CN113481185A

CN113481185A - Salt-tolerant beta-galactosidase GalNC2-13 and preparation method and application thereof

Info

Publication number: CN113481185A
Application number: CN202110897489.7A
Authority: CN
Inventors: 许波; 范琴; 黄遵锡; 吴倩
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2021-10-08
Anticipated expiration: 2041-08-05
Also published as: CN113481185B

Abstract

The invention discloses a salt-tolerant beta-galactosidase GalNC2-13 and a preparation method and application thereof, wherein the amino acid sequence of the beta-galactosidase GalNC2-13 is shown as SEQ ID NO.1, the amino acid sequence is 592 amino acids in total, the theoretical molecular weight is 67.83kDa, and the coding gene is shown as SEQ ID NO. 2. The beta-galactosidase GalNC2-13 has good NaCl stability, and has good application potential in food industry, synthesis of Galactooligosaccharides (GOS), report gene and the like.

Description

Salt-tolerant beta-galactosidase GalNC2-13 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a salt-tolerant beta-galactosidase GalNC2-13 and a preparation method and application thereof.

Background

Beta-galactosidase (EC 3.2.1.23) is capable of catalyzing the hydrolysis of lactose to galactose and glucose; secondly, the enzyme has a transglycosylation activity that catalyzes the formation of galactooligosaccharides from lactose. Lactose is a disaccharide, is abundant in mammalian milk and is essential for nutrition of newborn; can be hydrolyzed by lactase in the intestinal tract into absorbable glucose and galactose. The salt-tolerant beta-galactosidase has high enzyme activity in high salt concentration, and eliminates plant polysaccharide in the food industry of high salt process. In addition, Galactooligosaccharides (GOS) can be synthesized and used as a reporter gene. The GOS is produced by beta-galactosidase through the transglycosylation activity during the lactose hydrolysis process, is a non-digestible prebiotic ingredient in food, and is vital to human health, and the enzymatic preparation of GOS has the advantages of simplicity, high efficiency, large amount, less side reactions and the like.

Currently, the method of hydrolyzing lactose by beta-galactosidase is mainly adopted to prepare GOS. The GOS has the functions of promoting the proliferation of probiotics, preventing and treating constipation and the like; secondly, in the food industry as low-calorie sweeteners for fermented milk products, bread and beverages; the milk powder has wide application in various fields such as infant formula milk powder, baked food, pet food and the like. In addition, the problem of lactose intolerance can be solved. In addition, the milk can be stored for a long time at room temperature under acidic conditions and applied to various products without decomposition, so that the milk has very wide application prospect in the whey and milk processing markets. Therefore, the development of multifunctional beta-galactosidase is of great significance.

Disclosure of Invention

The invention aims to provide a salt-tolerant beta-galactosidase GalNC2-13, a preparation method and application thereof, and a construction method of the hydrolase, wherein the salt-tolerant beta-galactosidase GalNC2-13 not only has good salt-tolerant characteristics, but also has high-efficiency transglycosylation activity and high-efficiency lactose hydrolysis activity.

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

a salt-tolerant beta-galactosidase GalNC2-13, wherein the beta-galactosidase GalNC2-13 is derived from animal manure microorganism metagenome, the amino acid sequence of the beta-galactosidase GalNC is shown as SEQ ID NO.1, the beta-galactosidase GalNC total 592 amino acids have the theoretical molecular weight of 67.83 kDa.

The optimum action pH of the beta-galactosidase GalNC2-13 is 6.5, the beta-galactosidase is processed for 1h under the conditions of pH 6.0 and pH6.5, and the residual enzyme activities are 130% and 132% respectively; the optimal action temperature is 37 ℃, and the half-life period is achieved after the tolerance time of 0.5h is up to 37 ℃; the enzyme has better NaCl stability, and the enzyme activity reaches the maximum (about 2 times) when the NaCl concentration is 0.5 mol/L. Can reach more than 200 percent under the concentration of 0.5-1.0 mol/LNaCl; the activity of 190 percent is maintained under the concentration of 1.5mol/L NaCl; the concentration is kept above 130% under 2.0-2.5 mol/L; even at 3.0-5.0mol/L NaCl, the concentration can be maintained at 65-95%.

In another aspect of the invention, the coding gene of the salt-tolerant beta-galactosidase GalNC2-13 is provided, the nucleotide sequence of the coding gene is shown as SEQ ID NO.2, and the gene size is 1779 bp.

