Development of a strategy for the screening of α-glucosidase-producing microorganisms
Development of a strategy for the screening of α-glucosidase-producing microorganisms
Development of a strategy for the screening of α-glucosidase-producing microorganisms
Introduction
Bo ZhouЪ, Nan HuangЪ, Wei ZengЪ,
Hao Zhang, Guiguang Chen, and Zhiqun Liang* α-Glucosidases (EC 3.2.1.20) are glycoside hydrolases be-
longing to the glycoside hydrolase (GH) family, of enzymes
State Key Laboratory for Conservation and Utilization of Subtropical that connect activated glycosyl donors with acceptors. These
Agro-bioresources, Guangxi Microorganism and Enzyme Research
Center of Engineering Technology, College of Life Science and enzymes catalyze the formation of α ataly linkages (Shim-
Technology, Guangxi University, 100 Daxue Road, Nanning 530004, ba et al., 2009), producing isomaltosyl oligosaccharides, are
Guangxi, P. R. China mainly from family GH13, GH31 and less extent in family
GH4, GH63, GH97, and GH122 according to Carbohydrate-
(Received Jun 3, 2019 / Revised Dec 12, 2019 / Accepted Dec 16, 2019) Active Enzymes database (CAZy) (Bissaro et al., 2015; Gu-
tiérrez-Alonso et al., 2016; Hleap and Blouin, 2018). The
α-Glucosidase is a crucial enzyme for the production of iso- industrial value of α-glucosidase has been demonstrated by
maltooligosaccharide. In this study, a novel method compris- its ability to hydrolyze various substrates, such as maltose
ing eosin Y (EY) and α-D-methylglucoside (AMG) in glass and starch, to produce isomaltooligosaccharide (IMO), a
plates was tested for the primary screening of α-glucosidase- prebiotic with biological characteristics (Fernández Arrojo
producing strains. First, α-glucosidase-producing Aspergillus et al., 2007).
niger strains were selected on plates containing EY and AMG α-Glucosidase exhibits hydrolytic activity, hydrolyzing the
based on transparent zone formation resulting from the sol- terminal, non-reducing, (14)-linked α-glucose residues of
ubilization of EY by the hydrolyzed product. Conventional its substrates (Song et al., 2013; Tagami et al., 2015). Nume-
methods that use trypan blue (TB) and p-nitrophenyl-α-D- rous α-glucosidase-producing strains have been isolated and
glucopyranoside (pPNP) as indicators were then compared screened based on their hydrolytic properties, including Ba-
with the new strategy. The results showed that EY-containing cillus subtilis HTG (Krohn and Lindsay, 1991), Xanthophyllo-
plates provide the advantages of low price and higher speci- myces dendrorhous ATCC MYA-131 (Marín et al., 2006), and
ficity for the screening of α-glucosidase-producing strains. Cellvibrio japonicus Agd31B (Larsbrink et al., 2012). Con-
We then evaluated the correlation between the hydrolytic ac- ventional methods to screen for α-glucosidase-producing mi-
tivity of α-glucosidase and diffusion distance, and found that croorganisms are based on agar-containing plates, combined
good linearity could be established within a 6–75 U/ml en- with the addition of iodine or p-nitrophenyl-α-D-glucopyr-
zyme concentration range. Finally, the hydrolytic and trans- anoside (pNPG) (Suzuki et al., 1976; Zhou et al., 2009; Chen
glycosylation activities of α-glucosidase obtained from the et al., 2011). Although some excellent strains have been iso-
target isolates were determined by EY plate assay and 3,5- lated using these methods, they still have some limitations. For
dinitrosalicylic acid-Saccharomyces cerevisiae assay, respec- example, plates containing soluble starch or pNPG require
tively. The results showed that the diameter of the transparent the addition of an iodine solution or alkaline solutions (so-
zone varied among isolates was positively correlated with dium carbonate or sodium hydroxide) to provide an alkaline
α-glucosidase hydrolytic activity, while good linearity could environment, resulting in damage to the target strain. Even
also be established between α-glucosidase transglycosylation though trypan blue (TB) was introduced to replace iodine
activity and non-fermentable reducing sugars content. With (Chen et al., 2011), the reaction is still susceptible to interfer-
this strategy, 7 Aspergillus niger mutants with high yield of ence by amylase activity. Moreover, pNPG, commonly used
α-glucosidase from 200 obvious single colonies on the pri- as an indicator of α-glucosidase hydrolysis, is expensive and
mary screen plate were obtained. rapidly self-decomposes, which limits the scope of its appli-
cations. In addition, the clear zone displayed in blurred yellow
Keywords: α-D-methylglucoside, α-glucosidase, eosin y, hy- fluorescence produced through pNPG hydrolysis is difficult
drolysis, transglycosylation, Aspergillus niger to detect in medium that exhibits background color. There-
fore, a simple and specific method need be established to
overcome these limitations.
