the negatively charged substrate,
oxaloacetate. Another factor that may contribute to the high activity at low
temperature of DsCS includes an increase in the relative flexibility of the
small domain compared to the large one, because precise positioning of
the small domain after substrate binding is necessary for efficient catalysis.
However, the average crystallographic temperature (B) factor of the whole
DsCS molecule is much lower than that of PfCS. A number of other factors
which may be responsible for cold adaptation in DsCS have been identified:
(i) reduced subunit-interface interactions—especially no intersubunit ion-
pair networks, (ii) increased length of loops with higher numbers of
charged residues and fewer proline residues therein, and (iii) an increase
in solvent-exposed hydrophobic residues. It should be mentioned that an
increase of ion pairs within the subunit of cold-adapted DsCS in comparison
with the hyperthermophilic PfCS was observed, and this appears to be
associated with the resistance to cold denaturation.
Malate dehydrogenase—The three-dimensional structures of malate
dehydrogenase from the arctic bacterium Aquasprillium arcticum (AaMDH)
in three distinct forms—an apo form, a binary complex with NADH, and a
ternary complex with NAD/oxaloacetate—have been solved with 1.9-,
2.1-, and 2.5 A resolutions, respectively (Kim et al. 1999). These structures
have been compared with the available high-resolution structures of
thermophilic malate dehydrogenase from Thermusflavus (TfMDH), showing
61% sequence identity. The catalytic efficiency of AaMDH is 2-3 times
higher than that of mesophilic or thermophilic malate dehydrogenases at 4-
10°C, and the half-life inactivation of AaMDH at 55°C is only 10 min,
whereas TfMDH is fully active at 90°C for 1 hour. Comparison studies of the
3D structures of psychrophilic and thermophilic enzymes revealed several
structural factors that may be related to cold adaptation. Two of them have
been also observed for cold-active citrate synthase: more favorable
electrostatic potentials for substrate binding at and near the active site
region and the reduced intersubunit ion-pair interactions (see above). The
local flexibility of each MDH in an equivalent scale of thermal parameters
has been compared by introducing the "relative B-factor,” which has been
obtained by dividing the B-factors of all atoms in each enzyme by an
average B-factor of a whole molecule, considering the fact that
crystallographic temperature B-factors are sensitive to the difference in
resolution, packing, solvent content, and quality data. All main-chain and
most of the side-chain atoms interacting with NADH and oxaloacetate in
the AaMDH show up to 2-fold increased relative B-factors, reflecting their
increased relative flexibility. Two different methods have been used to
compare the relative flexibility of the active site regions in both MDHs. In
either case, the values that may represent the relative local flexibility for the
MDHs are higher in AaMDH than in TfMDH, suggesting that the active site
of the cold-active enzyme is relatively more flexible, which could facilitate
catalytic action at low temperatures. However, again as in the case of the
cold-active citrate synthase, the overall average B-factor for AaMDH is
significantly lower than that of its thermophilic counterpart. Nevertheless,
an increased local flexibility, estimated from crystallographic temperature
factors, has been experimentally observed for two cold-adapted proteins:
the higher relative flexibility of the small domain of citrate synthase and the
relatively flexible active site region of malate dehydrogenase.
Triose-phosphate isomerase—The three-dimensional structures of triose-
phosphate isomerase from the psychrophilic bacterium Vibrio marinus
(vTIM) in a complex with sulfate and with 2-phosphoglycolate have been
solved with 2.7 A resolution (Alvarez et al. 1998). They are the first
published experimental X-ray crystal structures of a cold-adapted enzyme.
vTIM is a very unstable protein with a half-life of only 10 min at 25°C,
showing the intrinsic characteristic of cold adaptation. The vTIM sequence
is most closely related to the sequence of mesophilic triose-phosphate
isomerase from Escherichia coli (eTIM) with 66% of sequence identity. The
catalytic activity of vTIM at 10°C is very similar to that of eTIM at 25°C.
Although the three-dimensional structures of both enzymes are similar, no
exhaustive structural comparison has been made in an attempt to find out
the structural factors of vTIM related to cold adaptation. Nevertheless, a
careful comparison of all known 45 sequences of TIM revealed that cold-
adapted vTIM has a unique residue in the phosphate binding helix,
Ala238, which is replaced by serine in all other TIM sequences. To find out
the implications of this unique sequence feature, Ala238 has been replaced
by serine, and the resultant mutant, the A238S mutant, has been
characterized. It turns out that this point mutation resulted in a decrease
in the catalytic efficiency at 10°C and an increase in the thermostability, as
evidenced by calorimetric studies. Therefore, alanine at this position should
Table 3. Structural properties of glutamate racemases
Parameters Source organisms of glutamate racemase

