CN113969290A

CN113969290A - Deep sea bacterium-derived alpha-glucosidase QsGH97a and coding gene and application thereof

Info

Publication number: CN113969290A
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: 2022-01-25
Anticipated expiration: 2041-11-16
Also published as: CN113969290B

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 from a bacterial genome library provided by the second ocean research institute of the national ocean administration, and the alpha-glucosidase QsGH97a is found to have better enzymological properties by expression and purification, can be used for industrial 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 industrial production of the alpha-glucosidase can be realized, the alpha-glucosidase gene is applied to the chemical industry for producing glucose in large quantities, and in addition, the excellent alkali resistance and salt resistance of the alpha-glucosidase gene make the alpha-glucosidase gene have a prospect in special industrial production.

Description

Deep sea bacterium-derived alpha-glucosidase QsGH97a and coding gene and application thereof

Technical Field

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

Background

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

The currently researched alpha-glucosidase is mainly from microorganisms, most of the industrially applied alpha-glucosidase is derived from aspergillus niger, but the disadvantages of low enzymatic activity and poor thermal stability limit the industrial application of the alpha-glucosidase, and the application search of the alpha-glucosidase is still not wide enough. Alpha-glucosidase from different organisms has different characteristics, so that the application range of the alpha-glucosidase is more distinctive, and therefore, the continuous development of the alpha-glucosidase with good characteristics to meet the industrial needs is vital. Alpha-glucosidase from marine microorganisms has new characteristics of temperature stability, metal resistance, salt tolerance, cold adaptability and the like, so that the mining of the alpha-glucosidase from the marine microorganisms, which can be better applied to industrial production, is a good strategy, not only can new enzyme be obtained so as to better understand the biochemical characteristics of the alpha-glucosidase, but also a reference is provided for industrially modifying the enzyme with other application values.

Disclosure of Invention

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

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

1. a gene encoding an α -glucosidase QsGH97a, the gene having a nucleotide sequence selected from any one of:

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

(2) the mutant gene which is obtained by substituting, adding and/or deleting one or more than two nucleotides in the sequence shown in SEQ ID NO.1 and retains the biological characteristics of the alpha-glucosidase QsGH97a protein can be obtained.

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 microorganism or a eukaryotic cell.

Further preferably, the host is Escherichia coli, Saccharomyces cerevisiae, or Chinese hamster ovary cells.

Still more preferably, the host is an e.

4. The amino acid sequence of the alpha-glucosidase QsGH97a obtained by the expression of the coding gene is shown as SEQ ID NO. 2; or away from glutamic acid residues 459 (Glu 459), 483 (Glu 483) and 378 (Glu 378) of the sequence shown in SEQ ID NO.2 (catalytic active center); substitution, addition and/or deletion of one or more amino acids from the amino acid sequences of glutamic acid residue 174 (Glu174) and glutamic acid residue 477 (Glu 477) (metal ion binding site) can give a mutant protein having the characteristics of α -glucosidase QsGH97 a.

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 carrier, host or alpha-glucosidase QsGH97a can be used for producing glucose 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 obtains the alpha-glucosidase QsGH97a gene from a metagenome library of deep sea sediments in the Pacific sea mountain provided by the second ocean research institute of the national ocean Bureau by screening, and the gene is derived from deep sea bacteria Qipengyuania seohaensis sp.SW-135. The expression of the purified alpha-glucosidase QsGH97a shows that the alpha-glucosidase QsGH97a has better enzymatic properties, can be used for producing glucose, and can be applied to food processing and chemical industry related to saccharide hydrolysis. The obtained alpha-glucosidase gene can be cloned into a proper host to realize heterologous expression, the purification method is simple, the industrial production of the alpha-glucosidase can be realized, the alpha-glucosidase gene is applied to the chemical industry for producing glucose in large quantities, and in addition, the excellent alkali resistance and salt resistance of the alpha-glucosidase gene make the alpha-glucosidase gene have a prospect in special industrial production.

