CN113481185B - Salt-tolerant beta-galactosidase GalNC2-13 and preparation method and application thereof - Google Patents

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

A salt-tolerant β-galactosidase GalNC2-13 and its preparation method and application

technical field

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

Background technique

β-galactosidase (EC 3.2.1.23) can catalyze the hydrolysis of lactose to generate galactose and glucose; secondly, the enzyme has the transglycosylation activity of catalyzing lactose to form galactooligosaccharides. Lactose is a disaccharide that is abundant in mammalian milk and is essential for neonatal nutrition; it is hydrolyzed by lactase in the gut into absorbable glucose and galactose. Salt-tolerant β-galactosidase has higher enzyme activity in high-salt concentration, and digests plant polysaccharides in the food industry with high-salt processes. In addition, galacto-oligosaccharides (GOS) can be synthesized and used as reporter genes. GOS is produced by β-galactosidase through the transglycosylation activity in the process of lactose hydrolysis. It is an indigestible prebiotic component in food and is essential to human health. The enzymatic preparation of GOS is simple, efficient and effective. Large amount, less side effects and other advantages.

At present, the method of hydrolyzing lactose with β-galactosidase is mainly used to prepare GOS. GOS has the functions of promoting the proliferation of probiotics, preventing and treating constipation; secondly, it is used in the food industry as a low-calorie sweetener for fermented milk products, bread and beverages; it is used in many fields such as infant formula milk powder, baked food and pet food. Wide range of applications. In addition, the problem of lactose intolerance can be solved. In addition, it can be stored at room temperature for a long time under acidic conditions and can be applied to various products without decomposing, so its application prospects in the whey and milk processing markets are very broad. Therefore, it is of great significance to develop multifunctional β-galactosidase.

Contents of the invention

The object of the present invention is to provide a salt-tolerant β-galactosidase GalNC2-13 and its preparation method and application, and also provide the construction method of the hydrolase, the salt-tolerant β-galactosidase GalNC2-13 of the present invention Not only has good salt tolerance, but also has high-efficiency transglycosylation activity and high-efficiency lactose hydrolysis activity.

In order to achieve the above-mentioned technical purpose, the present invention specifically adopts the following technical solutions:

A salt-tolerant β-galactosidase GalNC2-13, the β-galactosidase GalNC2-13 is derived from the metagenome of animal feces microorganisms, its amino acid sequence is shown in SEQ ID NO.1, a total of 592 amino acids, The theoretical molecular weight is 67.83kDa.

The optimum action pH of the β-galactosidase GalNC2-13 is 6.5, and the remaining enzyme activity is 130% and 132% respectively after being treated at pH 6.0 and pH 6.5 for 1 hour; the optimum action temperature is 37°C, at 37 The half-life of the enzyme can be tolerated for 0.5 h at ℃; the enzyme has good NaCl stability, and the enzyme activity reaches the maximum (about 2 times) when the NaCl concentration is 0.5 mol/L. It can reach more than 200% at the concentration of 0.5-1.0mol/L NaCl; maintain 190% activity at the concentration of 1.5mol/L NaCl; maintain more than 130% at the concentration of 2.0-2.5mol/L; even at the concentration of 3.0-5.0mol/L It can also be maintained at 65%-95% under LNaCl.

In another aspect of the present invention, the gene encoding the salt-tolerant β-galactosidase GalNC2-13 is provided, its nucleotide sequence is shown in SEQ ID NO.2, and the gene size is 1779bp.

In another aspect of the present invention, a recombinant expression vector comprising the gene encoding the salt-tolerant β-galactosidase GalNC2-13 is provided, and the recombinant expression vector is pEASY-E2/GalNC2-13.

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

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

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

1) Using the fecal microbial metagenomic DNA of the western black crested gibbon as a template, design primers F and R for PCR amplification to obtain the β-galactosidase gene;

2) recombining the β-galactosidase gene with the expression vector and transforming it into a host cell to obtain a recombinant strain, culturing the recombinant strain and inducing the expression of the recombinant β-galactosidase;

3) recovering and purifying the expressed β-galactosidase to obtain β-galactosidase GalNC2-13.