Gram-negative Low G+C content
proteobaderia Gram-positive baderia
Psy* Eco’ Car* Bpu*
Number of amino acids
total 2% 285 268 272
strongly basic (K, R)b 27 26 31 25
strongly acidic (D, E)b 30 35 28 30
hydrophobic (A, I, L, F, W, V)b 120 123 99 97
polar (N, C, Q, S, T, Y)b 81 53 70 72
Amino acid content (%)
proline 5.1 6.7 4.1 4.8
glycine 5.1 6.0 6.3 7.4
arginine 3.7 5.6 3.4 2.6
lysine 5.4 3.5 8.7 6.6
A ig/A rg+Lys ratio 0.4 0.7 0.3 0.3
Hydrophobidty indeces
GRAVY' -0.015 0.187 -0.059 -0.04
aliphatic index 105.1 105.8 98.6 101.4
P1 6.2 5.2 8.4 6.3
Charge at pH 7.0 -2.4 -8.2 3.8 -3.7
Insertions/deletionsd 5 /2 0 /0 1/1 0 /0
N-terminal extention'
(number of residues) 10 21 0 0
“Abbreviations: Psy, P sy ch ro ba cter sp. Ant300; Eco, Esch erich ia coli; Car, C a m o b a d e r iu m sp. St2;
Bpu, B acillus p u m ilu s.
bAmino acids are shown by one-letter notation.
‘Grand average of hydropathidty.
‘‘In comparison with mesophilic counterpart.
'N-terminal extension plays an important role for the regulation of Eco glutamate recemase
and is also present in the Psy enzyme. The Eco enzyme is strongly activated by the
presence of UDP-N-acetylmuramoyl-L-alanine, the precursor of a peptidoglycan. In contrast,
the enzymes from Gram-positive bacteria are not activated at all.
BpuGR are lower than those of any GRs from gram-negative bacteria;
therefore, a decrease in arginine contents could be essential as an adaptation
factor only for PsyGRbut not forCarGR. Thus, reasonable comparison can
be made only by studying a set of enzymes from organisms belonging to
similar taxonomic groups.
Alanine dehydrogenase studies—AlaDH catalyzes the reversible NAD+-
dependent deamination of L-alanine to pyruvate. In gram-positive bacteria,
AlaDH is of physiological importance for cell survival under extreme
conditions by participating in the sporulation process (Siranosian et al.
1993). On the other hand, AlaDH from non-spore-forming gram-negative
bacteria probably serves as a link between the carbon and amino acid
metabolism, providing the cell with both nitrogen and/or energy.
COLD-ACTTVE ENZYMES FROM COLD-ADAPTED MICROORGANISMS 15
To study the structural factors responsible for cold adaptation of AlaDHs,

AlaDH genes were cloned from two different species of cold-adapted
bacteria, Shewanella sp. AclO (gram-negative bacteria; SheAlaDH) and
Camobacterium sp. St2 (CarAlaDH) (Galkin et al. 1999). Multiple sequence
alignment and the corresponding phylogenetic tree of these AlaDH
sequences have been constructed with those of other known homologs
(Fig. 3B). As was the case for GRs, no discontinuous relationship among
the AlaDH genes due to unusual evolutionary processes was found,
allowing us to classify AlaDHs into two major clusters: AlaDHs from low
G+C gram-positive bacteria and those from the y-subdivision of gram
negative Proteobacteria. Among all AlaDHs thus far sequenced, SheAlaDH
showed the highest sequence similarity to that of AlaDH from the gram
negative bacterium Vibrio proteolyticus (VprAlaDH), and CarAlaDH was
most similar to AlaDHs from mesophilic and thermophilic Bacillus strains
(Galkin et al. 1999).
We then analyzed the enzymological properties of SheAlaDH and
CarAlaDH to find that these enzymes show features typical of cold-
adapted enzymes; both the optimal temperature for catalytic activity and
the temperature limit to retain thermostability were lower than the values
obtained for the mesophilic counterparts (Table 4 and Fig. 1) (Galkin et al.
1999). The k ,/K value for the SheAlaDH reaction was about three times
higher than that for VprAlaDH but much lower than that for AlaDH from
Bacillus subtilis (BsuAlaDH) (Galkin et al. 1999). This high catalytic efficiency
of BsuAlaDH may presumably be related to its absolute importance for
survival of this spore-forming bacterial host (see above). Because it has
been suggested that BsuAlaDH from spore-forming Bacillus subtilis is
more evolutionarily advanced in developing its catalytic power on a way
from ancient to present-day enzymes (Demetrius 1995), we can propose
that AlaDHs from gram-negative bacteria were subjected to less selective
pressure during the evolutionary process to maximize its catalytic
competence or that gram-negative bacteria could compensate such
difference in the catalytic efficiency by optimization of its expression and
intracellular concentration.
We then carried out a comparative analysis of the primary and tertiary
structures of AlaDHs for the identification of structural factors associated
with cold adaptation using two psychrotrophic AlaDHs (i.e., SheAlaDH
and CarAlaDH), two mesophilic AlaDHs {i.e., VprAlaDH and BsuAlaDH),
and AlaDH from the thermophilic bacterium Bacillus stearothermophilus
(BstAlaDH) (Table 4). Because the X-ray structure of AlaDH from the
cyanobacterium Phormidium lapideum (PlaAlaDH) was recently determined
at a high resolution (Baker et al. 1998), we constructed the homology-
based 3D structural models of these AlaDHs for comparison.
A 16S rDNA GR
B 16S rDNA AlaDH
16 141210 8 6 4 2 0 0 5 10 15 20
Figure 3. (A) Comparison of phylogenetic trees constructed on the basis of amino acid
sequences of bacterial glutamate racemase (GR, right) and 16S rDNA sequences of bacterial
hosts (left). (B) Comparison of phylogenetic trees constructed on the basis of amino add
sequences of bacterial alanine dehydrogenase (AlaDH, right) and 16S rDNA sequences of
their source strains (left). Roman numerals indicate dustering of sequences into two major
categories of low G +C gram-positive bacteria (I) and y-subdivision of gram-negative
Proteobacteria (II). A balanced dadogram was constructed from a matrix of pairwise genetic
distances generated by the CLUSTAL W method by using the MEGAUGN program. The
scale indicates percentages of sequence divergence. Abbreviations of spedes names are as
follows: Bpu, B acillus p u m ilu s ; Bsu, B acillus su b tilis; Bee, B a cillu s c ereu s; Shm, S taph ylo co ccus
h a em o ly ticu s; Bsp, B a cillu s sp h a ericu s; Lbr, Lactobacillus brev is; Ppe, P ed iococcus p en to sa ceu s; Lfe,
L a cto b a c illu s f e r m e n t u m ; Car, C a r n o b a c t e r iu m sp. St2; Mle, M y c o b a c t e r iu m le p r a e ; Met,
M y c o b a c te r iu m t u b e r c u lo s is ; Cgl, C o r y n e b a c t e r iu m g lu t a m i c u m ; Eco, E s c h e rich ia c o li; Hin,
H a e m o p h ilu s in flu e n z a e ; Psy, P sy ch ro b a cter sp. Ant300; Nme, N e isseria m e n in g it id is ; Zmo,
Z y m o m o n a s m o bilis; Hpy, H elico b a cter p y lo ri; Aae, A q u ife x a eo licu s; Syn, S y n ech o cy stis sp. PCC
6803; Vpr, V ibrio p ro teo ly ticu s; and She, Shexvanella sp. AclO.
Table 4. Structural properties of alanine dehydrogenases
Parameters Source organisms of alanine dehydrogenase