In another aspect of the invention, a recombinant expression vector containing the salt-tolerant beta-galactosidase GalNC2-13 encoding gene is provided, and the recombinant expression vector is pEASY-E2/GalNC 2-13.

In another aspect of the present invention, there is provided a recombinant strain comprising the salt-tolerant β -galactosidase GalNC2-13 encoding gene, including but not limited to escherichia coli, yeast, bacillus or lactobacillus, preferably recombinant strain BL21(DE3)/GalNC 2-13.

The invention clones beta-galactosidase coding gene by a PCR method, connects the beta-galactosidase coding gene GalNC2-13 with a plasmid pEASY-E2 to obtain a recombinant expression vector, and then transforms escherichia coli BL21(DE3) to obtain recombinant bacteria.

In another aspect of the present invention, there is provided a method for preparing the salt-tolerant β -galactosidase GalNC2-13, comprising the steps of:

1) taking Western black-crown ape excrement microorganism metagenome DNA as a template, designing primers F and R for PCR amplification to obtain a beta-galactosidase gene;

2) recombining beta-galactosidase gene and expression vector, then transforming to host cell to obtain recombinant strain, culturing the recombinant strain and inducing the expression of recombinant beta-galactosidase;

3) recovering and purifying the expressed beta-galactosidase to obtain the beta-galactosidase GalNC 2-13.

The nucleotide sequences of the primers F and R are shown in SEQ ID NO. 3-4.

In another aspect of the invention, the salt-tolerant beta-galactosidase GalNC2-13 has good salt tolerance, has good application potential in food industry processing, and can be used in high salt process for digesting plant polysaccharide, synthesizing galacto-oligosaccharide (GOS) and serving as a reporter gene.

The invention has the beneficial effects that:

the most suitable pH value of the salt-tolerant beta-galactosidase GalNC2-13 is 6.5, the salt-tolerant beta-galactosidase GalNC2-13 is treated for 1h under the conditions of pH 6.0 and pH6.5, and the residual enzyme activities are respectively 130% and 132%; the optimal action temperature is 37 ℃, and the half-life period is achieved after the tolerance time of 0.5h is up to 37 ℃; the enzyme has better NaCl stability, and the enzyme activity reaches the maximum (about 2 times) when the NaCl concentration is 0.5 mol/L. Can reach more than 200 percent under the concentration of 0.5-1.0 mol/LNaCl; the activity of 190 percent is maintained under the concentration of 1.5 mol/LNaCl; the concentration is kept above 130% under 2.0-2.5 mol/L; even under 3.0-5.0mol/L NaCl, the concentration can be maintained at 65% -95%. Km and Vmax of the enzyme are 4.796mmol/L and 1.022mmol/min respectively; pb²⁺Tween80, DTT and beta-mercaptoethanol have activating effect on the beta-mercaptoethanol, and the activity of the enzyme is respectively improved by 25 percent, 12 percent, 65 percent and 32 percent; sn (tin)²⁺、Na⁺、K⁺、Fe³⁺、Mg²⁺And glycerol had little effect on its enzymatic activity. The properties show that the salt-tolerant beta-galactosidase GalNC2-13 prepared by the invention has better application prospects in the food industry (such as the dairy industry), and in addition, in the synthesis of Galactooligosaccharides (GOS), the report gene and the like.

Drawings

FIG. 1 is an SDS-PAGE analysis of recombinant β -galactosidase GalNC2-13 expressed in E.coli provided by an embodiment of the invention, wherein M: low molecular weight protein Mark er; 1: crude enzyme after E.coli induction containing only pEASY-E2 vector; 2: unpurified recombinant β -galactosidase enzyme; 3: purified recombinant β -galactosidase enzyme;

FIG. 2 is the optimum pH of recombinant β -galactosidase provided by the examples of the present invention;

FIG. 3 is the pH stability of recombinant β -galactosidase provided by the examples of the present invention;

FIG. 4 shows the optimal temperature for recombinant β -galactosidase provided by the examples of the present invention;

FIG. 5 shows the temperature stability of recombinant β -galactosidase provided by the examples of the present invention;

FIG. 6 shows the effect of recombinant β -galactosidase NaCl provided by the examples of the present invention.

