CN113969290B

CN113969290B - Deep sea bacteria-derived alpha-glucosidase QsGH97a and encoding gene and application thereof

Info

Publication number: CN113969290B
Application number: CN202111356942.XA
Authority: CN
Inventors: 黄静; 吴开娟; 许学伟
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2023-06-30
Anticipated expiration: 2041-11-16
Also published as: CN113969290A

Abstract

The invention discloses an alpha-glucosidase QsGH97a from deep sea bacteria, and a coding gene and application thereof, wherein the alpha-glucosidase QsGH97a gene is obtained by screening a bacterial genome library provided by a second ocean research of the national ocean bureau, and the alpha-glucosidase QsGH97a is found to have better enzymatic properties by expression and purification, so that the alpha-glucosidase QsGH97a can be used for producing industrial glucose and can be applied to food processing and chemical industry related to carbohydrate hydrolysis. The obtained alpha-glucosidase gene can be cloned into a proper host to realize heterologous expression, the purification method is simple, the alpha-glucosidase can be industrially produced, and the alpha-glucosidase gene can be applied to the chemical industry for mass production of glucose.

Description

Deep sea bacteria-derived alpha-glucosidase QsGH97a and encoding gene and application thereof

Technical Field

The invention relates to alpha-glucosidase, in particular to alpha-glucosidase QsGH97a from deep sea bacteria, a coding gene thereof and application thereof in glucose production by saccharide hydrolysis, and belongs to the technical field of genetic engineering.

Background

Alpha-glucosidase is widely found in animals, plants and microorganisms and is capable of hydrolyzing not only the alpha-glucosidic bond at the non-reducing end of the polysaccharide to produce glucose, but also transglycosylation to produce isomaltooligosaccharides. According to the alpha-glucosidase amino acid sequence, it is predominantly distributed in GH families 4, 13, 31, 63, 97 and 122. The application of the enzyme is very wide, and in the medical field, alpha-glucosidase participates in a plurality of physiological functions including sugar metabolism in different life bodies, so that the enzyme can be used for treating diseases caused by lack of enzymes for decomposing maltose and glycogen; in the field of industrial production, the different characteristics of the alpha-glucosidase make the alpha-glucosidase have good application prospects, such as the property of hydrolyzing saccharides to generate glucose, and can be used for glucose production, so that more valuable products such as ethanol can be produced. In addition, the transglycosidase enzyme has transglycosidation properties that make it useful for the production of Isomaltooligosaccharides (IMOs) and the like.

The currently studied alpha-glucosidase is mainly from microorganisms, most of the industrially applied alpha-glucosidase is from aspergillus niger, but the disadvantages of low enzyme activity and poor thermal stability limit the industrial application of the alpha-glucosidase, and the application exploration of the alpha-glucosidase is still not wide enough. The α -glucosidase from different biological sources has different characteristics, making its application range more distinctive, and thus it is important to continuously develop α -glucosidase with good characteristics to meet industrial needs. The alpha-glucosidase from marine microorganisms has the novel characteristics of temperature stability, metallic resistance, salt tolerance, cold adaptation capability and the like, so that the mining of the alpha-glucosidase which can be better applied to industrial production from marine microorganisms is a good strategy, not only can the novel enzyme be obtained so as to better understand the biochemical characteristics of the alpha-glucosidase, but also provides a reference for enzymes with other application values in industrial transformation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide alpha-glucosidase QsGH97a from deep sea bacteria, and a coding gene and application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

1. a gene encoding α -glucosidase QsGH97a, said gene having any one of the following nucleotide sequences:

(1) Is consistent with the sequence shown in SEQ ID NO. 1;

(2) Substitution, addition and/or deletion of one or more than two nucleotides to the sequence shown in SEQ ID No.1 can obtain the mutant gene which codes for the protein QsGH97a of the alpha-glucosidase.

Preferably, the mutant gene has at least 90% homology with the sequence shown in SEQ ID NO. 1.

Further preferably, the mutant gene has at least 95% homology with the sequence shown in SEQ ID NO. 1.

Still more preferably, the mutant gene has at least 99% homology with the sequence shown in SEQ ID NO. 1.