■ /-subdivision of low G+C content
proteobacteria Gram-positive bacteria
Vpr* She* Bst* Bsu* Car*
Optimum growth temperature (°C) 37 20 57 37 20

Thermostability (°C)b 63 59 81 64 41
Optimum temperature 53-57 47-50 75-80 60-64 35-38
for activity (°C)
K J K (mM-V*)' 36 27 nd 47-140 nd
Amino acids
total 374 371 372 378 371
polar (N, C, Q, S, T, Y,)d 67 73 75 75 86
hydrophobic (A, I, L, F, W, V)d 156 151 143 154 140
arginine 13 15 18 11 5
lysine 20 17 18 21 26
A rg/Arg+Lys ratio 0.39 0.47 0.5 0.34 0.19
proline contents (%) 4.5 4.3 4.3 5.0 5.0
glycine contents (%) 8.8 9.4 10.8 10.1 10.1
P1 5.7 5.9 6.6 5.3 4.6
Charge pH 7.0 -9.2 -6.5 -2.1 -9.7 -19.1
Aliphatic index 101.5 106.4 97.5 104.3 97.2
Hydropathidty (GRAVY)" 0.053 0.228 0.053 0.113 0.094
Salt bridges' 117 87 132 99 60
Aromatic interactions' 36 30 54 42 48
Hydrogen bonds' 1892 1986 1936 1947 1962
Hydrophobic interactions* 2.2 2.1 2.2 2.2 2.1
"Abbreviations are: Vpr, V ib rio p r o t e o ly t ic u s ; She, S h ew a n e lla sp. AclO; Bst, B a c illu s
Bsu, B a cillu s su b tilis; Car, C a m o b a c te riu m sp. St2.
slea ro th erm o p h ilu s;
'Temperatures at which the enzyme loses 50% of its original activity after 30-min incubation.
'At 25°C. See (Galkin e l al. 1999) for details.
dAmino acids are shown by one-letter notation.
"Grand average of hydropathidty.
Total numbers in hexamer.
'Ratio of buried apolar to polar surface area.
We found that the total numbers of salt bridges clearly decrease in the
order of thermophilic through psychrotrophic AlaDHs when we compared
the 3D structures of AlaDHs from the same bacterial subgroups with each
other (SheAlaDH and VprAlaDH; and CarAlaDH, BsuAlaDH, and
BstAlaDH; see Table 4). Therefore, salt bridges should play an important
role in the stabilization of AlaDHs, and a decreased number of salt bridges
is an essential factor for the cold adaptation of both CarAlaDH and
SheAlaDH. Specifically, a very clear correlation between the molar ratio of
basic residues Arg/(Arg+Lys) (see Section 4) and protein thermostability
(see also Fig. 1) could be identified with AlaDHs from gram-positive
bacteria by primary-structure comparison studies (Table 4), and the

decreased number of arginine residues in CarAlaDH is indeed associated
with the loss of 5 and 8 potentially stabilizing ionic interactions per
subunit in comparison with BsuAlaDH and BstAlaDH, respectively.
However, the Arg/(Arg+Lys) ratio for the gram-negative SheAlaDH is
nearly the same as that for thermostable BstAlaDH and even higher than
those for two other mesophilic enzymes from both taxonomic divisions.
Therefore, the decreased number of arginine-mediated ionic interactions
could be responsible only for the thermolability of CarAlaDH and not for
that of SheAlaDH.
The slightly decreased hydrophobic interactions of the protein cores of
both cold-active AlaDHs, as estimated using a ratio of the buried apolar
surface area to the polar surface area of proteins, could be another structural
factor related to cold adaptation. However, this assumption cannot be
derived from comparisons using other parameters of protein
hydrophobicity, such as aromatic-aromatic interactions and the contents
of hydrophobic amino acids (Table 4): AlaDHs from gram-positive bacteria
have higher numbers of aromatic-aromatic contacts than those of enzymes
from gram-negative bacteria, and this trend seems to be independent from
both the host habitant temperatures and the thermostability of the proteins.
Although a decrease in proline content has frequently been observed
with cold adaptation of enzymes and proteins, variation of proline contents
in AlaDHs does not seem to be related to protein temperature adaptation
but, rather, to species specificity: the proline contents of AlaDHs from
gram-negative bacteria are lower than those from gram-positive ones.
To sum up, it can be stated that, as in the case of glutamate racemases,
many structural factors observed with cold-active AlaDHs have been
found to arise from taxonomic species specificity rather than temperature
adaptation. It is therefore difficult to correctly predict the structural
characteristics responsible for cold adaptation by primary- and tertiary-
structure comparison studies if evolutionary relationships of enzymes are
ignored.
6. Comparative Mutagenesis Studies and Rational Design of Cold-