FIG. 7 shows the stability of recombinant β -galactosidase NaCl provided by the examples of the present invention;

FIG. 8 is a UPLC analysis of galacto-oligosaccharide synthesized by beta-galactosidase according to the example of the present invention, wherein A is GOS standard and B is GOS which is a transglycosidic product of GalNC 2-13;

FIG. 9 is a UPLC analysis of beta-galactosidase hydrolyzed lactose according to an embodiment of the invention, wherein A is a glucose standard and B is a hydrolysate of GalNC 2-13.

Detailed Description

The technical solution of the present invention will be described clearly and completely with reference to the specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and take the full scope of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test materials and reagents

1. Bacterial strain and carrier: escherichia coli BL21(DE3) was purchased from Onck Biotechnology Ltd, and E.coli expression vector pEASY-E2 was purchased from Beijing Quanjin Biotechnology Ltd.

2. Genetically engineered operating enzymes, kits and other biochemical reagents: restriction enzyme, DNA polymerase, ligase and dNTP are purchased from TaKaRa company, and the DNA purification kit is OMEGA BIO-TEK company; others are all made-in-home reagents (all available from general Biochemical reagent company).

3. LB culture medium: peptone 10g, Yeast extract 5g, NaCl 10g, distilled water to 1000mL, natural pH (about 7). Solid media 2.0% (w/v) agar was added on the above basis.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

EXAMPLE 1 acquisition of the beta-galactosidase Gene GalNC2-13

1) Cloning of beta-galactosidase Gene GalNC2-13

The DNA fragment was expressed as GalNC2-13 DNA F: 5'-taagaaggagatatacatatggaattgatgcttgaaattaaaaataaag-3' and GalNC2-13 DNA R: 5'-gtggtggtggtggtgctcgagtcctaaatcgtgcttatcaag-3' is an upstream primer and a downstream primer, and PCR amplification is carried out by using a western black-crown ape stool microorganism metagenome as a template. The PCR reaction parameters are as follows: denaturation at 98 ℃ for 30s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 90s for 30 cycles. The PCR result obtained the target gene GalNC 2-13.

EXAMPLE 2 preparation of beta-galactosidase GalNC2-13

The beta-galactosidase gene GalNC2-13 prepared in example 1 was ligated with the plasmid pEASY-E2 to obtain a recombinant expression vector pEASY-E2/GalNC2-13, and E.coli BL21(DE3) was transformed to obtain a recombinant E.coli strain BL21(DE3)/GalNC 2-13. Escherichia coli strain BL21(DE3)/GalNC2-13 containing recombinant expression vector pEASY-E2/GalNC2-13 was inoculated in LB (100. mu.g/mL Amp) culture medium in an amount of 0.1% V/V and cultured at 37 ℃ and 180rpm for 12-16 hours. Then inoculating the activated bacterial liquid into fresh LB (containing 100. mu.g/mL Amp) culture solution in an amount of 1% (V/V), and shake-culturing at 37 deg.C, 180rpm, 37 deg.C and 180r/min for about 4-5h (OD)₆₀₀0.6-0.8), adding IPTG with the final concentration of 0.7mmol/L, and culturing at 20 ℃ and 180r/min for 16h in a shaking table to induce the generation of recombinant protein. Centrifuging at 4 deg.C and 5000r/min for 10min to collect thallus. After suspending the cells in a suitable amount of sterile water, the cells were disrupted under high pressure (35 KPSI). Freezing the above crushed cell liquid at 4 deg.C and 12000r/minAfter 10min, taking the supernatant and purifying the target protein by Nickel-NTA Agarose to obtain the salt-tolerant beta-galactosidase GalNC 2-13.

The purified protein GalNC2-13 was subjected to SDS-PAGE analysis, and the results are shown in fig. 1, fig. 1 is an SDS-PAGE analysis of recombinant β -galactosidase expressed in escherichia coli provided by an embodiment of the present invention, wherein M: low molecular weight protein Marker; 1: crude enzyme after E.coli induction containing pE ASY-E2 vector only; 2: unpurified recombinant β -galactosidase enzyme; 3: purified recombinant beta-galactosidase. As can be seen from FIG. 1, the recombinant β -galactosidase was expressed in E.coli and was purified by Nickel-NTA Agarose to give a single band.

