CN113637660B

CN113637660B - Beta-galactosidase GalNC3-89, and preparation method and application thereof

Info

Publication number: CN113637660B
Application number: CN202110898698.3A
Authority: CN
Inventors: 许波; 范琴; 黄遵锡; 唐湘华
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2023-09-08
Anticipated expiration: 2041-08-05
Also published as: CN113637660A

Abstract

The invention discloses a beta-galactosidase GalNC3-89, a preparation method and application thereof, wherein the amino acid sequence of the beta-galactosidase GalNC3-89 is shown as SEQ ID NO.1, the total molecular weight is 91.50kDa, and the coding gene is shown as SEQ ID NO. 2. The beta-galactosidase GalNC3-89 has good salt resistance, temperature stability and pH stability, high-efficiency transglycosylation activity and high-efficiency lactose hydrolysis activity, and has good application potential in food processing, bioethanol production and industrial dairy product preparation.

Description

Beta-galactosidase GalNC3-89, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to beta-galactosidase GalNC3-89, 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 transglycosylation activity which catalyzes the formation of galactooligosaccharides from lactose. Lactose is a disaccharide, is abundant in mammalian milk, and is vital to nutrition of newborns; can be hydrolyzed by lactase in intestinal tract to absorbable glucose and galactose. The salt-tolerant beta-galactosidase has higher enzyme activity in high salt concentration and digests plant polysaccharide in the food industry of high salt technology. In addition, galactooligosaccharides (GOS) can be synthesized and used as reporter genes. GOS is produced by beta-galactosidase through transglycosylation activity in lactose hydrolysis process, is a component of indigestible prebiotics in food, is vital to human health, and has the advantages of simplicity, high efficiency, large quantity, less side reaction and the like.

Currently, the GOS is prepared mainly by adopting a method of hydrolyzing lactose by beta-galactosidase. GOS has the functions of promoting the proliferation of probiotics, preventing and treating constipation and the like; second, use in the food industry as low calorie sweeteners for fermented milk products, breads and beverages; has wide application in the fields of infant formula milk powder, baked food, pet food and the like. In addition, lactose intolerance can be solved. In addition, the product can be stored and applied to various products for a long time under the acidic condition without decomposition, so that the product has a very broad application prospect in whey and milk processing markets. Therefore, the development of multifunctional beta-galactosidase has important significance.

Disclosure of Invention

The invention aims to provide a beta-galactosidase GalNC3-89, a preparation method and application thereof, and a construction method of the hydrolase, wherein the beta-galactosidase GalNC3-89 has good salt resistance, temperature stability and pH stability, high-efficiency transglycosylation activity and high-efficiency lactose hydrolysis activity.

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

beta-galactosidase GalNC3-89, wherein the beta-galactosidase GalNC3-89 is derived from animal feces metagenome, the amino acid sequence of the beta-galactosidase GalNC3-89 is shown as SEQ ID NO.1, and the total amino acid is 827 amino acids, and the theoretical molecular weight is 91.50kDa.

The optimal action pH of the beta-galactosidase is 6.5, and the residual enzyme activities are all above 70% when the beta-galactosidase is treated for 1h at the pH of 5.0 to 9.0; the optimal action temperature is 40 ℃, the enzyme activities are all over 90 percent after the treatment is carried out for 1 hour at 37 ℃ and 40 ℃ respectively; the enzyme has good NaCl stability, and maintains more than 100% of enzyme activity under 0.5-2.0mol/L NaCl; the enzyme activity can be maintained at 55% -99% even under 2.5-5.0mol/L NaCl.

In another aspect of the invention, a coding gene of the beta-galactosidase GalNC3-89 is provided, the nucleotide sequence of the coding gene is shown as SEQ ID NO.2, and the size of the coding gene is 2484bp.

In another aspect of the present invention, there is provided a recombinant expression vector comprising the gene encoding the β -galactosidase GalNC3-89, wherein said recombinant expression vector is pEASY-E2/GalNC3-89.