adapted Hydrolytic Enzymes
Site-directed mutagenesis of cold active enzymes, based on the sequence
and structural comparisons with mesophilic and thermophilic counterparts,
provides a powerful strategy to analyze the mechanism of protein cold
adaptation. We have already discussed the results of mutagenesis studies
of the cold-active triose-phosphate isomerase. In these studies, the protein
structure could be rigidified by restoring the hydrogen bonding network,
and an inverse correlation between thermostability and cold activity of
the enzyme was found (see Section 4). Below, we will discuss the results
of comparative mutagenesis studies on cold-adapted lipases and
proteinases in an attempt to clarify the relationships between their cold
activities and thermostabilities.
Lipase—Lipases catalyze the hydrolysis of acylglycerides and other
fatty acid esters and serve as versatile biocatalysts that are useful for a
wide variety of applications, for which enzyme thermostability is regarded
as one of the most important characteristics (Herbert 1992).
Recently, we have isolated lipase-producing cold-adapted
microorganisms from cold environments (Choo 1998, Kulakova 2000) and
cloned three distinct lipase genes. We successfully expressed these lipase
genes in E. coli cells, purified the recombinant enzymes to homogeneity,
and characterized them. They were found to be cold-active enzymes
because they exhibit high levels of activity at temperatures ranging from
5 to 25°C and low thermostabilities in comparison with their mesophilic
counterparts (Choo 1998, Kulakova 2000). One of these lipases, PsyLip
obtained from Antarctic Psychrobacter sp. strain Ant300, was found to be a
homolog of the hormone-sensitive lipase (HSL) group of the lipase/
esterase family (Hemila et al. 1994). Using the PsyLip gene, we then carried
out the following rational mutagenesis studies in an attempt to enhance
the thermostability of this enzyme.
We constructed a modeled 3D structure of PsyLip on the basis of the
published 3D structure of brefeldin A esterase (Wei et al. 1999), which is
also a member of the HSL group (Choo 1998, Kulakova 2000). Comparisons
of the primary and modeled 3D structures of PsyLip with those of the
mesophilic and thermophilic members of the HSL group have revealed
parameters which should be possibly involved in cold adaptation: a lower
Arg/(Arg+Lys) ratio, fewer ionic and aromatic interactions, and an
essentially lower proline content (see also Section 4). Particularly, one
proline residue that is highly conserved among the mesophilic and
thermophilic members was found to be substituted by glycine in PsyLip.
This glycine, Gly244, is located near the active site in the turn connecting
the P-sheet and the a-helix (Fig. 4A). Thus, this glycine was replaced by
proline by site-directed mutagenesis (Fig. 4B), and the resultant PsyLip
mutant was biochemically characterized. The results showed that the
thermostability of PsyLip was enhanced by replacement of Gly244 by
proline: the mutant enzyme retained almost 95% of its original activity
after incubation at 40°C for 60 min, while the wild-type enzyme lost about
80% of its activity. Moreover, this amino-acid substitution resulted in 2.6-
fold diminution in the overall catalytic efficiency (kai/Km) for the hydrolysis
of p-nitrophenyl valerate, along with a shift of the enzyme's substrate
specificity toward a preference for short acyl-chain substrates at the active
site. Probably, the active site region might be rigidified by the introduction
N>
O
Figure 4. (A) A modeled close-up view of an environment near the catalytic triad (Asp331, His361, and Ser216) and Gly244 of a lipase (PsyLip) from
Antarctic P sy ch ro ba cter sp. strain Ant300. (B) Active-site environment of an engineered PsyLip, where Gly244 has been replaced by proline.
of a proline residue, resulting in a reduced catalytic activity. Moreover, in