2. A vector carrying the above-mentioned coding gene.

3. Prokaryotic or eukaryotic hosts are transformed or transfected with the above vectors.

Preferably, the host comprises a microbial or eukaryotic cell.

Further preferred hosts are E.coli, saccharomyces cerevisiae or Chinese hamster ovary cells.

Still more preferably, the host is an E.coli bacterium.

4. The amino acid sequence of the alpha-glucosidase QsGH97a obtained by the expression of the encoding gene is shown as SEQ ID NO. 2; or for the sequence shown in SEQ ID NO.2, the sequence is far away from the 459 glutamic acid residue (Glu 459), the 483 glutamic acid residue (Glu 483) and the 378 glutamic acid residue (Glu 378) (catalytic active center); substitution, addition and/or deletion of one or more amino acids from the amino acid sequences of glutamic acid residue 174 (Glu 174) and glutamic acid residue 477 (Glu 477) (metal ion binding site) can give a mutant protein having the characteristics of α -glucosidase QsGH97a.

Preferably, the mutant protein has at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2.

Further preferably, the mutant protein has at least 95% homology with the amino acid sequence shown in SEQ ID NO. 2.

Still more preferably, the mutant protein has at least 99% homology with the amino acid sequence shown in SEQ ID NO. 2.

5. The vector, the host or the alpha-glucosidase QsGH97a can be applied to glucose production by decomposing saccharides and other industrial production.

Preferably, the saccharide is a substance whose final hydrolysate is glucose.

The invention has the beneficial effects that:

the invention screens and obtains the alpha-glucosidase QsGH97a gene from a Pacific ocean mountain deep sea sediment metagenomic library provided by the second ocean research institute of the national ocean bureau, and the alpha-glucosidase QsGH97a gene is derived from deep sea bacteria Qipengyuania seohaensis sp.SW-135. The expression and purification of the alpha-glucosidase QsGH97a find that the alpha-glucosidase QsGH97a has better enzymatic properties, can be used for glucose production, and can be applied to food processing and chemical industry related to carbohydrate hydrolysis. The obtained alpha-glucosidase gene can be cloned into a proper host to realize heterologous expression, the purification method is simple, the alpha-glucosidase can be industrially produced, and the alpha-glucosidase gene can be applied to the chemical industry for mass production of glucose.

An alpha-glucosidase gene qsgh97a having a nucleotide sequence as shown in SEQ ID NO. 1. The size of the alpha-glucosidase gene QsGH97a is 2037bp, and the base composition is as follows: 411A (20.18%), 354T (17.38%), 648C (31.81%) and 624G (30.63%), the amino acid sequence of which is shown in SEQ ID No.2, and the encoded protein has 678 amino acids with a size of 75.57KD. The highest homology was found by Blast alignment to the enzyme of the GH97 family derived from Qipengyuania sp.1ndw3, 83.13%. Amino acid sequence analysis of alpha-glucosidase QsGH97a shows that alpha-glucosidase QsGH97a has catalytic domains consisting of Glu 459, glu 483 and Glu 378, indicating that QsGH97a belongs to the alpha-glucosidase GH97 family. Taken together, qsGH97a should be a new member of the GH97 family.

According to the molecular cloning technology, firstly, the amplified alpha-glucosidase QsGH97a gene is connected to a target vector through DNA ligase, then the recombinant vector is transformed or transfected into a prokaryote or eukaryote host, the recombinant alpha-glucosidase QsGH97a is induced to be expressed, and finally, the alpha-glucosidase QsGH97a without a tag is obtained through enzyme digestion. The host includes microorganisms such as E.coli and the like, yeasts (Saccharomyces cerevisiae), mammalian cells (Chinese hamster ovary cells) and the like.