An alpha-glucosidase gene qsgh97a having the nucleotide sequence shown in SEQ ID No. 1. The size of the alpha-glucosidase gene QsGH97a is 2037bp, and the basic group composition is as follows: 411A (20.18%), 354T (17.38%), 648C (31.81%) and 624G (30.63%), and the amino acid sequence is shown in SEQ ID No.2, and the encoded protein has 678 amino acids with a size of 75.57 KD. Homology with the GH97 family enzyme derived from Qipengyuania sp.1ndw3 was found to be the highest 83.13% by Blast alignment. Amino acid sequence analysis of the alpha-glucosidase QsGH97a shows that the alpha-glucosidase QsGH97a has a catalytic domain consisting of Glu 459, Glu 483 and Glu 378, indicating that QsGH97a belongs to the alpha-glucosidase GH97 family. In summary, 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 prokaryotic or eukaryotic host, the recombinant alpha-glucosidase QsGH97a is generated through induced expression, and finally the alpha-glucosidase QsGH97a without the tag is obtained through enzyme digestion. Coli, yeast (saccharomyces cerevisiae), mammalian cells (chinese hamster ovary cells), and the like.

The vectors of the invention are well known to those skilled in the art and are experimentally validated as being feasible prokaryotic or eukaryotic expression vectors. The expression vector used in The invention is pSMT3(Li, J.et al. (2012), The RIP1/RIP3 minor forms a functional amplified signaling complex for programmed disruption. cell 150(2), 339. 350.), The alpha-glucosidase QsGH97a gene is inserted into The multiple cloning site of The vector pSMT3 to obtain a recombinant plasmid, and The recombinant plasmid is transformed into E.coli BL21(DE3) plus, and The alpha-glucosidase QsGH97a with high activity is induced and expressed by IPTG. The enzyme activity experiment result shows that the alpha-glucosidase QsGH97a can hydrolyze alpha-glucoside bonds and beta-galactose glucoside bonds, i.e. polysaccharide can be hydrolyzed 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 without adding metal ions, and the Michaelis constant K is_m＝0.21mM。

The temperature range of the alpha-glucosidase QsGH97a for exerting more than 50% of catalytic action is 20-50 ℃, and the optimal temperature is 45 ℃; the pH range is pH 6.0-pH 11.0, and the optimum pH is pH 8.0. Adding different concentrations of metal ions Ba²⁺Or Sr²⁺It can promote 3-7 times enzyme activity, and slightly enhance enzyme activity under the condition of 5% Tween 20 and 80%. Addition of 1mM Ba²⁺Or Sr²⁺Thereafter, the enzyme tolerance in high concentrations of detergent, organic solvent and NaCl was greatly enhanced.

Drawings

FIG. 1 is a diagram of the gel electrophoresis analysis of sodium dodecyl sulfate-polyacrylamide gel of purified alpha-glucosidase QsGH97 a.

FIG. 2 is a substrate specificity diagram of the alpha-glucosidase QsGH97 a. pNP. beta. Glu: p-nitrophenyl beta-D-glucoside; pNP. alpha. Glu: p-nitrophenyl alpha-D-glucoside, pNP alpha Arap: p-nitrophenyl alpha-L-arabinopyranoside; pNP. beta. Lac: p-nitrophenyl beta-D-lactoside; pNP. alpha. Gal: p-nitrophenyl alpha-D-galactoside; pNP. beta. Gal: p-nitrophenyl beta-D-galactoside; pNP β Man: 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 assay value was 100% when pNP. alpha.Glu was defined as the substrate.

FIG. 3 is a graph showing the optimum reaction temperature for the alpha-glucosidase QsGH97 a.

FIG. 4 is a graph showing the optimum reaction pH for the alpha-glucosidase QsGH97 a.

FIG. 5 is a graph showing the effect of metal ions on the activity of the alpha-glucosidase QsGH97 a.

FIG. 6 is a graph showing the effect of organic solvents on the activity of alpha-glucosidase QsGH97a, in which A is the effect of common organic solvents on the activity of the enzyme, and B is the addition of 1mM Ba²⁺Or Sr²⁺Influence of organic solvent (blank is standard reaction system, control is standard reaction system added with 1mM Ba)²⁺Or Sr²⁺And the other is that different organic solvents are added into the control system respectively).

FIG. 7 is a graph showing the effect of detergent on the activity of the enzyme α -glucosidase QsGH97a, wherein,a is the effect of detergent on enzyme activity and B is the addition of 1mM Ba²⁺Or Sr²⁺Effect of post-detergent on enzyme Activity (blank as Standard reaction System, control as Standard reaction System 1mM Ba²⁺Or Sr²⁺And the other is a control system added with different detergents respectively).