the mutant enzyme, positioning of the substrate with a long acyl chain at
the active site may become more difficult due to decreased flexibility. As
in the case with triose-phosphate isomerase, the results of this mutagenesis
studies show an intimate inverse relationship between thermostability
and cold activity.
Serine proteinoses—Subtilases are members of the superfamily of
subtilisin-like serine proteinases. Over 200 sequences of subtilases are
currently known, and three-dimensional structures of subtilisin Carlsberg
(Neidhart and Petsko 1988), subtilisin BPN' (Hirono et al. 1984), subtilisin
Savinase (Betzel et al. 1992), thermitase (Gros et al. 1991), and proteinase K
(Betzel et al. 1988) have been reported. A wealth of data available for
subtilases makes members of this family attractive targets for studying the
structure-function relationships of proteinases.
Several cold-active members of subtilases have recently been found
from cold-adapted bacteria. One of such subtilases, termed "subtilisin
S39" secreted by Antarctic Bacillus sp. strain TA 39, has been purified to
homogeneity and characterized (Narinx et al. 1997). At 5°C, this enzyme
displays a 7-fold higher specific activity for azocasein hydrolysis than
subtilisin Carlsberg, a mesophilic member of the family. It also shows
much lower thermostability than subtilisin Carlsberg. Thus, the enzyme
displays the usual properties of cold-adapted enzymes. The analysis of
the modeled structure of subtilisin S39 revealed some structural differences,
which potentially enhance the structural flexibility of the enzyme to result
in high catalytic activity at low temperatures as well as low thermostability.
Site-directed mutagenesis was then performed with the aim to rigidify the
protein molecule by restoring either salt bridges or aromatic-aromatic
interactions, by introducing an additional disulfide bridge, and/or by
increasing the affinity of one of Ca2+-binding sites in the enzyme. Very
interesting results were obtained with one of such alterations, i.e., the
substitution of Thr85 by aspartic acid. This amino-acid substitution resulted
in a 100-fold increase in the enzyme’s affinity for Ca2+and an enhancement
of enzyme thermostability. Moreover, the specific activity of this mutant
toward azocasein was found to be 1.7-3 times higher than that of wild-
type enzyme (Narinx et al. 1997).
Finally, we will refer to site-directed mutagenesis studies of serine
alkaline proteinase (SapSh) from Antarctic bacteria Shewanella sp. strain
AclO, which is also one of subtilases (Kulakova 2000). The biochemical
characterization of SapSh revealed that it is also a cold-adapted enzyme.
SapSh is five times more active than subtilisin Carlsberg at temperatures
ranging from 5 to 15°C and is far less stable than the mesophilic subtilisin.
The optimal temperature for the catalytic activity of SapSh (40-45°C) was
much lower than that of subtilisin Carlsberg (60-65°C).
The homology-based modeled structure of SapSh was built using the

known crystal structures of subtilisin BPN', subtilisin Carlsberg, and
thermitase as templates. The analysis of their modeled structure revealed
several features which may be responsible for the cold adaptation of
SapSh, i.e., fewer aromatic-aromatic interactions, more interactions with
solvent due to the increase in the number of acidic residues on the
molecular surface, and particularly fewer salt bridges. Only one salt
bridge was found in the modeled structure of SapSh; on the other hand,
five and ten salt bridges were found in those of mesophilic subtilisin BPN’
and thermophilic thermitase, respectively. Therefore, loss of ionic
interactions should be particularly important for the observed
thermolability of SapSh. This, in turn, implies that the incorporation of
additional salt bridges to restore the ionic network in the SapSh structure
should increase the structural rigidity, resulting in the enhanced
thermostability of SapSh. We have tested this prediction by site-directed
mutagenesis studies (Kulakova 2000).
Among mutations introduced in SapSh, remarkable stabilization of
SapSh was achieved with substitution of Asn251* by glutamic add, and
the optimal temperature of activity of the mutant was higher by 5°C than
that of the wild type (an asterisk indicates the residue number on the basis
of alignment of the amino acid sequences of SapSh and subtilisin BPN’).
The structural model of the SapSh mutant suggested that enhancement of
the thermostability of SapSh by the Asn251*Glu substitution should arise
from the ability of Arg247* to form an additional salt bridge with newly
introduced Glu251*( along with a preexisting salt bridge between Arg247*
and Glul97* (Fig. 5). In this particular case, VMx values at low and
moderate temperatures of the mutant were lower than the values of the
wild-type enzyme (Table 5). However, the overall catalytic effidency
(V__/ K J of the mutant enzyme was even higher than that of the wild-
type enzyme because of the improved Michaelis constant of the mutant
(Table 5).
Table 5. Optimum temperatures and kinetic parameters of wild-type and N251*E mutant
SapShs.
K. V™ V '-./K n
(°C) (mg ml-1) ( s 1) (ml mg’1 s"1)
wild-type 45
4°C 0.21 1.7 8.6
20°C 0.29 5.8 20.8
N251*E 50
4°C 0.049 1.1 23.1
20°C 0.10 3.4 34.0
Azocasein was used as a substrate.
•Numbered on the basis of subtilisin BPN'.
Figure S. A modeled dose-up view of Arg247*-mediated salt bridges with Glu251* and Glul97*
in an engineered serine alkaline proteinase (SapSh) from an Antarctic bacterium Sh ew anella
sp. strain AclO. Asterisks indicate the residue number on the basis of alignment of amino add
sequences of SapSh and mesophilic subtilisin BPN'.
In conclusion, the results of site-directed mutagenesis studies presented

above provide good evidence for the possibility to create enzymes with
enhanced thermal stability and unchanged, or even improved, catalytic
efficiency at low and moderate temperatures. The rational design of
mutations could open a new perspective for the biotechnological application
of cold-active enzymes.
7. Concluding Remarks
X-ray crystallography as well as site-directed mutagenesis studies have
provided strong evidence that the cold adaptation of enzymes from cold-
adapted organisms is based on altered intramolecular and intermolecular
interactions that may enhance the flexibility of their molecular structures.
The data presented here show that not ail cold-adapted enzymes necessarily
use the same strategy for adaptation of their structures to low temperatures.
Because there is a possibility that species-specific differences between
source organisms may be erroneously assigned as structural characteristics
responsible for protein cold adaptation by means of structure comparison