Vectors of the invention are well known to those skilled in the art and experimentally validated as viable prokaryotic or eukaryotic expression vectors. The expression vector used in The present invention is pSMT3 (Li, J.et al (2012). The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed interna. Cell 150 (2), 339-350.), the α -glucosidase QsGH97a gene is inserted into The multiple cloning site of The vector pSMT3 to obtain a recombinant plasmid, and transformed into E.coli BL21 (DE 3) plus, and The highly active α -glucosidase QsGH97a is expressed by IPTG induction. The result of enzyme activity experiment shows that alpha-glucosidase QsGH97a can hydrolyze alpha-glucoside bond and beta-galactose glucoside bond, namely can hydrolyze polysaccharide to generate glucose or galactose, wherein the catalytic activity is highest when the substrate is p-nitrophenol alpha-D-glucopyranoside (pNP alpha Glu), the enzyme activity Vmax is 0.64U/mg when no metal ion is added, and the Michaelis constant K _m ＝0.21mM。

The temperature range of the alpha-glucosidase QsGH97a exerting more than 50% of catalysis is 20-50 ℃, and the optimal temperature is 45 ℃; the pH range is pH 6.0-pH 11.0, and the optimal pH is pH 8.0. Adding metal ions Ba with different concentrations ²⁺ Or Sr ²⁺ Can promote 3-7 times of enzyme activity, and slightly enhance enzyme activity under conditions of 5% Tween 20 and 80. 1mM Ba was added ²⁺ Or Sr ²⁺ After that, the enzyme tolerance in high concentrations of detergent, organic solvent and NaCl is greatly enhanced.

Drawings

FIG. 1 is a diagram of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified alpha-glucosidase QsGH97a.

FIG. 2 is a graph of substrate specificity of alpha-glucosidase QsGH97a. pnpβglu: p-nitrophenyl beta-D-glucoside; pnpαglu: p-nitrophenyl alpha-D-glucoside, pNP alpha Arap: p-nitrophenyl alpha-L-arabinopyranoside; pnpNPβLac: p-nitrophenyl beta-D-lactoside; pnpαgal: p-nitrophenyl alpha-D-galactoside; pNP βGal: p-nitrophenyl beta-D-galactoside; pnpNPβMan: p-nitrophenyl alpha-D-mannoside; pNP. Beta. Xyl: p-nitrophenyl beta-D-xyloside; pNP beta Fuc: p-nitrophenyl beta-D-fucoside; pNP. Beta. Cel: p-nitrophenyl beta-D-cellobioside. The measured value was 100% when the substrate was defined as pNPαGlu.

FIG. 3 is a graph showing the optimum reaction temperature of α -glucosidase QsGH97a.

FIG. 4 is a graph showing the pH optimum for the reaction of alpha-glucosidase QsGH97a.

FIG. 5 is a graph showing the effect of metal ions on the activity of α -glucosidase QsGH97a.

FIG. 6 is a graph showing the effect of an organic solvent on the activity of α -glucosidase QsGH97a, wherein A is the effect of a common organic solvent on the enzyme activity, and B is the addition of 1mM Ba ²⁺ Or Sr ²⁺ The effect of the organic solvent on the enzyme activity (blank is standard reaction system, contrast is standard reaction system, 1mM Ba is added ²⁺ Or Sr ²⁺ The other is to add different organic solvents into the control system respectively).

FIG. 7 is a graph showing the effect of detergent on the activity of alpha-glucosidase QsGH97a, wherein A is the effect of detergent on enzyme activity, and B is the addition of 1mM Ba ²⁺ Or Sr ²⁺ Influence of post-detergent on enzyme Activity (blank for Standard reaction System, control for Standard reaction System 1mM Ba was added ²⁺ Or Sr ²⁺ The other was to add different detergents separately to the control system).

FIG. 8 is a graph showing the effect of alpha-glucosidase QsGH97a on NaCl, wherein A is the effect of NaCl aqueous solution on enzyme activity, and B is the addition of 1mM Ba ²⁺ Or Sr ²⁺ The influence of the aqueous NaCl solution on the enzyme activity (blank is the standard reaction system, and control is the standard reaction system, 1mM Ba is added ²⁺ Or Sr ²⁺ The other is to add NaCl with different concentration into the control system).

Detailed Description

The invention is further illustrated in the following figures and examples, which are provided for the purpose of illustration only and are not intended to be limiting.