In another aspect of the invention, recombinant strains are provided comprising the gene encoding the beta-galactosidase GalNC3-89, including but not limited to E.coli, yeast, bacillus or Lactobacillus, preferably recombinant strain BL21 (DE 3)/GalNC 3-89.

The invention clones beta-galactosidase GalNC3-89 coding gene by PCR method, connects the coding gene with plasmid pEASY-E2 to obtain recombinant expression vector, and then converts colibacillus BL21 (DE 3) to obtain recombinant bacterium.

In another aspect of the present invention, there is provided a method for preparing the beta-galactosidase GalNC3-89, comprising the steps of:

1) Taking the metagenome DNA of the stool microorganisms of the great apes of the Sichuan crown as a template, designing primers F and R for PCR amplification to obtain a beta-galactosidase gene;

2) The beta-galactosidase gene is recombined with an expression vector and then is transformed into a host cell to obtain a recombined strain, and the recombined strain is cultivated and induced to express the recombined beta-galactosidase;

3) Recovering and purifying the expressed beta-galactosidase to obtain the beta-galactosidase GalNC3-89.

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

In another aspect of the invention, there is provided the use of said beta-galactosidase GalNC3-89 in food processing, bioethanol production and for the preparation of industrial dairy products.

In the food processing process, the beta-galactosidase GalNC3-89 can be used for digesting plant polysaccharide and synthesizing galacto-oligosaccharide (GOS) by a high-salt process and can be used as a reporter gene; in chemical processes, monosaccharides after hydrolysis of lactose can be used to produce bioethanol; in the industrial dairy products, the problem of environmental pollution caused by excessive whey generated in the production process is solved; furthermore, in the dairy industry, it is used to produce low lactose milk.

The beneficial effects of the invention are as follows:

the optimal action pH of the beta-galactosidase is 6.5, and the residual enzyme activities of the beta-galactosidase are all above 70 percent after the beta-galactosidase is treated for 1h at the pH of 5.0 to 9.0; the optimal action temperature is 40 ℃, the enzyme activities are all over 90 percent after the treatment is carried out for 1 hour at 37 ℃ and 40 ℃ respectively; the enzyme has good NaCl stability, and maintains more than 100% of enzyme activity under 0.5-2.0mol/L NaCl; the enzyme activity can be maintained at 55% -99% even under 2.5-5.0mol/L NaCl. Km and Vmax of the enzyme are 1.935mmol/L and 0.8948mmol/min respectively; na (Na) ⁺ 、Fe ³⁺ 、Pb ²⁺ Tween80 and TritonX-100 activated GalNC3-89 to increase enzyme activity by 10%, 14%, 28%, 31% and 9%, respectively; the activity of the rest metal ions and the chemical reagent is inhibited to different degrees. The properties show that the beta-galactosidase prepared by the invention has better application potential in the food industry, the synthesis of galacto-oligosaccharides (GOS), the application as a reporter gene and the like. Secondly, the beta-galactosidase can effectively utilize monosaccharide after lactose hydrolysis to produce bioethanol, and solves the problem of environmental pollution caused by excessive whey generated in the production process of industrial dairy products; in addition, the method can be used for producing low-lactose milk in the dairy industry and has good application prospect.

Drawings

FIG. 1 is a SDS-PAGE analysis of recombinant beta-galactosidase GalNC3-89 expressed in E.coli according to an embodiment of the invention, wherein M: a low molecular weight protein Marker;1: e.coli-induced crude enzyme containing pEASY-E2 vector alone; 2: unpurified recombinant beta-galactosidase; 3: purified recombinant β -galactosidase;

FIG. 2 is an optimum pH for recombinant β -galactosidase provided in an embodiment of the invention;

FIG. 3 is a graph showing the pH stability of recombinant β -galactosidase provided by an example of the present invention;

FIG. 4 is an optimum temperature for a recombinant β -galactosidase provided by an embodiment of the invention;

FIG. 5 is a graph showing the temperature stability of recombinant β -galactosidase provided by an example of the present invention;

FIG. 6 is an illustration of the effect of recombinant beta-galactosidase NaCl provided by an example of the present invention.