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

In another aspect of the present invention, the salt-tolerant β-galactosidase GalNC2-13 has good salt-tolerant characteristics, and has good application potential in food industry processing, specifically, it can be used in high-salt processes to Digest plant polysaccharides, synthesize galacto-oligosaccharides (GOS) and serve as reporter genes.

The beneficial effects of the present invention are:

The optimal pH of the salt-tolerant β-galactosidase GalNC2-13 of the present invention is 6.5, and it is treated at pH 6.0 and pH 6.5 for 1 hour, and its remaining enzyme activity is 130% and 132% respectively; the optimal action temperature is 37°C, tolerate 0.5h at 37°C to achieve a half-life; the enzyme has good NaCl stability, and the enzyme activity reaches the maximum (about 2 times) when the NaCl concentration is 0.5mol/L. It can reach more than 200% at the concentration of 0.5-1.0mol/L NaCl; maintain 190% activity at the concentration of 1.5mol/L NaCl; maintain more than 130% at the concentration of 2.0-2.5mol/L; even at 3.0-5.0mol/L It can also be maintained at 65%-95% under NaCl. The Km and Vmax of the enzyme are 4.796mmol/L and 1.022mmol/min respectively; Pb ²⁺ , Tween80, DTT and β-mercaptoethanol can activate it, increasing the enzyme activity by 25%, 12% and 65% respectively , 32%; Sn ²⁺ , Na ⁺ , K ⁺ , Fe ³⁺ , Mg ²⁺ and glycerol had almost no effect on its enzyme activity. The above properties show that the salt-tolerant β-galactosidase GalNC2-13 prepared by the present invention has good applications in the food industry (such as the dairy industry), and secondly, in the synthesis of galacto-oligosaccharides (GOS) and as reporter genes. prospect.

Description of drawings

Fig. 1 is the SDS-PAGE analysis of the recombinant β-galactosidase GalNC2-13 expressed in Escherichia coli provided by the embodiment of the present invention, wherein, M: low molecular weight protein Marker; 1: contains only pEASY-E2 vector Crude enzyme induced by Escherichia coli; 2: Unpurified recombinant β-galactosidase; 3: Purified recombinant β-galactosidase;

Fig. 2 is the optimal pH of the recombinant β-galactosidase provided by the embodiment of the present invention;

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

Fig. 4 is the optimum temperature of the recombinant β-galactosidase provided by the embodiment of the present invention;

Fig. 5 is the temperature stability of the recombinant β-galactosidase provided by the embodiment of the present invention;

Fig. 6 is the effect of the recombinant β-galactosidase NaCl provided by the embodiment of the present invention.

Fig. 7 is the stability of the recombinant β-galactosidase NaCl provided by the embodiment of the present invention;

Figure 8 is the UPLC analysis of galacto-oligosaccharides synthesized by β-galactosidase in the embodiment of the present invention, wherein, A is the GOS standard product, and B is the transglycoside product GOS of GalNC2-13;

Fig. 9 is a UPLC analysis of lactose hydrolyzed by β-galactosidase according to an embodiment of the present invention, wherein A is a glucose standard and B is a hydrolyzate of GalNC2-13.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Apparently, the described embodiments are only a part of the embodiments of the present invention and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Test materials and reagents

1. Strains and vectors: the strain Escherichia coli BL21 (DE3) was purchased from Qingke Biotechnology Co., Ltd., and the E.coli expression vector pEASY-E2 was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

2. Enzymes, kits and other biochemical reagents for genetic engineering operations: restriction endonucleases, DNA polymerases, ligases and dNTPs were purchased from TaKaRa Company, DNA purification kits were from OMEGA BIO-TEK Company; others were domestically produced Reagents (both can be purchased from common biochemical reagent companies).

3. LB medium: Peptone 10g, Yeast extract 5g, NaCl 10g, add distilled water to 1000mL, pH natural (about 7). The solid medium was added with 2.0% (w/v) agar on the basis of the above.