Example 1

Cloning of alpha-glucosidase gene qsgh97a and construction of recombinant plasmid

Deep sea sediment samples were collected from the pacific sea mountain edge area. Bacterial genomic libraries are provided by the national ocean of Cooperation units second ocean research institute.

The primers used for constructing the recombinant prokaryotic expression vector comprise:

an upstream primer: 5'-GGCGGATGATCCGCCATATCGCCCTCTTCATC-3', as shown in SEQ ID NO. 3;

a downstream primer: 5'-AAACTCGAGTCACCCCTGCGGCACGAACTCG-3' as shown in SEQ ID NO. 4.

The restriction enzyme sites are BamHI and XhoI respectively, the selected expression vector is pSMT3 vector, the cloned target gene fragment and the vector are subjected to restriction enzyme digestion after the correct size of the strip is determined by agarose electrophoresis, the restriction enzyme sites are BamHI and XhoI respectively, and then the restriction enzyme digestion products are recovered. T4 DNA ligase was used and the fragment to vector molar ratio was 7:1 were placed at 16℃overnight, the recombinant vector obtained was transformed into E.coli DH 5. Alpha (full gold, china) and plated on LB agar medium plates containing 50. Mu.g/mL kanamycin resistance to screen positive clones. After sequencing and verification to be accurate, the obtained product is stored in a refrigerator at the temperature of-80 ℃. By comparing the homology of the inserted target gene sequence with known alpha-glucosidase gene sequences in the database by Blastx (http:// blast. Ncbi. Lm. Nih. Gov /), the highest homology with the enzyme from the GH97 family of Qiangyuania sp.1NDW3 was found to be 83.13% and its registration number in the GenBank database was QZD92199.1. The size of the alpha-glucosidase gene qsgh97a is 2037bp (the nucleotide sequence is shown as SEQ ID NO. 1), and the base composition is: 411A (20.18%), 354T (17.38%), 648C (31.81%) and 624G (30.63%), the amino acid sequence of which is shown in SEQ ID NO.2, the encoded protein comprises 678 amino acid residues, with a size of 75.57kDa. The alpha-glucosidase QsGH97a belongs to the GH97 family, and amino acid sequence analysis shows that the alpha-glucosidase QsGH97a has catalytic domains consisting of Glu 459, glu 483 and Glu 378, and forms an alpha-glucosidase QsGH97a catalytic center, which shows that the QsGH97a belongs to the alpha-glucosidase GH97 family.

Taken together, qsGH97a should be a new member of the GH97 family.

Example 2

Transformation of recombinant plasmid of alpha-glucosidase gene qsgh97a

Positive clones sequenced correctly were placed in LB liquid medium for culture at 37℃and then plasmid was extracted using plasmid extraction kit (Axygen, USA) to obtain recombinant plasmid. E.coli strain BL21 (DE 3) plus (full gold, china) competent cells taken out from a refrigerator at-80 ℃ are subpackaged into EP tubes by 30 μl, 1 μl of plasmid is added, after incubation on ice for 30min, heat shock is performed for 1min at 42 ℃ and 2min on ice, then 500 μl of LB medium is added, shaking culture is performed for 1 hour at 37 ℃ and then 200 μl of supernatant is removed by centrifugation, and the remaining bacterial liquid is uniformly spread on LB agar plates containing 50 μg/mL kanamycin resistance.