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

FIG. 8 is a UPLC analysis of galactooligosaccharides synthesized by β -galactosidase according to the examples of the invention, wherein A is GOS standard and B is the transglycosylation product GOS of GalNC 3-89;

FIG. 9 is a UPLC analysis of lactose hydrolyzed by beta-galactosidase according to an embodiment of the invention, wherein A is glucose standard and B is the hydrolysate of GalNC3-89.

Detailed Description

The following description of the present invention will be made more complete and clear with reference to the detailed description of embodiments of the invention, it being understood that the embodiments described are only some of the embodiments of the invention and all of the embodiments are presented. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Test materials and reagents

1. Strains and vectors: strain Escherichia coli BL (DE 3) was purchased from the prime biotechnology company, and the e.coli expression vector pEASY-E2 was purchased from the beijing holohol biotechnology company.

2. Genetic engineering operating enzymes, kits and other biochemical reagents: restriction enzymes, DNA polymerase, ligase and dNTPs are purchased from TaKaRa, and the DNA purification kit is OMEGA BIO-TEK; the other are all domestic reagents (all are available from common biochemical reagent company).

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

Description: the molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.

Example 1 beta-galactosidase Gene GalNC3-89 acquisition

With GalNC3-89 DNA F:5'-taagaaggagatatacatatggaattggcccggacggaattcaaactg-3' and GalNC3-89 DNA R:5'-gtggtggtggtggtgctcgagtttcacggaaatttccaac-3' is an upstream and downstream primer, and PCR amplification is carried out by using the Western black crown gibbon fecal microorganism metagenomic DNA as a template. The PCR reaction parameters are as follows: denaturation at 98℃for 30s, annealing at 55℃for 15s, extension at 72℃for 90s,30 cycles. The PCR result shows that the target gene GalNC3-89 is obtained.

Example 2 preparation of beta-galactosidase GalNC3-89

The beta-galactosidase gene GalNC3-89 prepared in example 1 was ligated with plasmid pEASY-E2 to obtain recombinant expression vector pEASY-E2/GalNC3-89, and large E.coli BL21 (DE 3) was transformed to obtain recombinant E.coli strain BL21 (DE 3)/GalNC 3-89. E.coli strain BL21 (DE 3)/GalNC 3-89 containing recombinant expression vector pEASY-E2/GalNC3-89 was inoculated into LB (100. Mu.g/mL Amp) medium at 0.1% V/V, and cultured at 37℃and 180rpm for 12-16 hours. The activated bacterial cells were then inoculated into fresh LB (100. Mu.g/mL Amp) medium at 1% (V/V) and shake-cultured at 37℃and 180rpm at 37℃and 180r/min for about 4-5 hours (OD) ₆₀₀ =0.6 to 0.8), IPTG was added at a final concentration of 0.7mmol/L and cultured at 20 ℃ for 16 hours under a 180r/min shaker to induce recombinant protein production. And (5) centrifuging at 4 ℃ for 10min at 5000r/min to collect thalli. After the cells were suspended with an appropriate amount of sterile water, the cells were crushed under high pressure (35 KPSI). And (3) subjecting the crushed cell fluid to refrigerated centrifugation at 12000r/min at 4 ℃ for 10min, taking a supernatant, and purifying target proteins by using a Nickel-NTA Agarose to obtain salt-tolerant beta-galactosidase GalNC3-89.