studies, attention should be paid to the taxonomic similarities of source
organisms to obtain a rational set of counterpart enzymes: cold-adapted
and usual ones. In other words, a reasonable comparative analysis of cold-
adapted and mesophilic enzymes can be obtained through the studies of
enzymes from source organisms belonging to the same taxonomic group.
On the basis of this sort of reasonable comparison, a rational design of
enzymes with improved characteristics of biotechnological importance
can be done through protein engineering approaches. Thus, it may be
possible to construct cold-adapted enzymes with high activity at cold
temperatures in order to apply them to a cold environment without
sacrificing protein stability. Moreover, the studies will open horizons for
a better understanding of the mechanisms of adaptation of living organisms
to the cold environments on earth.
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E n g i n e e r i n g o f M i c r o b i a l D i o x y g e n a s e s f o r th e
D e g ra d a tio n o f X e n o b io tic C o m p o u n d s
fun Hirose1and Kensuke Furukawa2
'Department of Applied Chemistry, Faculty of Engineering, Miyazaki University,

Miyazaki 889-2192, Japan
2Department of Biosdence and Biotechnology, Faculty of Agriculture,
Kyushu University, Fukuoka 812-8581, Japan
In tro d u c tio n
Over the past century, a large variety and a large quantity of hazardous
chemical compounds were produced in industry, and some of these were
released into the environment, resulting in serious environmental problems.
Bioremediation is one of the most promising approaches to cleaning up
such polluted soil, sediment or water, in which catabolic functions of
microorganisms are explored and engineered. Microbial degradation of
such xenobiotic compounds can often be initiated by oxygenation. In this
reaction, one or two atoms of molecular oxygen can be incorporated into
the substrates. The enzymes involved in such oxygenation are called
monooxygenase and dioxygenase (Dox), respectively. However, once
chlorines are substituted into the substrates, these compounds become
extremely resistant to microbial attack. Figure 1 shows the microbial
catabolic pathway of biphenyl/polychlorinated biphenyls (PCB), which is
one of the best-characterized degradation pathways to date. In this reaction
two dioxygenases (Doxes) are involved; the first Dox introduces two
hydroxyl groups into the biphenyl ring and the second Dox cleaves the
hydroxylated ring. The former reaction requires NADH as an electron
donor, but the latter does not require an external reductant. Similar catabolic
pathways catalyzed by Doxes are also well documented with respect to
the degradation of benzene, toluene, naphthalene and phenanthrene. The
first Doxes involved in the initial oxygenation are of particular importance
because this reaction destabilizes the aromatic ring and initiates the
degradation of aromatic compounds. This review deals with the
engineering of the initial Doxes, focusing on the acquision of new and
novel degradation capabilities for PCB and trichloroethylene (TCE).
s
Initial dioxygenation Second dioxygenation

Figure 1. Oxidative degradation of a polychlorinated biphenyl (PCB) by bacteria.

Exploring the Variety of Random
Documents with Different Content
HON. BEEKMAN WINTHROP
Copyright, Harris-Ewing, ’08.
THE SICILIAN AND CALABRIAN

EARTHQUAKE
“Messina and Reggio destroyed by an earthquake” flashed over
the wires and appeared in our press the last days of the year. The
terrible news, with its story of the fearful loss of life and property,
seemed too appalling to be true. The world, though stunned by its
magnitude, was yet to learn that no pen could describe the horrors of
a disaster unparalleled in modern history, and that only those who
saw the scene of devastation soon after the catastrophe have any
realization of its terrible results. As for those who lived through the
earthquake and escaped, the mental fear and physical agony they
had undergone left their minds dazed and blank. When some
realization of the truth dawned upon the world a wave of sympathy
was awakened everywhere. It is especially for such times of disaster
that the Red Cross has its being, and the call for help was
immediately issued from headquarters at Washington. The President
and Governors of States were notified that our National Society was
ready to receive and transmit the contributions our people were glad
to make for suffering Italy. President Roosevelt, in his cables to the
King of Italy, expressing his own and his countrymen’s sympathy,
stated that the “American Red Cross has issued an appeal for the
sufferers.” Many Governors of States issued proclamations, asking
that all contributions be sent through the American Red Cross. How
promptly and how generously, our people expressed their sympathy
in tangible shape is known everywhere. Glad were we in America to
do what we could to help our suffering fellow-men in beautiful and
well-loved Italy. Something of what the American Red Cross, our
national member of that greatest of all institutions of international
brotherhood, has been able to do with the contributions it has
received is told in this Bulletin by those who in Italy have helped to
administer the funds. In all of this work the Society has had the most
valuable and untiring assistance of Mr. Lloyd Griscom, the American
Ambassador at Rome. It cannot too strongly express its appreciation
of all that he has accomplished in the line of careful and prompt use
of the money it has sent. What our Red Cross has accomplished has
been done with a sincere desire to be of help, with a deep
appreciation of the complex and difficult problem Italy has had and
still has to face, and with the hope that the wounds of this beautiful
country, so recently devastated by this terrible calamity, may soon be
healed and the people re-established in a happy and prosperous life.
MAJ.-GEN. GEORGE W. DAVIS
ERNEST P. BICKNELL
CONTRIBUTIONS TO THE ITALIAN