Example 3

Expression and purification of alpha-glucosidase QsGH97a

Positive clones transformed into E.coli strain BL21 (DE 3) plus competent cells were picked up and cultured to OD at 37℃in 500mL LB liquid medium containing 50. Mu.g/mL kanamycin resistance ₆₀₀ To 0.8-1.0, IPTG (isopropyl-. Beta. -D-thiogalactoside) was added at a final concentration of 0.5mM at 16℃and cultured on a shaker at 200rpm for 20 hours, after which cells were collected by centrifugation at low temperature and resuspended in Buffer A solution (50 mM Tris-HCl, pH 8.0, 500mM sodium chloride, 10mM imidazole, 1% by volume glycerol) and the resuspension was subjected to disruption with an ultrasonic disrupter. The supernatant was collected by low temperature centrifugation, and after incubation with Ni-NTA medium for 0.5h, it was purified by gravity column. Because the expressed recombinant protein contains His tag, the recombinant protein can be affinity adsorbed on a nickel column, and the recombinant protein can be obtained by gradient elution of imidazole solutions (10 mM, 20mM and 250 mM) with different concentrations, and then the size of the protein can be detected by SDS-PAGE. Measuring the concentration of a sample with correct protein size by SDS-PAGE, and then mixing the protein sample with UThe mass ratio of lp1 enzyme (sumo tag for cleaving recombinant protein) was 500:1, and dialyzing overnight at 4 ℃ to reduce the imidazole concentration. And (3) passing the digested sample through a nickel column again, collecting the effluent sample, and verifying the size of the protein again through SDS-PAGE to finally obtain pure alpha-glucosidase QsGH97a with the molecular weight of about 75.6kDa, wherein the molecular weight is consistent with the predicted size (figure 1). Protein concentration was determined using coomassie brilliant blue dye to give 10mg protein.

Example 4

Enzymatic actives of alpha-glucosidase QsGH97a

Enzymatic kinetic detection of alpha-glucosidase QsGH97a

The enzymatic activity of QsGH97a was measured by the p-nitrophenol method. 100. Mu.L of the system comprising 5. Mu.L of enzyme solution at a mass concentration of 0.6mg/mL, 5. Mu.L of p-nitrophenol-. Alpha. -D-glucoside (Source Blodgett., shanghai) at a final concentration of 0.01mM,0.02mM,0.05mM,0.1mM,0.2mM,0.5mM,1.0mM,1.5mM,2.0mM, respectively, and 90. Mu.L of 20mM Tris-HCl buffer (pH 8.0) were continuously measured for absorbance OD at 45℃using a microplate reader (Thermo Scientific Multiskan FC, USA) ₄₀₅ For 2 minutes, the inactivated enzyme solution was used as a control for zeroing. Fitting the data with software GraphPad to obtain an alpha-glucosidase activity Vmax of 0.64U/mg, michaelis constant K _m =0.21 mM. The standard curve for pNP was used to calculate the amount of pNP produced. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1. Mu. Mol of pNP per minute.

Example 5

Determination of optimal substrate for alpha-glucosidase QsGH97a

The reaction system is the same as above, comprising: 90. Mu.L of 20mM Tris-HCl buffer (pH 8.0), 5. Mu.L of different substrates with a final concentration of 1mM, and 5. Mu.L of pure enzyme with a mass concentration of 0.6mg/mL, the OD of the reaction was determined at 45℃for 2min ₄₀₅ . The measurement substrates are as follows: p-nitrophenol-beta-D-glucoside, p-nitrophenol-alpha-L-arabinopyranoside, p-nitrophenol-beta-D-lactoside, p-nitrophenol-beta-D-galactoside, p-nitrophenol-alpha-D-galactoside, p-nitrophenol-beta-D-mannoside, p-nitrophenol-beta-D-xyloside, p-nitro-phenol-beta-D-fucoside, p-nitrophenol-beta-D-cellobioside. The measurement result shows that the alpha-glucosidase QsGH97a has the maximum catalytic activity on p-nitrophenol-alpha-D-glucoside, has the catalytic activity of 12.5% on p-nitrophenol-beta-D-galactoside, and has no catalytic activity on the rest substrates (figure 2). In summary, the most suitable substrate for α -glucosidase QsGH97a is p-nitrophenol- α -D-glucoside.

Example 6

Analysis of optimal reaction conditions for alpha-glucosidase QsGH97a

The optimum reaction temperature of the alpha-glucosidase QsGH97a is determined in the range of 4-70 ℃. The system is as follows: 5. Mu.L of enzyme solution having a mass concentration of 0.6mg/mL, 5. Mu.L of p-nitrophenol-alpha-D-glucoside having a final concentration of 1mM and 90. Mu.L of 20mM Tris-hydrochloric acid buffer (pH 8.0), OD after 2 minutes of reaction was measured at 20℃30℃35℃40℃45℃50℃55℃60℃and 70℃respectively ₄₀₅ . The measurement results show that QsGH97a has activity in the reaction temperature range of 20-60 ℃, and the optimal reaction temperature is 45 ℃ (figure 3).