SDS-PAGE analysis of the purified protein GalNC3-89 is carried out, and the result is shown in FIG. 1, and FIG. 1 is the SDS-PAGE analysis of recombinant beta-galactosidase expressed in Escherichia coli, wherein M: a low molecular weight protein Marker;1: e.coli-induced crude enzyme containing only pEASY-E2 vector; 2: unpurified recombinant beta-galactosidase; 3: purified recombinant beta-galactosidase. As can be seen from FIG. 1, the recombinant β -galactosidase was expressed in E.coli and was purified as a single band by Nickel-NTA Agarose.

Example 3 determination of the Properties of beta-galactosidase GalNC3-89

Enzymatic activity assay methods reference Zhang Wenhong (Zhang Wenhong, 2019): recombinant beta-galactosidase enzyme activity was determined with p-nitrophenyl-beta-D-galactopyranoside (pNPGal). P-nitrophenyl-beta-D-galactopyranoside (pNPGal) was dissolved in pH6.5 buffer to prepare a substrate solution with 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 with proper dilution multiple, accurately reacting for 10min, immediately adding 1mL of 1mol/L of Na ₂ CO ₃ The reaction was terminated and developed. 200 mu L of the reaction solution is added into a 96-well plate, and the OD of the reaction solution is measured by an enzyme-labeled instrument ₄₂₀ And 50. Mu.L of the 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 to release 1. Mu. Mol of pNP per minute under the optimal reaction conditions of the enzyme.

1) Determination of optimum pH and pH stability of beta-galactosidase GalNC3-89

Determination of optimal pH of enzyme: the purified beta-galactosidase GalNC3-89 of example 2 was assayed for enzyme activity in pH3.0-12.0 (pH 3.0-7.0:0.1mol/L citrate-disodium hydrogen phosphate buffer; pH8.0-12.0:0.2mol/L glycine-sodium hydroxide buffer) buffer at 37 ℃.

Determination of pH stability of enzyme: the enzyme was incubated at 37℃in buffer solutions of different pH3.0-12.0 for 1h. According to the enzyme activity determination method, the residual enzyme activity is determined under the optimal reaction conditions. The relative activity of the enzyme at each pH was calculated using the highest activity as 100%.

Referring to fig. 2 and 3, fig. 2 shows the optimal pH of the salt-tolerant β -galactosidase provided by the example of the present invention, and fig. 3 shows the pH stability of the β -galactosidase provided by the example of the present invention. As can be seen from fig. 2 and 3, the optimal pH of the beta-galactosidase provided by the present invention is 6.5; the residual enzyme activities of the enzyme are all above 70% after being treated for 1h at pH 5.0 and pH 9.0 respectively.

2) Determination of the optimum temperature and the temperature stability of beta-galactosidase

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

Temperature stability measurement of enzyme: the enzymatic reaction was carried out at pH6.5 and 40℃at 10 minutes intervals by measuring at 37℃and 40℃for 1 hour under pH6.5, and untreated enzyme solution was used as a control.

The results are shown in fig. 4 and 5. Fig. 4 shows the optimum temperature of the beta-galactosidase provided by the embodiment of the invention, and fig. 5 shows the temperature stability of the beta-galactosidase provided by the embodiment of the invention. The results show that: the salt-tolerant beta-galactosidase has an optimal temperature of 40 ℃ and is stable at 37 ℃ and 40 ℃.

3) Influence of NaCl on beta-galactosidase and determination of NaCl tolerance

Determination of the NaCl influence of the enzyme: the enzymatic reaction was carried out at 40℃and pH6.5,0.5-5 mol/L NaCl.

Determination of NaCl stability of the enzyme: adding NaCl with different concentrations into a standard enzyme reaction system under the optimal action condition of the enzyme to ensure that the final concentration is 0.5-5mol/L, carrying out constant-temperature water bath at 40 ℃ for 1h, and measuring the residual enzyme activity under the conditions of 40 ℃ and pH value of 6.5. Untreated enzyme solution was used as a control.