α-Glucosidase from different organisms and GH families
exhibit a wide variety of catalytic properties (Gutiérrez-
Alonso et al., 2016; Hleap and Blouin, 2018), which have
†
These authors contributed equally to this work. been exploited for the production of glucose, IMO (Basu et
*For correspondence. E-mail: zqliang@gxu.edu.cn; Tel.: +86-0771-327118 al., 2016), and alkyl glucose (Tanaka et al., 2002) based on
1; Fax: +86-0771-3271181
Copyright G2020, The Microbiological Society of Korea
the specific hydrolytic and transglucosylation activities of
164 Zhou et al.
Fig. 1. The integrated screening process of α-glucosidase-producing strains. Step 1: primary screening; Step 2: microculture; Step 3: reaction with maltose
solution; Step 4: cultivation of yeast and consume fermentable sugar; Step 5: DNSS assay and EY assay; Step 6: obtain strains.
Strategy for the screening of α-glucosidase-producing strains 165
The yield of non-fermentable reducing sugars, including and a Waters Sugar-Pak 1 column (10 μm, 6.5 mm × 300 mm)
isomaltose, panose, and isomaltotriose, was measured by 3,5- as previously reported (Chen et al., 2011). Glucose, maltose,
dinitrosalicylic acid-S. cerevisiae (DNSS) assay to determine isomaltose, maltotriose, panose, and isomaltotriose were used
the transglycosylation activity of α-glucosidase (Chen et al., as the standards.
2011). In brief, 30% maltose was mixed into each sample
and incubated for 1 h at 37°C. The reaction was terminated
by placing the sample in a boiling water bath for 10 min, and Results
then a 9-fold volume of yeast culture was added to consume
the fermentable sugars. The yield of non-fermentable redu- Optimization of primary screening conditions
cing sugars was measured by DNS assay after incubation at α-Glucosidase specifically catalyzes the hydrolysis of α-glu-
30°C for 15 h with shaking (200 rpm). A standard curve was
cosidic bond from the non-reducing end of its substrates
generated by plotting the average blank-corrected absorb- (Charron et al., 1986). Eosin Y, a macromolecular complex,
ance of glucose at 540 nm versus its concentration in mi- is well known for its ability to dye proteins and compounds
cromole per L (0.25–2.5 μmol/L). One unit of transglycosy-
through electrostatic interactions (Jones et al., 1984; Lin et
lation activity was defined as the synthesis of 1 micromole al., 1991; Jin et al., 2018). In this report, AMG, a α-glucosi-
non-fermentable reducing sugars per min. dase-specific substrate that can be degraded to glucose and
Non-fermentable reducing sugar yield was also analyzed by
methanol by this enzyme, was used to detect target strains.
ion-exchange chromatography using an HPLC system equip- The α-glucosidase-catalyzed hydrolysis of AMG is illustrated
ped with an Ecosil NH2 column (5 μm, 4.6 mm × 250 mm)
(A)
(B) (C)
Fig. 3. (A) A comparison of the transparent zone on the primary screen plate. From left to right are deionized water, α-amylase (100 U/ml), α-glucosidase
(25 U/ml, 50 U/ml, 75 U/ml, and 100 U/ml). The control was deionized water placed 50 μl into the hole (4 mm in diameter). (B) α-Glucosidase-producing
colonies in isolation plates containing acidic dye EY (0.3%, w/v). (C) The correlation between inhibition rate of strains, germination time of spores, and
concentration of EY. The data represent mean values of triplicate and independent experiments.
Strategy for the screening of α-glucosidase-producing strains 167
in Fig. 2. Following AMG degradation by α-glucosidase, trast, with a dye concentration of 0.4%, the medium was too
eosin Y was dissolved by methanol, a product of AMG hy- dark to visualize the transparent zone in the early stages of
drolysis, and a transparent zone formed around α-glucosi- growth. Based on these results, 0.3% eosin Y was selected as
dase-producing colonies in the plate. In addition, transglu- the optimal screening concentration. By doing so, it is possi-
cosidase L (100 U/ml) was also pipetted into EY plates (Fig. ble to simultaneously detect more than 100 colonies on each
3A), and reflected the hydrolytic activity of α-glucosidase. plate. Therefore, this plate method is suitable for screening
In contrast, deionized water and α-amylase (100 U/ml) did a large number of samples.