The optimal reaction pH of the alpha-glucosidase QsGH97a is determined in the range of pH 3.0-pH 12.0. The system is as follows: 5. Mu.L of enzyme solution with a mass concentration of 0.6mg/mL, 5. Mu.L of p-nitrophenol-alpha-D-glucoside with a final concentration of 1mM and 90. Mu.L of 20mM buffer with different pH. The buffers used were: 20mM citrate-sodium citrate buffer (pH 3.0-pH 6.0), 20mM disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 6.0-pH 8.0), 20mM Tris-HCl buffer (pH 7.5-pH 9.0) and 20mM glycine-sodium hydroxide buffer (pH 9.0-pH 12.0). The measurement results show that the alpha-glucosidase QsGH97a has activity in the pH range of 6.0-11.0, and the optimal reaction pH is pH 8.0 (FIG. 4).

Example 7

Enzymatic stability analysis of alpha-glucosidase QsGH97a

The reaction system for analyzing the effect of metal ions on the activity of alpha-glucosidase QsGH97a is as follows: 5. Mu.L of enzyme solution with a mass concentration of 0.6mg/mL, 5. Mu.L of p-nitrophenol-alpha-D-glucoside with a final concentration of 1mM and 90. Mu.L of 20mM Tris-hydrochloric acid buffer (pH 8.0) were added with 1mM or 10mM Co containing metal ions, respectively ²⁺ 、Cu ²⁺ 、Ca ²⁺ 、Mg ²⁺ 、Zn ²⁺ 、Sr ²⁺ 、Mn ²⁺ 、Ni ²⁺ 、Ba ²⁺ 、K ⁺ 、Na ⁺ Is reacted at 45℃for 2min to determine the OD ₄₀₅ The relative activities of enzymes in the different metal ions were calculated using the enzyme activity of the control group as 100%. The measurement result shows that Ba ²⁺ And Sr ²⁺ Can strongly improve the activity of QsGH97a, co ²⁺ 、Cu ²⁺ 、Zn ²⁺ 、Mn ²⁺ And EDTA can strongly inhibit the activity of QsGH97a, ca ²⁺ 、Mg ²⁺ 、Ni ²⁺ 、K ⁺ And Na (Na) ⁺ Has little effect on the activity of the enzyme (FIG. 5).

The reaction system for analyzing the influence of the organic solvent on the activity of the alpha-glucosidase QsGH97a is as follows: 5. Mu.L of an enzyme solution having a mass concentration of 0.6mg/mL, 5. Mu.L of p-nitrophenol-. Alpha. -D-glucoside having a final concentration of 1mM and 20mM Tris-HCl buffer (pH 8.0), 5% or 15% by volume of an organic solvent (isopropanol, acetonitrile, ethanol, methanol, acetone, dimethyl sulfoxide and glycerol) were added, and the OD after 2 minutes of the reaction was measured at 45 ℃ ₄₀₅ The relative activities of the enzymes in the different organic solvents were calculated using the enzyme activity of the control group as 100%. The measurement results showed that glycerol and dimethyl sulfoxide can inhibit the activity of the enzyme, 15% of glycerol and dimethyl sulfoxide can completely inhibit the activity of the enzyme, and methanol and ethanol can inhibit part of the activity of the enzyme, which indicates that the alpha-glucosidase QsGH97a has poor stability in common organic solvents (A in FIG. 6), but when 1mM Ba is added into the reaction system ²⁺ Or Sr ²⁺ After that, the enzyme generally retains 200% or more of its activity in 15% of an organic solvent (B in FIG. 6).