EXAMPLE 3 determination of the Properties of the salt-tolerant beta-galactosidase GalNC2-13

Enzyme activity assay methods refer to zhangwenhong (zhangchenghong, 2019): the remaining recombinant β -galactosidase enzyme activity was determined as p-nitrophenyl- β -D-galactopyranoside (pNPGal). p-nitrophenyl-beta-D-galactopyranoside (pNPGal) was dissolved in a buffer solution of pH6.5 to prepare a substrate solution having a final concentration of 2 mmol/L. Taking 450 mu L of pNPGal solution, preheating for 5min at 37 ℃, adding 50 mu L of enzyme solution diluted by proper times, accurately reacting for 10min, and immediately adding 1mL of Na with 1mol/L₂CO₃The reaction was terminated and developed. 200. mu.L of the above reaction solution was put in a 96-well plate, and OD of the reaction solution was measured with a microplate reader₄₂₀And 50. mu.L of inactivated enzyme solution was added as a blank. Definition of enzyme activity unit: one enzyme activity unit (U) is the amount of enzyme required to hydrolyze pNPGal per minute to release 1. mu. mol pNP under the optimal reaction conditions of the enzyme.

1) Determination of optimum pH and pH stability of salt-tolerant beta-galactosidase GalNC2-13

Determination of the optimum pH of the enzyme: the salt-tolerant beta-galactosidase GalNC2-13 purified in example 2 was assayed for enzyme activity in a buffer of pH3.0-12.0(pH 3.0-7.0: 0.1mol/L citric acid-disodium hydrogenphosphate buffer; pH 8.0-12.0: 0.2mol/L glycine-sodium hydroxide buffer) at 37 ℃.

Determination of the pH stability of the enzyme: the enzyme was incubated at 37 ℃ for 1h in buffer solutions of varying pH 3.0-12.0. And (3) determining the residual enzyme activity under the optimal reaction condition according to an enzyme activity determination method. The relative activity of the enzyme at each pH value was calculated with the highest activity as 100%.

Referring to fig. 2 and fig. 3, fig. 2 shows the optimum pH of the salt-tolerant β -galactosidase provided by the embodiment of the present invention, and fig. 3 shows the pH stability of the salt-tolerant β -galactosidase provided by the embodiment of the present invention. As can be seen from FIGS. 2 and 3, the optimum pH of the salt-tolerant beta-galactosidase provided by the present invention is 6.5; after 1h of treatment at pH 6.0 and pH6.5, the residual enzyme activities are 130% and 132%, respectively.

2) Determination of optimum temperature and temperature stability of salt-tolerant beta-galactosidase

Determination of optimum temperature of enzyme: the activity of beta-galactosidase at different temperatures (0-60 ℃) was measured at pH6.5 and the relative activity of the enzyme at each temperature was calculated as the maximum activity 100%.

Temperature stability assay of enzymes: the enzyme solution was incubated at pH6.5 at 30 ℃, 37 ℃ and 40 ℃ for 1 hour, and the enzyme reaction was carried out at pH6.5 and 30 ℃ every 10 minutes, using untreated enzyme solution as a control.

The results are shown in FIGS. 4 and 5. Fig. 4 is the optimum temperature of the salt-tolerant beta-galactosidase provided by the embodiment of the invention, and fig. 5 is the temperature stability of the salt-tolerant beta-galactosidase provided by the embodiment of the invention. The results show that: the optimum temperature of the salt-tolerant beta-galactosidase is 37 ℃, and the salt-tolerant beta-galactosidase keeps stable at the conditions of 30 ℃ and 37 ℃.

3) Determination of NaCl influence and NaCl tolerance of salt-tolerant beta-galactosidase

NaCl effect assay of the enzyme: the enzymatic reaction is carried out at 37 ℃ and pH6.5 under the condition of 0.5-5mol/L NaCl.

NaCl stability assay of the enzyme: adding NaCl with different concentrations under the optimal action condition of the enzyme and in a standard enzyme reaction system to ensure that the final concentration is 0.5-5mol/L, carrying out constant-temperature water bath at 37 ℃ for 1h, and then measuring the residual enzyme activity at 37 ℃ and pH 6.5. Untreated enzyme solution was used as a control.

The results are shown in FIGS. 6 and 7. FIG. 6 is the NaCl tolerance effect of the salt-tolerant beta-galactosidase provided by the embodiments of the present invention, and FIG. 7 is the NaCl tolerance of the salt-tolerant beta-galactosidase provided by the embodiments of the present invention. The results show that: the enzyme activity reached a maximum (about 2-fold) at a NaCl concentration of 0.5 mol/L. The concentration of NaCl can reach more than 200 percent under the condition of 0.5-1.0 mol/L; the activity of 190 percent is maintained under the concentration of 1.5mol/L Na Cl; the concentration is kept above 130% under 2.0-2.5 mol/L; even under 3.0-5.0mol/L NaCl, the concentration can be maintained at 65% -95%.