Explanation: For the molecular biology experimental methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kit and product manual.

The acquisition of embodiment 1β-galactosidase gene GalNC2-13

1) Cloning of β-galactosidase gene GalNC2-13

Using GalNC2-13 DNA F: 5’-taagaaggagatatacatatggaattgatgcttgaaattaaaaataaag-3’ and GalNC2-13 DNA R: 5’-gtggtggtggtggtgctcgagtcctaaatcgtgcttatcaag-3’ as upstream and downstream primers, PCR amplification was performed using the fecal microbial metagenome of the western black crested gibbon as a template. The PCR reaction parameters were: denaturation at 98°C for 30s, annealing at 55°C for 15s, extension at 72°C for 90s, 30 cycles. The PCR results obtained the target gene GalNC2-13.

Preparation of embodiment 2β-galactosidase GalNC2-13

The β-galactosidase gene GalNC2-13 prepared in Example 1 was connected to the plasmid pEASY-E2 to obtain the recombinant expression vector pEASY-E2/GalNC2-13, and transformed into large E. coli BL21 (DE3) to obtain recombinant E. coli strain BL21 ( DE3)/GalNC2-13. Take the Escherichia coli strain BL21(DE3)/GalNC2-13 containing the recombinant expression vector pEASY-E2/GalNC2-13, and inoculate it in LB (containing 100 μg/mL Amp) culture medium according to the amount of 0.1% V/V), at 37°C , 180rpm culture 12-16h. Then inoculate the activated bacterial solution into fresh LB (containing 100 μg/mL Amp) culture solution with 1% (V/V) amount, and culture it on a shaker at 37°C, 180rpm, 180r/min for about 4-5h (OD ₆₀₀ =0.6-0.8), after adding IPTG with a final concentration of 0.7mmol/L and culturing for 16h at 20°C under a shaker at 180r/min to induce the production of recombinant protein. The cells were collected by centrifugation at 5000 r/min for 10 min at 4°C. After suspending the cells with an appropriate amount of sterile water, the cells were crushed under high pressure (35KPSI). After the broken cell liquid was refrigerated and centrifuged at 12000r/min for 10min at 4°C, the supernatant was taken and the target protein was purified with Nickel-NTA Agarose to obtain the salt-tolerant β-galactosidase GalNC2-13.

Carry out SDS-PAGE analysis to described purified protein GalNC2-13, see Fig. 1 for the result, Fig. 1 is the SDS-PAGE analysis of the recombinant β-galactosidase expressed in Escherichia coli provided by the embodiment of the present invention, wherein, M : low molecular weight protein marker; 1: crude enzyme induced by Escherichia coli containing only pE ASY-E2 vector; 2: unpurified recombinant β-galactosidase; 3: purified recombinant β-galactosidase. It can be seen from Figure 1 that the recombinant β-galactosidase was expressed in Escherichia coli, and purified by Nickel-NTA Agarose into a single band.

Example 3 Determination of properties of salt-tolerant β-galactosidase GalNC2-13

The enzyme activity determination method refers to Zhang Wenhong (Zhang Wenhong, 2019): p-nitrophenyl-β-D-galactopyranoside (pNPGal) was used to determine the enzymatic activity of the remaining recombinant β-galactosidase. p-Nitrophenyl-β-D-galactopyranoside (pNPGal) was dissolved in pH 6.5 buffer to prepare a substrate solution with a final concentration of 2 mmol/L. Take 450 μL of pNPGal solution, preheat at 37°C for 5 minutes, add 50 μL of enzyme solution diluted to an appropriate multiple, and react accurately for 10 minutes, immediately add 1 mL of 1mol/L Na ₂ CO ₃ to terminate the reaction and develop color. Add 200 μL of the above reaction solution to a 96-well plate, measure the OD ₄₂₀ of the reaction solution with a microplate reader, and add 50 μL of inactivated enzyme solution as a blank control. Definition of enzyme activity unit: One enzyme activity unit (U) is the amount of enzyme required to hydrolyze pNPGal to release 1 μmol pNP per minute under the optimal reaction conditions of the enzyme.