FIG. 8 is a graph showing the effect of the alpha-glucosidase QsGH97a on NaCl tolerance, where A is the effect of aqueous NaCl solution on enzyme activity and B is the addition of 1mM Ba²⁺Or Sr²⁺Influence of aqueous NaCl solution on enzyme Activity (blank as standard reaction System and control as standard reaction System with 1mM Ba²⁺Or Sr²⁺And the other is that NaCl with different concentrations is added into the control system respectively).

Detailed Description

The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.

Example 1

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

Samples of deep sea sediments were collected from the marginal area of the pacific seashore. The bacterial genomic library was provided by the second oceanographic institute of the national oceanographic agency of cooperative units.

The primers 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 double enzyme digestion after agarose electrophoresis to determine that the size of the band is correct, the restriction enzyme sites are BamHI and XhoI respectively, and then the restriction enzyme products are reclaimed by tapping respectively. Using T4 DNA ligase and following a fragment to vector molar ratio of 7: 1 was allowed to stand at 16 ℃ overnight, the obtained recombinant vector was transformed into E.coli DH5 α (all-trans gold, China), and then plated on LB agar medium plates containing 50. mu.g/mL kanamycin resistance to screen positive clones. After the sequencing verification is accurate, the mixture is stored in a refrigerator at the temperature of minus 80 ℃. Homology of the inserted gene sequence of interest with the known alpha-glucosidase gene sequences in the database was found by Blastx (http:// blast. ncbi. nlm. nih. gov /), with the highest homology of 83.13% with the enzyme of GH97 family derived from Qipengyuania sp.1NDW3, which has been registered with QZD92199.1 in the GenBank database. 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 as follows: 411A (20.18%), 354T (17.38%), 648C (31.81%) and 624G (30.63%), whose amino acid sequence is shown in SEQ ID NO.2, and the encoded protein contains 678 amino acid residues and is 75.57kDa in size. The amino acid sequence analysis of the alpha-glucosidase QsGH97a belongs to the GH97 family, and shows that the catalytic domain consisting of Glu 459, Glu 483 and Glu 378 forms the catalytic center of the alpha-glucosidase QsGH97a, and the QsGH97a belongs to the alpha-glucosidase GH97 family.

In summary, QsGH97a should be a new member of the GH97 family.

Example 2

Transformation of recombinant plasmid of alpha-glucosidase gene qsgh97a

And (3) placing the positive clonal bacteria with correct sequencing into an LB liquid culture medium to be cultured at 37 ℃, and then extracting the plasmid by using a plasmid extraction kit (Axygen, USA) to obtain the recombinant plasmid. E.coli strain BL21(DE3) plus (whole gold, China) competent cells taken out of a refrigerator at-80 ℃ are dispensed into an EP tube by 30 mul, 1 mul of plasmid is added, after incubation on ice for 30min, heat shock is carried out at 42 ℃ for 1min, incubation on ice for 2min, then 500 mul of LB culture medium is added, shaking culture is carried out at 37 ℃ for 1 hour, 200 mul of supernatant is removed by centrifugation, and the residual bacterial liquid is uniformly coated on an LB agar plate containing 50 mug/mL kanamycin resistance.

Example 3

Expression and purification of alpha-glucosidase QsGH97a

Positive clones transformed into E.coli strain BL21(DE3) plus competent cells on plates were picked and cultured in 500mL of LB liquid medium containing 50. mu.g/mL kanamycin resistanceCultured in medium at 37 ℃ to OD₆₀₀To 0.8 to 1.0, IPTG (isopropyl-. beta. -D-thiogalactoside) was added to a final concentration of 0.5mM at 16 ℃ and cultured on a shaker at 200rpm for 20 hours, and then the cells were collected by centrifugation at low temperature and resuspended in Buffer A solution (50mM Tris-hydrochloric acid, pH 8.0, 500mM sodium chloride, 10mM imidazole, 1% glycerol by volume), and the resuspended solution was placed on ice and disrupted using an ultrasonicator. The supernatant was collected by low temperature centrifugation and purified by gravity column after incubation with Ni-NTA medium for 0.5 h. As the expressed recombinant protein contains His label, the recombinant protein can be adsorbed on a nickel column in an affinity way, and is subjected to gradient elution by imidazole solutions (10mM, 20mM and 250mM) with different concentrations, the recombinant protein can be obtained by collecting eluent, and then the size of the protein is detected by SDS-PAGE. After the concentration of a sample with correct protein size detected by SDS-PAGE is determined, the mass ratio of the protein sample to Ulp1 enzyme (sumo label for cutting recombinant protein) is 500: 1 and dialyzed overnight at 4 ℃ to reduce the imidazole concentration. The digested sample was again passed through a nickel column, the eluted sample was collected and the protein size was again verified by SDS-PAGE to give pure α -glucosidase QsGH97a with a molecular weight of about 75.6kDa, consistent with the predicted size (FIG. 1). Protein concentration was determined by Coomassie Brilliant blue dye method to yield 10mg of protein.