4) Determination of kinetic parameters of recombinant beta-galactosidase

Kinetic parameters were determined at pH6.5, temperature 37 ℃ and first-order reaction time with pNPGal at different concentrations as substrate (0.2-5.2mmol/L), and Km and Vmax values were calculated according to the Lineweaver-Burk method. The Km and Vmax of the enzyme were determined to be 4.796mmol/L and 1.022mmol/min, respectively, at 37 ℃ and pH 6.5.

5) Determination of influence of different metal ions and chemical reagents on activity of recombinant beta-galactosidase

Various metal ions (Na)⁺、K⁺、Fe²⁺、Fe³⁺、Cu²⁺、Ag⁺、Ca²⁺、Zn²⁺、Co²⁺、Mn²⁺、Ni2⁺、Al3⁺、Li⁺、Mg²⁺、Sn²⁺、Pb²⁺、Hg²⁺) And chemical reagents (SDS, EDTA, guanidine hydrochloride, Tween80, Triton X100, DTT, glycerol, acetic acid, ethanol, methanol, PEG4000, ethyl acetate, urea, beta-mercaptoethanol) are added into the enzymatic reaction system, so that the final concentrations are respectively 10mmol/L and 1% (V/V), and the activity of the beta-galactosidase is measured under the condition of the optimal action of the enzyme. The enzyme activity without metal ions and chemical reagents is taken as a reference. See table 1 for results.

TABLE 1 Effect of chemical reagents on the Activity of recombinant beta-galactosidase

As can be seen from Table 1, Pb²⁺Tween80, DTT and beta-mercaptoethanol have activating effect on the beta-mercaptoethanol, and the activity of the enzyme is respectively improved by 25 percent, 12 percent, 65 percent and 32 percent; sn (tin)²⁺、Na⁺、K⁺、Fe³⁺、Mg²⁺And glycerol had little effect on its enzymatic activity. Zn²⁺And Mn²⁺Completely inhibit the activity of the metal ions, and the rest metal ions or chemical agents have different degrees of inhibition effect on the metal ions or the chemical agents.

6) UPLC assay for recombinant beta-galactosidase transglycosylation activity

Adding the recombinant beta-galactosidase into 25% (W/V) lactose solution, reacting for 24h at 37 ℃ and pH6.5, boiling for 5min immediately to terminate the reaction, centrifuging at 12000r/min for 10min, and taking the supernatant for UPLC analysis.

Results referring to fig. 8, recombinant β -galactosidase was able to convert lactose to GOS in its entirety at 24 h.

7) UPLC assay for recombinant beta-galactosidase hydrolyzed lactose products

Adding the recombinant beta-galactosidase into 5% (W/V) lactose solution, reacting for 12h at 37 ℃ and pH6.5, immediately boiling for 5min to terminate the reaction, then centrifuging for 10min at 12000r/min, and taking the supernatant to perform UPLC analysis.

Results referring to fig. 9, recombinant β -galactosidase was able to hydrolyze lactose to glucose in total at 12 h.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> university of Yunnan Master

<120> salt-tolerant beta-galactosidase GalNC2-13 and preparation method and application thereof

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<170> SIPOSequenceListing 1.0

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<211> 592

<212> PRT

<213> beta-galactoside (GalNC2-13)

<400> 1

Met Leu Glu Ile Lys Asn Lys Glu Phe Tyr Met Asp Gly Lys Pro Phe

1 5 10 15

Lys Ile Tyr Ser Gly Ala Met His Tyr Phe Arg Ile Leu Pro Glu Tyr

20 25 30

Trp Glu Asp Arg Leu Thr Lys Leu Lys Leu Ala Gly Phe Asn Thr Val

35 40 45

Glu Thr Tyr Val Cys Trp Asn Leu His Glu Pro Lys Pro Asn Glu Phe

50 55 60

Cys Phe Asp Gly Met Leu Asp Ile Val Arg Phe Val Glu Thr Ala Lys

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Lys Val Gly Leu Tyr Cys Ile Val Arg Pro Gly Pro Tyr Ile Cys Ala