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

Determination of the optimal pH of the enzyme: the salt-tolerant β-galactosidase GalNC2-13 purified in Example 2 was measured at 37°C for pH 3.0-12.0 (pH 3.0-7.0: 0.1mol/L citric acid-hydrogen phosphate Disodium buffer; pH8.0-12.0: 0.2mol/L glycine-sodium hydroxide buffer) Enzyme activity in the buffer.

Determination of the pH stability of the enzyme: the enzyme was incubated at 37° C. for 1 hour in buffer solutions with different pH values ranging from 3.0 to 12.0. According to the enzyme activity assay method, the residual enzyme activity was determined under the optimum reaction conditions. Taking the highest activity as 100%, calculate the relative activity of the enzyme at each pH value.

The results are shown in Fig. 2 and Fig. 3, Fig. 2 is the optimal pH of the salt-tolerant β-galactosidase provided by the embodiment of the present invention, and Fig. 3 is the pH stability of the salt-tolerant β-galactosidase provided by the embodiment of the present invention . It can be seen from Fig. 2 and Fig. 3 that the optimum pH of the salt-tolerant β-galactosidase provided by the present invention is 6.5; when treated at pH 6.0 and pH 6.5 for 1 hour, the remaining enzyme activities are 130% and 132% respectively.

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

Determination of the optimal temperature of the enzyme: at pH 6.5, the activity of β-galactosidase at different temperatures (0-60° C.) was measured, and the relative activity of the enzyme at each temperature was calculated based on the highest activity of 100%.

Determination of the temperature stability of the enzyme: under the condition of pH 6.5, measure at 30°C, 37°C and 40°C for 1 hour, carry out enzymatic reaction at pH 6.5 and 30°C every 10 minutes, use untreated enzyme solution as comparison.

See Figure 4 and Figure 5 for the results. Figure 4 shows the optimum temperature of the salt-tolerant β-galactosidase provided by the examples of the present invention, and Figure 5 shows the temperature stability of the salt-tolerant β-galactosidase provided by the examples of the present invention. The results showed that the optimum temperature of salt-tolerant β-galactosidase was 37℃, and it remained stable at 30℃ and 37℃.

3) NaCl influence of salt-tolerant β-galactosidase and determination of NaCl tolerance

Determination of the effect of NaCl on the enzyme: the enzymatic reaction was carried out at 37°C, pH 6.5, and 0.5-5mol/L NaCl.

Determination of the NaCl stability of the enzyme: Add different concentrations of NaCl to the standard enzyme reaction system under the optimal action conditions of the enzyme so that the final concentration is 0.5-5mol/L. , pH 6.5 conditions to determine the remaining enzyme activity. Untreated enzyme solution was used as a control.

See Figure 6 and Figure 7 for the results. Fig. 6 shows the effect of NaCl on the salt-tolerant β-galactosidase provided by the example of the present invention, and Fig. 7 shows the NaCl tolerance of the salt-tolerant β-galactosidase provided by the example of the present invention. The results showed that the enzyme activity reached the maximum (about 2 times) when the NaCl concentration was 0.5mol/L. At a concentration of 0.5-1.0mol/L NaCl, it can reach more than 200%; at a concentration of 1.5mol/L NaCl, it can maintain 190% activity; at a concentration of 2.0-2.5mol/L, it can maintain above 130%; even at a concentration of 3.0-5.0 It can also be maintained at 65%-95% under mol/LNaCl.

4) Determination of kinetic parameters of recombinant β-galactosidase

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

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

Various metal ions (Na ⁺ , K ⁺ , Fe ²⁺ , Fe ³⁺ , Cu ²⁺ , Ag ⁺ , Ca ²⁺ , Zn ²⁺ , Co ²⁺ , Mn ²⁺ , Ni2 ⁺ , Al3 ⁺ , Li ⁺ , Mg ²⁺ , Sn ²⁺ , Pb ²⁺ , Hg ²⁺ ) and chemical reagents (SDS, EDTA, guanidine hydrochloride, Tween80, Triton X100, DTT, glycerol, acetic acid, ethanol, methanol, PEG4000, ethyl acetate Esters, urea, β-mercaptoethanol) were added to the enzymatic reaction system, so that the final concentrations were 10mmol/L and 1% (V/V) respectively, and the activity of β-galactosidase was measured under the optimal enzyme action conditions . Take the enzyme activity without adding metal ions and chemical reagents as a reference. See Table 1 for the results.