The reaction system for analyzing the effect of detergent on the activity of α -glucosidase QsGH97a is: 5. Mu.L of an enzyme solution having a mass concentration of 0.6mg/mL, 5. Mu.L of p-nitrophenol-. Alpha. -D-glucoside having a final concentration of 1mM and 20mM Tris-HCl buffer (pH 8.0), a detergent having a volume percentage of 1% or 5%, respectively, was added, comprising: SDS, tween 20, tween 80, triton X-114 or Triton X-100), and OD after 2min of reaction at 45℃was measured ₄₀₅ In comparison with the enzyme activity of the groupThe relative activities of the enzymes in the different detergents were calculated at 100%. The results of the assay showed that Triton X-114 slightly inhibited the enzyme activity, while Tween 20 and Tween 80 enhanced the activity, triton X-100 had little effect on the enzyme activity (FIG. 7A), again with the addition of 1mM Ba ²⁺ Or Sr ²⁺ After this, qsGH97a was more tolerant to 5% detergent (B in FIG. 7).

The reaction system for analyzing the effect of NaCl concentration on the activity of alpha-glucosidase QsGH97a is as follows: 5. Mu.L of enzyme solution with a mass concentration of 0.6mg/mL, 5. Mu.L of p-nitrophenol-alpha-D-glucoside with a final concentration of 1mM and 90. Mu.L of 20mM Tris-hydrochloric acid buffer (pH 8.0), a series of NaCl solutions (1M, 2M, 3M, 4M and 5M) with a concentration were added, and the OD after 2min of the reaction was measured at 45 DEG C ₄₀₅ The relative activities of the enzymes in NaCl at different concentrations were calculated with the enzyme activity of the control group being 100%. The measurement result shows that the alpha-glucosidase QsGH97a has a certain salt tolerance, and can maintain about 20% of enzyme activity (A in FIG. 8) in 5M NaCl aqueous solution, and 1mM Ba ²⁺ Or Sr ²⁺ The addition of (C) allows the enzyme to remain about 50% active in 5M aqueous NaCl solution (B in FIG. 8).

While the foregoing description of the embodiments of the present invention has been presented with reference to the drawings, it is not intended to limit the scope of the invention, but rather, various modifications or variations can be made by those skilled in the art without the need of inventive effort on the basis of the technical solutions of the present invention.

Sequence listing

<110> university of south-middle school

<120> alpha-glucosidase QsGH97a from deep sea bacteria, and coding gene and application thereof