not produce a transparent zone. Clear zones, representing
solubilization of the acidic dye, appeared around the colo- Comparison of TB, pNPG, and EY plates
nies when A. niger F86 was grown on primary screening plates
Fourteen mutants were randomly screened for α-glucosidase
(Fig. 3B), indicating that the α-glucosidase hydrolytic acti-
hydrolytic activity on TB, pNPG, and EY plates to test the
vity of the samples can be determined by the size of the trans-
efficacy of the developed plate assay. As shown in Fig. 4A,
parent zone.
all 14 isolates produced clear zones when grown on TB plates,
Because eosin Y is an acidic dye and can inhibit the growth
indicating that the soluble starch had been hydrolyzed. The
of microorganisms, the initial concentration of dye required
diameters of the clear zones on the TB plates were similar
optimization. The dye inhibition rate was less than 6% at
among the different strains. However, the results for the α-
EY concentrations ranging from 0.01% to 0.5% (Fig. 3C).
glucosidase hydrolytic activity of these fourteen strains on
However, the time of germination and growth of Aspergillus
the TB plates showed that there were three positive mutants
niger strain F86 was delayed in an EY-concentration-depen-
(O13, TE61, and TE16; Table 1) distributed in the plates of
dent manner, and the spore production time increased with
the third and fourth columns, and the rest were either false
increasing concentration of the dye. Compared with the con-
or negative mutants. Moreover, the α-glucosidase produced
trol (without dye), the germination time increased from 18–27
by the A. niger F86 strain has been proved cannot hydrolyze
h at an EY concentration of 0.5%, and the time required for
soluble starch (Zhang et al., 2011). The amylase difference of
spore production increased from 40–72 h. At dye concen-
all strains was less than 0.32 U/ml, and the activity of F86
trations below 0.25%, the medium in the plates was too trans-
amylase was 3.58 U/ml (date not shown). These results in-
parent to observe the formation of transparent zones. In con-
dicated that the use of TB plates to screen for α-glucosidase-
producing strains was not so reliable.
Table 1. The hydrolytic activity and transglycosylation activity of mutants These experiments were repeated on pNPG plates (Fig. 4B)
from isolated α-glucosidase-producing Aspergillus niger strains and EY plates (Fig. 4C). All the strains showed specific hy-
Strain Hydrolytic activitya (U/ml) Transglycosylation activityb (U/ml) drolysis zones on the plates, and the clear zones matched the
H9-30 12.52 ± 0.88 318.18 ± 4.71 results of hydrolytic activity. However, the blurred ranges
TE61 12.42 ± 0.72 312.37 ± 5.12 generated by PNP were difficult to distinguish under natu-
O13 11.73 ± 0.28 305.16 ± 6.83 ral light. A maltose analog (pNPG) was added to the plate as
TE7 11.13 ± 0.21 300.83 ± 2.74 a substrate and the production of PNP, which generates a
TW16 11.36 ± 0.66 299.53 ± 6.88
yellow color, can be detected by spectroscopy in an alkaline
N32 11.09 ± 0.87 288.28 ± 2.13
environment containing sodium carbonate or sodium hyd-
TE16 11.07 ± 0.42 286.47 ± 4.42
roxide (Krolicka et al., 2018). Therefore, it is only commonly
used for detection of enzyme properties and rarely added to
F86c 8.43 ± 0.33 220.66 ± 5.77
a
Determined by pNPG assay
plate culture medium for preliminary screening of α-gluco-
b
Determined by DNSS assay sidase-producing microorganisms owing to spontaneous
c
Original strain
168 Zhou et al.
decomposition, high price, and limited conditions (Li et al., (Fig. 3A). This suggests that the concentration of AMG was
2018). These disadvantages can be overcome by using AMG relatively low compared to enzyme activity and exceeded the
instead of pNPG as a specific substrate of α-glucosidase. In detection range of the assay. In addition, the detection limit
addition, compared with the 50 USD/g market price (Sigma- of α-glucosidase was 0.07 U/ml which calculated by the quan-
Aldrich) for pNPG, the cost of AMG is 0.2 USD/g, which tities’ results (S/N=3). These results above suggested that the
reduces the screening cost in a large extent. EY plate assay could be applied to rapidly determine the hy-
drolytic activity of α-glucosidase during the screening pro-
Correlation between traditional methods and the developed cess for α-glucosidase producing strains.