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Glu Trp Asp Phe Gly Gly Leu Pro Ala Trp Leu Leu Lys Asp Lys Asn

100 105 110

Met Gln Ile Arg Cys Cys Tyr Pro Asp Tyr Leu Ala Cys Val Glu Arg

115 120 125

Phe Tyr Lys Ala Leu Leu Pro Arg Leu Val Ser Leu Leu Glu Thr Asn

130 135 140

Gly Gly Asn Ile Ile Ala Met Gln Val Glu Asn Glu Tyr Gly Ser Tyr

145 150 155 160

Gly Asn Asp Lys Asp Tyr Leu Arg Phe Val Glu Lys Leu Met Met Asp

165 170 175

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

180 185 190

Met Leu Ser Gly Gly Ser Leu Pro His Ile Tyr Lys Val Leu Asn Phe

195 200 205

Gly Ser Lys Ala Lys Thr Ala Phe Gly Cys Leu Lys Asp Phe Glu Asn

210 215 220

Asp Gly Pro Asn Met Cys Gly Glu Phe Trp Cys Gly Trp Phe Asp His

225 230 235 240

Trp Arg Asp Ile His His Thr Arg Asp Ala Ala Ser Val Gly Lys Glu

245 250 255

Ile Lys Asp Phe Leu Asp Ile Gly Ala Ser Phe Asn Phe Tyr Met Phe

260 265 270

His Gly Gly Thr Asn Phe Gly Phe Thr Ala Gly Ala Asn His Asn Pro

275 280 285

Gly Lys Gly Tyr Glu Pro Thr Ile Thr Ser Tyr Asp Tyr Cys Ala Leu

290 295 300

Leu Asn Glu Trp Gly Asp Tyr Thr Pro Ala Tyr His Glu Val Arg Lys

305 310 315 320

Ile Leu Cys Glu Asn Gln Gly Ile Glu Met Arg Gln Leu Pro Pro Ser

325 330 335

Pro Ala Leu Gln Ser Ile Gly Glu Val Lys Leu Thr Glu Phe Ala Pro

340 345 350

Leu Phe Gly Asn Leu Asp Asn Ile Ala Glu Lys His Arg Ala Ala Val

355 360 365

Pro Glu Ser Met Glu Tyr Phe Asp Gln Asn Phe Gly Leu Ile Tyr Tyr

370 375 380

Glu Thr Ile Leu Ser Gly Lys Tyr Asp Ile Ser Pro Ile Glu Phe Lys

385 390 395 400

Asn Val His Asp Phe Gly Tyr Val Tyr Phe Asp Ser Lys Leu Lys Lys

405 410 415

Arg Ile Asp Arg Thr Gln Tyr Thr Glu Pro Lys Lys Gly Leu Lys Ala

420 425 430

Leu Leu Gly Leu Lys Lys Glu Asp Lys Phe Leu Met Pro Ala Leu Lys

435 440 445

Gly Glu Arg Lys Ile Gly Val Leu Val Asp Ala Met Gly Arg Val Asn

450 455 460

Tyr Gly Glu His Met Ile Asp Arg Lys Gly Met Thr Asp Ile Tyr Ile

465 470 475 480

Gly Asn Gln Arg Gln Met Gly Tyr Asp Val Tyr Thr Met Pro Leu Asp

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Asn Leu Glu Lys Leu Val Tyr Gly Ser Ala Ser Asp Ser Leu Pro Val

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Phe Met Lys Gly Glu Phe Thr Ala Asp Ser Lys Ala Asp Cys Phe Val

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His Leu Asp Gly Phe Lys Lys Gly Tyr Val Trp Val Asn Gly Phe Asn

530 535 540

Leu Gly Arg Tyr Trp Ser Val Gly Pro Gln Lys Ser Leu Tyr Leu Pro

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Gly Ala Leu Leu Lys Asp Glu Asn Glu Ile Ile Val Leu Glu Met Glu

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

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<210> 2

<211> 1779

<212> DNA

<213> beta-galactosidase coding gene (GalNC2-13)