The influence of table 1 chemical reagent on the activity of recombinant β-galactosidase

It can be seen from Table 1 that Pb ²⁺ , Tween80, DTT and β-mercaptoethanol can activate it, increasing the enzyme activity by 25%, 12% and 65%, 32% respectively; Sn ²⁺ , Na ⁺ , K ⁺ , Fe ³⁺ , Mg ²⁺ and glycerol had almost no effect on its enzyme activity. Zn ²⁺ and Mn ²⁺ completely inhibited its activity, and other metal ions or chemical reagents inhibited it to varying degrees.

6) UPLC determination of transglycosylation activity of recombinant β-galactosidase

Add recombinant β-galactosidase to 25% (W/V) lactose solution, react at 37°C and pH 6.5 for 24 hours, immediately boil for 5 minutes to stop the reaction, then centrifuge at 12000r/min for 10 minutes, and take the supernatant Perform UPLC analysis.

The results are shown in Figure 8, the recombinant β-galactosidase can completely convert lactose into GOS within 24 hours.

7) UPLC determination of lactose products hydrolyzed by recombinant β-galactosidase

Add recombinant β-galactosidase to 5% (W/V) lactose solution, react at 37°C and pH 6.5 for 12 hours, immediately boil for 5 minutes to terminate the reaction, then centrifuge at 12000r/min for 10 minutes, and take the supernatant Perform UPLC analysis.

The results are shown in Figure 9, the recombinant β-galactosidase can completely hydrolyze lactose into glucose within 12 hours.

Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variants, the scope of the invention is defined by the appended claims and their equivalents.

sequence listing

<110> Yunnan Normal University

<120> A salt-tolerant β-galactosidase GalNC2-13 and its preparation method and application

<160> 4

<170> SIP Sequence Listing 1.0

<210> 1

<211> 592

<212> PRT

<213> β-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

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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

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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

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

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

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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

515 520 525

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> β-galactosidase encoding gene (GalNC2-13)

<400> 2

atgcttgaaa ttaaaaataa agaattttat atggacggta agccctttaa aatatactca 60

ggtgctatgc actattttcg catcctgccc gaatattggg agatagatt 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 ctaccatac gagctatgat 900

tattgtgcac tgcttaatga atggggtgat tatacccccg cttatcacga ggtgagaaaa 960

atactctgtg aaaatcaggg catagaaatg agacagctgc cgccgtcacc tgctttgcag 1020

tcaatcggtg aggtaaagct tactgaattt gcgccgcttt ttggtaatct tgacaatatt 1080

gccgaaaagc acagcagc tgtgcctgag agtatggaat atttcgacca gaatttcggc 1140

ctgaatttatt 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

<400> 3

taagaaggag atatacatat ggaattgatg cttgaaatta aaaataaag 49

<210> 4

<211> 42

<212>DNA

<213> Artificial Sequence

<400> 4

gtggtggtgg tggtgctcga gtcctaaatc gtgcttatca ag 42

Claims (5)

1. A salt-tolerant β-galactosidase GalNC2-13, characterized in that the amino acid sequence of the β-galactosidase GalNC2-13 is shown in SEQ ID NO.1. 2. The coding gene of the salt-tolerant β-galactosidase GalNC2-13 according to claim 1, characterized in that, the coding gene is as shown in SEQ ID NO.2. 3. A recombinant vector, characterized in that it comprises the coding gene according to claim 2. 4. A recombinant bacterium, characterized in that it comprises the coding gene according to claim 2. 5. The application of the salt-tolerant β-galactosidase GalNC2-13 according to claim 1 in food processing.

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