method A calibration curve for the pNPG assay was generated based
Correlation between spectrophotometric and EY plate assays : on the average blank-corrected absorbance of PNP versus its
An equal volume of transglucosidase L (0–200 U/ml) was concentration in micromole per liter (Fig. 5B) which showed
added to the holes in EY plates to test the correlation between a good correlation coefficient of 0.99965. The correlation
hydrolytic activity and diffusion distance. Analyses of the between the traditional pNPG assay and the EY plate assay
diffusion distance and hydrolytic activity (Fig. 5A) indicated was explored to determine whether the results of the EY plate
that the relationship between enzyme activity and diffusion assay could represent the hydrolytic activity of the strains.
distance was not a positive linearity singly. It was not in pro- Twenty-eight mutants with clear transparent zones on the
portion with the enzyme activity between 0–4 U/ml, while EY plates were randomly screened out and the correlation
a good linear relationship could be observed between 6–75 efficiency between the spectrophotometric and EY plate as-
U/ml, with a linear correlation coefficient greater than 0.99. says was examined. As shown in Fig. 5C, there was a positive
The linear relationship was lost again at 75 U/ml, and a blur- correlation (Adj. R-Square = 0.93261) between the diffusion
red and irregular transparent circle could be seen at 100 U/ml distance and hydrolytic activity. The results suggested that
(A) (B)
(C)
(A) (B)
Fig. 6. (A) The standard curve of DNS assay. (B) The correlation of twenty-eight random mutants were selected to investigate in non-fermentable reducing
sugars and transglycosylation activity. The data represent mean values of triplicate and independent experiments.
the EY plate assay was effective for the screening of α-gluco- α-glucosidase-secreting microorganisms. Second, this me-
sidase-producing microorganisms. thod can be used to screen a large number of samples more
Correlation between non-fermentable reducing sugars and economically than conventional methods. Finally, α-gluco-
transglycosylation activity : To further define the transgly- sidase hydrolytic and transglycosylation activity can be ra-
cosylation activity of the enzyme, 28 colonies were also se- pidly detected when combined with a DNSS assay, thereby
lected to measure the transglycosylation activity and the cor- improving screening efficiency and throughput. In brief, this
relation efficiency between the HPLC and DNSS assays was strategy can be used for the rapid and cost-effective screen-
examined. The calibration curve of the DNSS assay is shown ing of strains producing α-glucosidase of high yield and dif-
in Fig. 6A. As demonstrated in Fig. 6B, the yield of non-fer- fering catalytic properties.
mentable reducing sugars and transglycosylation activity pre- Numerous dyes, such as methylene blue (Nishikawa and
sented a good correlation (Adj. R-Square = 0.99714). This re- Ogawa, 2002) and neutral red (Zeng et al., 2013), are com-
sult suggested that the DNSS assay was suitable for the quan- monly used for the screening of microorganisms. To the
tification of α-glucosidase transglycosylation activity and best of our knowledge, this is the first study to use a combi-
could replace the HPLC assay. nation of EY and AMG to screen for α-glucosidase-produc-
ing strains. In the report of Nishikawa and Ogawa (Nishi-
Screening for α-glucosidase-producing microorganisms using kawa and Ogawa, 2002), the growth of the single colony was
the developed strategy inhibited by acidic dye. However, in our study, this inhibi-
Using this screening strategy, approximately 6,000 single tion was not observed, even at a 0.5% EY concentration. In
A. niger F86 strain mutants producing α-glucosidase were the primary screen, a linear relationship was maintained be-
identified on the EY plates. According to the size of the trans- tween the diffusion distance and hydrolytic activity rang-
parent circles, 200 strains were selected and cultured in fer- ing from 6 U/ml to 75 U/ml. However, this relationship was
mentation medium, and 7 colonies with high α-glucosidase lost (Fig. 5A) as the enzymatic activity exceeded 75 U/ml, and
activity were shown in Table 1. Of which, A. niger H9-30 an irregular, gelatinized, hydrolytic circle was observed at
showed the highest hydrolytic and transglycosylation activi- 100 U/ml (Fig. 3A). This suggests that, at 100 U/ml, the en-
ties, which were 48.5% and 44.2% higher than those of the zymatic activity exceeded the detection range due to a lack
original strains, respectively. of substrate (Lee et al., 2001). Linearity was reestablished when
the concentration of AMG in the medium was increased.