<400> 2

atgcttgaaa ttaaaaataa agaattttat atggacggta agccctttaa aatatactca 60

ggtgctatgc actattttcg catcctgccc gaatattggg aggatagatt aacaaagctt 120

aagcttgccg gttttaatac agtagaaacc tatgtctgtt ggaatctgca cgagccaaag 180

ccgaatgaat tttgctttga cggaatgctt gatatcgtaa gatttgttga aactgcaaaa 240

aaggtcggtt tatactgtat tgtccgtccc ggtccctaca tatgtgccga gtgggatttc 300

ggaggcctgc ctgcgtggct tttaaaggac aagaatatgc agattcgctg ctgctatcct 360

gattatcttg cttgtgttga gagattttat aaggcacttc tgccaaggct tgtttcgctg 420

cttgaaacaa atggcggcaa tattattgca atgcaggttg aaaatgagta cggttcttac 480

ggcaatgata aggattatct gcgctttgtt gaaaagctga tgatggactg cggtattgat 540

gttctgtatt ttacatcaga cggcaattgg aagaatatgc tttcaggcgg ttcactcccg 600

catatttata aggtgctgaa tttcggctct aaggcgaaaa cggcttttgg ctgtctgaag 660

gattttgaaa atgacggacc gaatatgtgc ggcgaattct ggtgcggctg gtttgaccat 720

tggagagata ttcaccatac aagagatgca gcatccgttg gcaaagagat taaggatttt 780

cttgatattg gtgcaagctt taatttctat atgttccatg gcggtacgaa ttttggcttt 840

actgcaggtg caaaccataa tcccggcaag ggttatgagc ctaccattac gagctatgat 900

tattgtgcac tgcttaatga atggggtgat tatacccccg cttatcacga ggtgagaaaa 960

atactctgtg aaaatcaggg catagaaatg agacagctgc cgccgtcacc tgctttgcag 1020

tcaatcggtg aggtaaagct tactgaattt gcgccgcttt ttggtaatct tgacaatatt 1080

gccgaaaagc acagagcagc tgtgcctgag agtatggaat atttcgacca gaatttcggc 1140

ctgatttatt atgaaaccat tctgagcgga aaatatgata tttcaccgat tgaattcaag 1200

aatgttcacg atttcggtta tgtatacttc gattcaaaac tgaaaaagag aattgacaga 1260

acacaatata cggagccgaa aaaaggtctc aaggctcttt tgggtttgaa gaaagaagat 1320

aaattcctta tgcctgcact gaaaggtgaa aggaaaatcg gtgtgcttgt tgatgctatg 1380

ggcagagtga attacggtga gcatatgatt gaccgaaagg gtatgacaga tatctatatc 1440

ggcaatcaaa gacagatggg ctatgatgtt tacacaatgc cgcttgataa tcttgaaaag 1500

cttgtatatg gctcagcatc agacagcctg cctgtattta tgaagggcga atttactgct 1560

gattcaaagg cagattgctt tgttcatctt gacggcttta aaaagggcta tgtctgggtt 1620

aacggcttta atctcggcag atattggagt gtcggaccgc agaaatcttt atatcttccg 1680

ggtgcacttc tcaaggatga aaatgagatt atagttcttg aaatggaggg ctttaacaag 1740

cctgcggttt ctattcttga taagcacgat ttaggataa 1779

<210> 3

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

taagaaggag atatacatat ggaattgatg cttgaaatta aaaataaag 49

<210> 4

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtggtggtgg tggtgctcga gtcctaaatc gtgcttatca ag 42

Claims

1. A salt-tolerant beta-galactosidase GalNC2-13, wherein the amino acid sequence of the beta-galactosidase GalNC2-13 is shown as SEQ ID NO. 1.

2. The salt-tolerant beta-galactosidase GalNC2-13 encoding gene of claim 1, wherein the encoding gene is represented by SEQ ID No. 2.

3. A recombinant vector comprising the coding gene of claim 2.

4. A recombinant bacterium comprising the coding gene according to claim 2.

5. The process for the preparation of the salt-tolerant β -galactosidase GalNC2-13 of claim 1, comprising the steps of:

1) taking western black-crown ape excrement microorganism metagenome DNA as a template, designing primers F and R shown in S EQ ID NO. 3-4 for PCR amplification, and obtaining a beta-galactosidase coding gene;

2) recombining a beta-galactosidase coding gene with an expression vector, then transforming the recombined gene into a host cell to obtain a recombined strain, culturing the recombined strain and inducing the expression of the recombined beta-galactosidase;

6. Use of the salt-tolerant β -galactosidase GalNC2-13 of claim 1 in food processing.