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<213> Artificial Sequence

<400> 2

Met Ile Arg His Ile Ala Leu Phe Ile Gly Ala Ser Leu Leu Pro Ile

1 5 10 15

Ala Ala Ala Gln Ala Glu Thr Val Thr Ser Pro Asp Gly Arg Ile Ala

20 25 30

Val Thr Leu Asp Ala Asp Gly Glu Gly Ile Pro Tyr Tyr Glu Val Thr

35 40 45

Arg Asp Gly Val Pro Val Ile Ala Lys Ser Asn Leu Gly Phe Ala Phe

50 55 60

Thr Asp Ala Asp Pro Met Arg Arg Asn Phe Glu Val Val Ala Phe Arg

65 70 75 80

Glu Glu Ser His Ala Ser Thr Trp Glu Gln Pro Trp Gly Glu Arg Gln

85 90 95

Trp Val Gln Asp Ile His Asn Glu Leu Ala Leu Thr Phe Arg Gln Arg

100 105 110

Asp Glu Asp Ala Arg Glu Ile Thr Val Arg Met Arg Val Phe Asp Asp

115 120 125

Gly Leu Gly Phe Arg Val Glu Phe Pro Glu Thr Glu Arg Gln Pro Val

130 135 140

Tyr Arg Ile Ala Glu Glu Leu Thr Glu Phe Asn Ile Ala Ala Asp Gly

145 150 155 160

Thr Ala Trp Ser Ile Pro Ala Gly Asp Trp Asn Arg Tyr Glu Tyr Leu

165 170 175

Tyr Ser Lys Thr Pro Val Ser Ala Leu Ser Thr Val His Thr Pro Val

180 185 190

Thr Met Val Leu Asp Asn Gly Leu His Leu Ser Phe His Glu Ala Ala

195 200 205

Leu Ile Asp Tyr Ser Gly Met Trp Leu Arg Arg Ile Asp Asp Thr Arg

210 215 220

Phe Arg Ala Gln Leu Ala Pro Ser Pro Arg Gly Pro Lys Val Ile Arg

225 230 235 240

Glu Gly Ala Phe Thr Thr Pro Trp Arg Thr Ile Gln Ile Ala Gly Gly

245 250 255

Pro Lys Gly Leu Phe Glu Ser Asn Ile Ile Leu Asn Leu Asn Glu Pro

260 265 270

Asn Lys Leu Gly Asp Val Ser Trp Phe Thr Pro His Lys Tyr Ile Gly

275 280 285

Ile Trp Trp Glu Met His Leu Asp Asp Ser Ser Trp Ala Ser Gly Glu

290 295 300

Lys His Gly Ala Thr Thr Ala Asn Ala Lys Arg His Ile Asp Phe Ala

305 310 315 320

Ala Glu Asn Gly Phe Arg Gly Val Leu Ile Glu Gly Trp Asn Leu Gly

325 330 335

Trp Asp Gly Thr Trp Phe Gly Asn Gly Arg Glu Phe Asp Phe Thr Thr

340 345 350

Ala Tyr Pro Asp Phe Asp Ile Val Glu Val Ala Arg Tyr Ala Arg Glu

355 360 365

Lys Gly Val Arg Ile Ile Gly His His Glu Thr Gly Gly Asn Ile Lys

370 375 380

Val Tyr Glu Glu Gln Leu Glu Asp Ala Met Ala Phe Tyr Glu Ser Leu

385 390 395 400

Gly Ile Asp Ala Val Lys Ser Gly Tyr Val Ala Asp Ala Gly Gly Ile

405 410 415

Ile Ala Pro Ala Gly Glu Gly Gly Met Gly Glu Thr Phe Val Trp His

420 425 430

Asp Gly Gln Glu Met Val Arg His His Leu Lys Val Val Glu Glu Ala

435 440 445

Ala Glu His Lys Ile Val Met Asn Pro His Glu Pro Ile Lys Asp Thr

450 455 460

Gly Leu Arg Arg Thr Tyr Pro Asn Trp Val Ser Arg Glu Gly Ala Arg

465 470 475 480

Gly Ala Glu Tyr Asp Ala Trp Ala Val Pro Lys Asn Asp Pro Gly His

485 490 495

Val Pro Glu Leu Ile Phe Thr Arg Met Leu Ser Gly Pro Met Asp Tyr

500 505 510

Thr Pro Gly Val Phe Ser Leu Glu Gly Arg Gly Ala Thr Ala Pro Asp

515 520 525

Leu Pro Ser Thr Leu Ala Arg Gln Leu Ala Phe Tyr Val Ala Ile Tyr

530 535 540

Ser Pro Ile Gln Met Val Ala Asp Leu Pro Glu Asn Leu Ala Lys Tyr

545 550 555 560

Pro Arg Ala Leu Asp Phe Val Lys Arg Val Pro Ala Asp Trp Ala Glu

565 570 575

Ser Leu Leu Val Asp Gly Lys Val Gly Glu Phe Ala Ile Ile Ala Arg

580 585 590

Arg Asp Arg Asp Thr Leu Asn Trp Tyr Val Gly Ala Val Thr Asp Ala

595 600 605

Glu Glu Arg Val Ala Ser Val Pro Leu Ser Phe Leu Asp Pro Gly Lys

610 615 620

Ser Tyr Ala Ala Thr Ile Trp Arg Asp Gly Ala Lys Ala Asp Gly Leu

625 630 635 640

Gly Glu Asp Arg His Ala Met Glu Val Glu Thr Met Thr Val Thr Ala

645 650 655

Asp Gln Thr Leu Asp Leu Arg Leu Ala Pro Ala Gly Gly Phe Ala Ile

660 665 670

Glu Phe Val Pro Gln Gly

675

<210> 3

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 3

ggcggatgat ccgccatatc gccctcttca tc 32

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 4

aaactcgagt cacccctgcg gcacgaactc g 31

Claims

1. The alpha-glucosidase QsGH97a is characterized in that the amino acid sequence is shown as SEQ ID NO.2 and is obtained by expressing the coding gene shown as SEQ ID NO. 1.

2. Use of the α -glucosidase QsGH97a according to claim 1 for the production of isomaltooligosaccharides or for the decomposition of polysaccharides to produce glucose.