Example 4

Enzymatic activity property of alpha-glucosidase QsGH97a

Enzymatic kinetic detection of alpha-glucosidase QsGH97a

The enzymatic activity of QsGH97a was determined by the p-nitrophenol method. A100. mu.L system comprised 5. mu.L of an enzyme solution having a mass concentration of 0.6mg/mL, 5. mu.L of p-nitrophenol-alpha-D-glucoside (Yuanshii, Shanghai) having 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-hydrochloric acid buffer (pH 8.0), and the absorbance OD was continuously measured at 45 ℃ using a microplate reader (Thermo Scientific Multiskan FC, USA)₄₀₅For 2 minutes, the inactivated enzyme solution was used as a control for zeroing. The data are fitted by software GraphPad, the obtained activity Vmax of the alpha-glucosidase is 0.64U/mg, and the Mie constant K is_m0.21 mM. pNP standard curve for measurementThe amount of pNP produced was calculated. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1. mu. mol pNP per minute.

Example 5

Determination of the optimum substrate for the alpha-glucosidase QsGH97a

The reaction system is as above, comprising: mu.L of 20mM Tris-hydrochloric acid buffer (pH 8.0), 5. mu.L of a different substrate at a final concentration of 1mM, and 5. mu.L of pure enzyme at a mass concentration of 0.6mg/mL, OD was measured at 45 ℃ for 2min of the reaction₄₀₅. The assay substrates were: p-nitrophenol-beta-D-glucoside, p-nitrophenol-alpha 0-L-arabinopyranoside, p-nitrophenol-alpha 2-D-lactoside, p-nitrophenol-beta-D-galactoside, p-nitrophenol-alpha 1-D-galactoside, p-nitrophenol-beta-D-mannoside, p-nitrophenol-beta-D-xyloside, p-nitrophenol-beta-D-fucoside, p-nitrophenol-beta-D-cellobioside. The determination result shows that the alpha 3-glucosidase QsGH97a has the maximum catalytic activity on p-nitrophenol-alpha 4-D-glucoside, has 12.5 percent of catalytic activity on p-nitrophenol-beta-D-galactoside and has no catalytic activity on the rest substrates (figure 2). In summary, the most suitable substrate for the alpha-glucosidase QsGH97a is p-nitrophenol-alpha-D-glucoside.

Example 6

Analysis of optimum reaction conditions for alpha-glucosidase QsGH97a

The optimum reaction temperature of the alpha-glucosidase QsGH97a was determined in the range of 4 ℃ to 70 ℃. The system is as follows: 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 90. mu.L of a 20mM Tris-hydrochloric acid buffer solution (pH 8.0) were subjected to OD measurement at 20 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 70 ℃ for 2min after the reaction₄₀₅. The results of the assay showed that QsGH97a was active at a reaction temperature range of 20-60 ℃ with an optimum reaction temperature of 45 ℃ (FIG. 3).

The optimum reaction pH for the alpha-glucosidase QsGH97a was determined in the pH 3.0-pH 12.0 range. The system 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 90. mu.L of 20mM buffers having different pH values. The buffers used were: 20mM citric acid-sodium citrate buffer (pH 3.0-pH 6.0), 20mM disodium hydrogenphosphate-potassium dihydrogenphosphate buffer (pH 6.0-pH 8.0), 20mM Tris-hydrochloric acid buffer (pH 7.5-pH9.0), and 20mM glycine-sodium hydroxide buffer (pH 9.0-pH 12.0). The results of the assay showed that the α -glucosidase, QsGH97a, was active at pH 6.0 to pH 11.0, with an optimum reaction pH of 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 the alpha-glucosidase QsGH97a is as follows: 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 90. mu.L of a 20mM Tris-hydrochloric acid buffer solution (pH 8.0), to which 1mM or 10mM, respectively, of Co containing a metal ion, was added²⁺、Cu²⁺、Ca²⁺、Mg²⁺、Zn²⁺、Sr²⁺、Mn²⁺、Ni²⁺、Ba²⁺、K⁺、Na⁺Salt solution or ethylenediaminetetraacetic acid (EDTA), and determining OD 2min after reaction at 45 deg.C₄₀₅The relative activities of the enzymes in the different metal ions were calculated with the enzyme activity of the control group as 100%. The measurement result showed that Ba²⁺And Sr²⁺Can strongly improve the activity of QsGH97a, Co²⁺、Cu²⁺、Zn²⁺、Mn²⁺And EDTA strongly inhibits the activity of QsGH97a, Ca²⁺、Mg²⁺、Ni²⁺、K⁺And Na⁺The effect on the activity of the enzyme was not great (FIG. 5).