Both the AMG content and detection range greatly influ-
Discussion ence its practical application, which should be considered
before screening. In this report, 4% was chosen as the optimal
Compared with conventional screening methods (Suzuki et AMG concentration and, under this condition, enzymatic
al., 1976; Nakao et al., 1994; Fernández-Arrojo et al., 2007; activity can be detected within a range of 6–75 U/ml in the
Ganzlin and Rinas, 2008; Chen et al., 2011), the screening EY plate assay.
approach reported in this study presented several important The diffusion speed and distance can be influenced by tem-
advantages. First, our method could effectively isolate α- perature (Zeng et al., 2013). In the experiment, there was a
glucosidase-producing strains on EY plates, while the single better linear relationship between diffusion distance and
and unique carbon source prevented contamination by non- enzyme activity at 37°C. Additionally, this linear relationship
170 Zhou et al.
could be affected by the concentration of agar based on the 60 h; moreover, the detection time was reduced to less than
degree of crosslinking. In this study, 37°C and 2% agar were 3 h (data not shown), and the chromogenic reaction could
determined as the optimal conditions for the EY plate assay. not be detected because of the spontaneous degradation of
Trypan blue is an acidic dye that is widely used to detect cell pNPG; (3) both pNPG and the associated alkaline environ-
membrane integrity and cellular survival (Tennant, 1964; ment are harmful to microorganisms, which is not con-
Jauregui et al., 1981; Strober, 2001). In addition, a blue com- ducive to the selection of target strains for further study. In
plex can be formed by combining polysaccharides, and a contrast, the novel EY plate method proposed in this study
transparent circle appears following the hydrolysis of poly- could overcome these shortcomings, is simple to operate, and
saccharide substrates by the corresponding enzymes (Ma et is more suitable for high-throughput screening.
al., 2007). Trypan blue is used as an indicator in the screen- In this study, two rapid characterization methods were used
ing of polysaccharide hydrolase-producing strains, such as to distinguish and screen for α-glucosidase exhibiting diffe-
those that producing cellulase, amylase, and α-glucosidase rent catalytic properties. Hydrolytic activity can be examined
(Ma et al., 2000; Margesin et al., 2003; Chen et al., 2011). by the EY plate assay proposed in this report. DNS is an im-
Interestingly, starch is also used as a substrate to screen for portant method to measure reducing sugar levels (Tanaka
α-glucosidase and amylase, and also through the visualiza- et al., 2002). Non-fermentable reducing sugars and transgly-
tion of transparent areas (Ma et al., 2000; Chen et al., 2011). cosylation activity presented a good correlation, as determined
However, most reported α-glucosidases show a preference by HPLC and DNSS assays. This indicates that the DNSS
for hydrolyzing oligosaccharides with a degree of 2-6 poly- assay can represent transglycosylation activity through the
merization and have a low or no affinity for polysaccharide levels of non-fermentable reducing sugars. Importantly, this
substrates (Nakao et al., 1994; Hostinová et al., 2005). In this step can significantly increase the throughput and efficiency
work, amylase interference was observed in the TB plates of sample analysis relative to HPLC and TLC assays.
and false-positive strains were screened out. This suggested In summary, we developed a specific, simple, and conve-
that using TB plates to screen for α-glucosidase-producing nient plate method for the screening of α-glucosidase-pro-
strains is likely to be unreliable. In contrast, the EY plates ducing microorganisms. In this study, in a primary screen,
showed strong specificity and reliability, and α-amylase can 200 colonies surrounded by clear, transparent zones were
also be excluded with the EY assay. Moreover, we also ob- selected, from which seven strains with high enzymatic ac-
served a good relationship between transparent zones and tivity were screened out. Our results indicated that this
hydrolytic activity. This indicates that the hydrolytic activity strategy can be widely used to screen for α-glucosidase-pro-
of target strains on EY plates can be determined based on ducing microorganisms.
the size of the transparent zones produced by the activity
of specific enzymes, and that EY plates are better than TB
plates for the screening of α-glucosidase-producing micro- Acknowledgments
organisms.
p-Nitrophenyl-α-d-glucopyranoside in solution is a widely This work was supported by the National Natural Science
used assay (pNPG assay) to determine the hydrolytic activity Foundation of China (31560448, 31760452); the Natural
of α-glucosidase, for which it has a high affinity (Km is low) Science Foundation of Guangxi (2016GXNSFAA380130,).
(Saha and Zeikus, 1991; Coleri et al., 2009; Hu et al., 2011).
Furthermore, pNPG can be degraded by α-glucosidase to
produce PNP, which fluoresces yellow in alkaline conditions, Conflict of Interest
and its concentration shows a good linear relationship with
the fluorescence intensity at 405 nm, as shown in Fig. 5B (Dej- The authors declare that have not any conflict of interest in
Adisai and Pitakbut, 2015). A low detection limit was esti- this work.
mated at between 6.6 × 10-4 U/ml to 7.6 × 10-4 U/ml based
on the reports of Li and Tang (Tang et al., 2017; Li et al.,
2019). According to the calibration curve (Fig. 5B), a quan- References
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