RED CROSS
Knowing that the Italian Red Cross was especially well organized
for carrying on hospital relief work, because of its field hospitals,
fourteen hospital trains and equipment for two ships’ hospitals,
besides an active personnel, the American Red Cross transmitted to
it through our Ambassador at Rome $320,000 to be applied to its
relief work in the earthquake district. The Italian Red Cross, in two
previous Calabrian earthquakes and at the time of the Vesuvian
eruption, maintained a number of hospitals and relief stations. At the
time of the latter disaster the American Red Cross received about
$12,000, which was transmitted to the Italian Red Cross. Later a
special report was made by this Society of the relief work it
performed at that time. A report of the relief operations in Southern
Italy will doubtless be issued sometime in the future, but this must
not be expected too soon, as experience has taught how long drawn
out is relief work after serious disasters. Baron Mayor des Planches,
the Italian Ambassador at Washington, in speaking of the Italian Red
Cross, said:
CHARLES L. MAGEE.
“As the representative of the Italian Government, I desire to

give the strongest indorsement of the Italian Red Cross, with
which the American Red Cross is in the most intimate
relation, and to say that my Government places absolute
confidence in this great national organization.”
On January 4, the following cablegram was received from Count
Taverna:
“The Italian Red Cross tenders sincerest thanks to

American Red Cross for conspicuous contribution of
1,538,500 Italian lire, received through American Ambassador
in Rome, toward the relief of the distressed districts of
Reggio, Calabria and Messina, and begs to express its keen
appreciation of the feelings of solidarity and warm sympathy
with the stricken populations, which have prompted their
generous act.
“COUNT TAVERNA, President Italian Red Cross.”
Since this despatch was received further remittances have been

made, bringing the total of the American Red Cross contributions to
the Italian Red Cross up to $320,000.
ROBERT W. DE FOREST
THE AMERICAN RED CROSS

ORPHANAGE
Hundreds of little
children were left
fatherless and
motherless amidst the
ruins of Messina and
Calabria. Scores of
them were even too
young to be able to give
any information in
regard to themselves or
their families. For years
these must be cared for,
and having been left
without property or
relatives, must be so
educated that, after
reaching mature years,
they will be able to
support themselves.
Queen Helena. Helpless childhood
appeals strongly to
everyone, and the Red
Cross, which after great calamities aims when the first temporary aid
is over, to rehabilitate and place again upon their feet the victims of
the disasters, was ready to accept the suggestion of the Italian
Government that some of the funds entrusted to its administration by
the American people should be devoted to the maintenance of an
agricultural colony in Sicily or Calabria for the care of a hundred or
more of the orphaned children. In national relief the American Red
Cross does not permit the use of its emergency funds for the
purpose of any permanent endowments, but in international relief it
believes it wisest to act under the suggestion of the American
diplomatic representative, the Government and relief committees in
the country where the disaster occurs. Therefore, when Mr. Griscom,
the Ambassador at Rome, after consulting with the Italian
Government, asked that such an agricultural orphanage colony be
maintained by a donation from the American Red Cross, the
suggestion was promptly complied with. Two hundred and fifty
thousand dollars are to be devoted to this purpose.
REAR-AD. PRESLEY M. RIXEY
The colony will be situated in Sicily or Calabria, and will consist of

model farms, where scientific agricultural instructions will be given by
agents of the Royal University of Agriculture. The Italian Government
will furnish the land, and the Italian National Relief, under the
patronage of Queen Helena will provide the buildings. It will be called
“The American Red Cross Orphanage,” and the American
Ambassador is to be an ex-officio member of its governing
committee. It is to be a lay institution, and not ecclesiastical. A yearly
budget of its expenses will be published, which must meet the
approval of the Minister of the Interior, who at present is also the
Prime Minister. A number of the poor women left widows and
dependent by the earthquake, and who in many cases also lost their
little children, will be given employment at this orphanage, and the
care of other little children will help to lift this sorrow from their
hearts. From these women the children will receive again much of
that mother-love and care of which this terrible disaster has robbed
them.
SURG.-GEN. WALTER WYMAN
Speaking of this orphanage, Mr. Griscom writes on February 19 to

the chairman of the Central Committee of the American Red Cross:
“I can assure you that this generous gift of the American
Red Cross has made a profound impression in Italy. I made
the formal presentation to Her Majesty, the Queen, on the
16th instant, and Her Majesty was overcome with emotion
and for a moment at loss to express herself. Finally she made
a beautiful speech and poured forth her admiration for the
organization of the American Red Cross.”
Ambassador Griscom, under date of February 18, forwarded to the