The reaction system for analyzing the influence of organic solvent on the activity of alpha-glucosidase QsGH97a is as follows: 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-hydrochloric acid buffer solution (pH 8.0), adding an organic solvent (isopropyl alcohol, acetonitrile, ethanol, methanol, acetone, dimethyl sulfoxide and glycerol) in an amount of 5% or 15% by volume, and measuring the OD 2min after the reaction at 45 ℃₄₀₅Relative activities of the enzymes in different organic solvents were calculated with the enzyme activity of the control group as 100%. The test result shows that the glycerol and the dimethyl sulfoxide can inhibit the activity of the enzyme,15% of glycerol and dimethyl sulfoxide can completely inhibit the activity of the enzyme, methanol and ethanol can inhibit partial enzyme activity, which shows that the stability of the alpha-glucosidase QsGH97a in common organic solvent is poor (A in figure 6), but when 1mM of Ba is added into the reaction system²⁺Or Sr²⁺After that, the enzyme can generally maintain more than 200% of activity in 15% of organic solvent (B in FIG. 6).

The reaction system for analyzing the effect of detergent on the activity of the α -glucosidase, QsGH97a, was: mu.L of an 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 20mM Tris-hydrochloric acid buffer solution (pH 8.0) are added with 1% or 5% by volume of detergent respectively, and the detergent comprises: SDS, Tween 20, Tween 80, Triton X-114 or Triton X-100), and the OD 2min after the reaction is measured at 45 ℃₄₀₅Relative activities of the enzymes in the different detergents were calculated with the enzyme activity of the control group as 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, and Triton X-100 had little effect on the enzyme activity (A in FIG. 7), again with the addition of 1mM Ba²⁺Or Sr²⁺Later, 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: 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 90. mu.L of a 20mM Tris-hydrochloric acid buffer solution (pH 8.0), a series of NaCl solutions (1M, 2M, 3M, 4M, and 5M) were added, and OD 2min after the reaction was measured at 45 ℃₄₀₅Relative activities of the enzymes in NaCl at different concentrations were calculated, taking the enzyme activity of the control group as 100%. The measurement result shows that the alpha-glucosidase QsGH97a has certain salt tolerance, and can maintain about 20% of enzyme activity (A in figure 8) in 5M NaCl aqueous solution and 1mM Ba²⁺Or Sr²⁺The enzyme was added so that it still maintained about 50% activity in 5M aqueous NaCl (B in FIG. 8).

Although the embodiments of the present invention have been described with reference to the accompanying drawings, the scope of the present invention is not limited thereto, and various modifications and variations which do not require inventive efforts and which are made by those skilled in the art are within the scope of the present invention.

Sequence listing

<110> university of south-middle school

<120> deep sea bacterium-derived alpha-glucosidase QsGH97a, and coding gene and application thereof

<130> 2021

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2037

<212> DNA

<213> Artificial Sequence

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atgatccgcc atatcgccct cttcatcggt gcctcgctcc tgccgatcgc tgcggcgcaa 60