State Department for transmission to the American Red Cross two
letters from the Countess Spaletti Rasponi, the President of the
Patronato Regina Elena, and from the Honorable Bruno Chimirri,
President of the “Comitato di Vigilanza,” respectively, expressing the
gratitude of the Committee and Council of the Patronato Regina
Elena for the gift of $250,000, for the establishment of the
Orphanage. The letters referred to follow:
MAJ.-GEN. R. M. O’REILLY
Copyright, Harris-Ewing, ’08
“Excellency:
“The Council of the ‘Opera Nazionale di Patronato Regina
Elena,’ having known of the conspicuous offer of 1,300,000
lire made by the American National Red Cross in favor of the
children whom the recent earthquake has thrown into the
condition of orphans, has passed a vote of thanks to the
officers and to Your Excellency, to whose influential interest it
is due if so important a part of the funds collected in America
has been devoted to our institution.
“And I, interpreting the desire of the Council, warmly and
specially beg Your Excellency to kindly transmit to the
meritorious American Red Cross the expression of our
profound and heartfelt gratitude toward all the noble and great
American nation, not inferior to any other in all the
manifestations of human genius and solidarity.
“With the assurances of my highest consideration,
“The President,
(Signed) “COUNTESS SPALETTI RASPONI.”
HON. ROBERT BACON
“Mr. Ambassador:
“I have the honor to offer you the warmest thanks of the
Committee and Council of the ‘Opera Nazionale di Patronato
Regina Elena’ for the generous offer which you have made on
behalf of the Calabrian and Sicilian orphans.
“I beg you to be good enough to be interpreter of our very
grateful sentiments to the American Red Cross, which has
completed, with its splendid gift, its relief work in Calabria and
Sicily.
“The Agricultural Colony, which will be named American
Red Cross Orphanage,’ will perpetuate the remembrance of
this charity, and will contribute to render continually more
close the ancient ties of sympathy and friendship which unite
Italy with your mighty Republic, ties which you called attention
to in your brilliant speech on the occasion of the centenary of
the great President Lincoln.
“Accept, Mr. Ambassador, the assurances of my high
consideration.
(Signed) “B. CHIMIRRI.
“To His Excellency,
“Hon. Lloyd C. Griscom,
“Ambassador of the United States of America, Rome.”
MED. DIRECTOR J. C. WIRE
HOUSES FOR ITALY

Our own experiences
after serious disasters
in the United States
have taught us that in
nearly all of such cases
one of the most serious
problems to be met is
the providing of shelter
for the thousands—
sometimes hundreds of
thousands of victims.
Italy has had this same
serious problem to meet
after the late
unparalleled disaster in
Sicily and Calabria. The American Ambassador at Rome was
requested by the State Department to consult with the Italian
Government as to the best use to be made of the $500,000 left by
the Congressional appropriation of $800,000, after the supplies on
the Navy ships, Celtic and Culgoa, which were sent to the scene of
the disaster, had been paid for. The reply came in the nature of a
request that this fund be expended in the purchase and providing of
materials for houses. This suggestion has been admirably carried
out by the Navy Department, which has purchased and shipped, fully
prepared, materials for the immediate erection of 2,500 houses,
including window sashes, doors, etc., and the charter of four ships
for their transportation. Some eight expert carpenters and a large
number of tools have been sent on these vessels, that the erection
of these houses may go on promptly.

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Library of Congress Cataloging-in-Publication Data

Bioremediation/ editors, Milton Fingerman, Rachakonda Nagabhushanam.

TD1 9 2 .5.B55683 2003

ISBN Set 1-57808-010-X

© 2003, Copyright Reserved

All rights reserved. No part of this publication may be reproduced, stored

Published by Science Publishers, Inc., Enfield, NH, USA

T h is v o lu m e is th e e ig h th in th e o n g o in g s e rie s w e h a v e e n title d Recent

C h e m ic a l a n d B io lo g ic a l T ra n s fo rm a tio n o f P a r a ly ti c S h ellfish 319

Audrey Galkin', Ljudmila Kulakova1, Toru Nakayama2,

1. Cold-adapted Microorganisms as Sources of Cold-active Enzymes

Isolate 16S rDNA Accession Similar organism Gram staining

2. General Properties of Cold-adapted Enzymes

Table 2. Catalytic properties of hydrolases from cold-adapted and mesophilic organisms

Enzyme Source E. Reference

a-amylase A ltero m o n a s h a lo p la n d i& (Antarctica) 187 73 (Feller et al. 1992)

a n d th e r m o p h ilic c o u n t e r p a r ts (G e r d a y et al. 1 9 9 7 ). It s h o u ld b e m e n tio n e d

Figure 1. Thermal stabilities of alanine dehydrogenases from psychrotrophic microorganisms

recently been claimed that the general thermolability of cold-active enzymes

3. Potential Applications of Cold-active Enzymes in Biotechnology

and fish-paste products, (iii) In the fruit-juice-making processes, cold-

|FDH | jAlaPH | D-ATA

COs - 'NADH ♦ H * — Pyruvate «--------^ D -A m in o acids

Figure 2. Enantioselective production of D-amino acids by a multienzyme system consisting

Additives in laundry detergents—Hydrolases such as proteinases, amylases,

washing is conventionally done with tap water at cold or moderate

enzyme to efficiently hydrolyze triglycerides such as soybean oil during

4. Structural Characteristics Associated with Cold-active Enzymes

and therefore decrease the backbone entropy of unfolding (Hardy et al.

hyperthermostable citrate synthase from archaeon Pyrococcusfuriosus (PfCS)

Table 3. Structural properties of glutamate racemases

Parameters Source organisms of glutamate racemase

To study the structural factors responsible for cold adaptation of AlaDHs,

B 16S rDNA AlaDH

Table 4. Structural properties of alanine dehydrogenases

Parameters Source organisms of alanine dehydrogenase

Vpr* She* Bst* Bsu* Car*

Optimum growth temperature (°C) 37 20 57 37 20

bacteria by primary-structure comparison studies (Table 4), and the

6. Comparative Mutagenesis Studies and Rational Design of Cold-

of a proline residue, resulting in a reduced catalytic activity. Moreover, in

The homology-based modeled structure of SapSh was built using the

In conclusion, the results of site-directed mutagenesis studies presented

responsible for protein cold adaptation by means of structure comparison

fun Hirose1and Kensuke Furukawa2

'Department of Applied Chemistry, Faculty of Engineering, Miyazaki University,