gcggaaacgg tcacctcgcc cgacgggcgt atcgcggtaa cgctagacgc ggacggcgaa 120

ggcattccct attacgaagt cacgcgcgac ggcgtgccgg tcatcgccaa atcgaacctc 180

ggcttcgctt tcaccgacgc cgacccgatg cgccgcaatt tcgaagtggt cgcctttcgc 240

gaggaaagcc acgccagcac ctgggagcag ccgtgggggg agcgccagtg ggtgcaggac 300

attcacaatg aactggccct caccttccgc cagcgcgatg aagacgcgcg cgagattacc 360

gtgcgtatgc gtgtgttcga cgatggcctc ggcttccgcg tcgaattccc ggaaacggaa 420

cgccagcccg tctatcgcat cgcggaagaa ctgaccgaat tcaacatcgc tgccgatggc 480

acggcatggt cgattcccgc tggcgactgg aaccgctacg aatatctcta ttcgaagacc 540

ccagtgagcg cgctctctac cgtgcacacg ccggtgacga tggtgctcga caacggcctg 600

cacctctctt tccacgaggc ggcactgatc gactattcgg gcatgtggct gcgccgcatc 660

gacgacaccc gttttcgcgc ccagctggcg ccatcgccgc gcggtcccaa ggtaatccgc 720

gaaggcgcct tcaccacgcc gtggcgcacg atccagatcg cgggcgggcc taagggcctg 780

ttcgaaagca acatcatcct caacctcaac gagcccaaca agctcggcga cgtcagctgg 840

ttcaccccgc acaagtacat cggcatctgg tgggaaatgc acctcgatga cagcagctgg 900

gcaagcggtg aaaagcacgg cgcgacgacc gcaaacgcca agcggcacat cgacttcgcc 960

gcagagaacg gctttcgcgg cgtcctgatc gaaggctgga acctgggctg ggacggcacc 1020

tggttcggca atggccgcga gttcgatttc accaccgcct atcccgattt cgatatcgtc 1080

gaagtcgccc ggtacgcgcg ggaaaagggc gtgcgcatca tcggccacca cgaaacgggc 1140

ggcaatatca aggtatacga agaacaactc gaagacgcga tggccttcta tgaaagcctc 1200

ggcatcgatg cggtgaagag cggttatgtc gccgatgctg gcgggatcat cgcgcctgcc 1260

ggtgaaggcg gcatgggcga gaccttcgtc tggcacgacg ggcaggagat ggtccgccat 1320

cacctgaagg tcgttgaaga ggcagccgag cacaagatcg tcatgaatcc gcacgaaccc 1380

atcaaggaca ccggccttcg ccggacctat ccgaactggg tgagccgcga gggcgctcgc 1440

ggcgctgaat acgatgcctg ggcggttccg aagaacgatc cgggccatgt gccggaactc 1500

attttcacgc ggatgctgtc gggtccgatg gactatacgc ctggcgtgtt ttcgctcgaa 1560

gggcgcggtg cgaccgcccc tgacctgccg agcacccttg cgcggcaact ggcattctac 1620

gtagcgatct actcgccgat ccagatggtc gccgacctgc cggaaaacct ggcgaagtat 1680

ccgcgcgcgc tcgacttcgt aaagcgggtt ccggccgact gggcagaaag ccttctcgtc 1740

gatggcaaag tgggcgaatt cgccatcatt gcccgccgcg atagggatac gctgaactgg 1800

tatgttggcg cagttaccga tgcagaggaa cgtgttgcat cggtcccact gtccttcctc 1860

gatcccggca agtcctatgc cgcaacgatc tggcgtgacg gcgcgaaagc cgatggactt 1920

ggcgaggatc gccatgcgat ggaggtcgag accatgaccg taaccgccga ccagacgctc 1980

gatctccgcc ttgcacccgc aggcggcttc gcgatcgagt tcgtgccgca ggggtga 2037

<210> 2

<211> 678

<212> PRT

<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. A gene encoding an α -glucosidase QsGH97a, wherein the gene has a nucleotide sequence selected from any one of:

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

2. A vector carrying the coding gene of claim 1.

3. A prokaryotic or eukaryotic host transformed or transfected with the vector of claim 2.

4. The alpha-glucosidase QsGH97a obtained by expression of the coding gene of claim 1, wherein the amino acid sequence is shown as SEQ ID NO. 2; or the sequence shown in SEQ ID NO.2, namely the amino acid sequence far away from the positions of Glu 459, Glu 483, Glu 378, Glu174 and Glu 477 is substituted, added and/or deleted by one or more than two amino acids, so that the mutant protein with the characteristics of the alpha-glucosidase QsGH97a can be obtained.

5. Use of the vector of claim 2, the host of claim 3 or the α -glucosidase of claim 4, QsGH97a for isomaltooligosaccharide production or for polysaccharide breakdown to produce glucose.

6. Use according to claim 5, characterized in that the saccharide is a substance whose final hydrolysate is glucose.

7. Use according to claim 6, wherein the α -1, 4-glycosidic linkage is the α -1, 4-glycosidic linkage of the non-reducing end of the polysaccharide.