The results are shown in fig. 6 and 7. FIG. 6 shows the NaCl effect of the beta-galactosidase provided by the example of the present invention, and FIG. 7 shows the NaCl tolerance of the salt-tolerant beta-galactosidase provided by the example of the present invention. The results show that: the enzyme activity reached half-life when the reaction system contained 3.5mol/L NaCl. After incubation at 40℃for 1h at 0.5-5.0mol/L NaCl, galNC3-89 retains 117% of the enzyme activity at 0.5mol/L NaCl, respectively; the activity of the catalyst is maintained to be more than 100% under the condition of 1.0-2.0mol/L NaCl; maintaining 55% -99% under 2.5-5.0mol/L NaCl.

4) Kinetic parameter determination of recombinant beta-galactosidase

Kinetic parameters were determined at pH6.5, temperature 40℃and primary reaction time using different concentrations of pNPGal as substrate (0.1-0.9 mmol/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 1.935mmol/L and 0.8948mmol/min, respectively, at 40℃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 ⁺ 、Fe ²⁺ 、Fe ³⁺ 、Cu ²⁺ 、Ag ⁺ 、Ca ²⁺ 、Zn ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Ni ²⁺ 、Al ³⁺ 、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, urea and beta-mercaptoethanol) are added into an enzymatic reaction system to make the final concentration of the reagent be 10mmol/L and 1% (V/V) respectively, and the activity of the beta-galactosidase is measured under the condition of optimal action of the enzyme. The results are shown in Table 1, with reference to the enzyme activity without metal ions and chemical reagents.

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

As can be seen from Table 1, na ⁺ 、Fe ³⁺ 、Pb ²⁺ Tween80 and Triton X-100 activated Gal NC3-89 to increase enzyme activity by 10%, 14%, 28%, 31% and 9%, respectively; the activity of the rest metal ions and the chemical reagent is inhibited to different degrees.

6) UPLC assay for transglycosylation Activity of recombinant beta-galactosidase

Recombinant beta-galactosidase is added into 25% (W/V) lactose solution, reacted for 24 hours at 37 ℃ and pH6.5, immediately boiled for 5min to terminate the reaction, centrifuged for 10min at 12000r/min, and the supernatant is taken for UPLC analysis.

As a result, see FIG. 8, recombinant β -galactosidase was able to convert lactose to GOS at 24 h.

7) UPLC assay of recombinant beta-galactosidase hydrolysis lactose products

Recombinant beta-galactosidase is added into 5% (W/V) lactose solution, reacted for 12 hours at 37 ℃ and pH6.5, immediately boiled for 5 minutes to terminate the reaction, centrifuged for 10 minutes at 12000r/min, and the supernatant is taken for UPLC analysis.

As a result, see FIG. 9, recombinant β -galactosidase was able to hydrolyze lactose completely to glucose at 12 h.

Although 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 alterations can be made to 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 teachers and students

<120> beta-galactosidase GalNC3-89, preparation method and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 827

<212> PRT

<213> beta-galactosidase (GalNC 3-89)

<400> 1

Met Lys Arg Leu Ser Phe Thr Ala Thr Leu Leu Leu Thr Ala Phe Ser

1 5 10 15

Ala Phe Ala Ala Arg Thr Glu Phe Lys Leu Glu Lys Gly Trp Arg Phe

20 25 30

Thr Arg Glu Asp His Ala Glu Ala Val Arg Pro Asp Phe Asp Asp Ser

35 40 45

Ala Trp Gln Arg Val Thr Val Pro His Asp Trp Ala Ile Tyr Gly Pro

50 55 60

Phe Asp Ile Gly Asn Asp Pro Gln Phe Val Ala Ile Glu Gln Asp Gly

65 70 75 80

Glu Thr Val Pro Ser Leu Lys Ala Gly Arg Thr Gly Gly Leu Pro Val

85 90 95

Val Gly Pro Gly Trp Tyr Arg Ile Arg Phe Asp Val Pro Asp Phe Ala

100 105 110

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

115 120 125

Arg Val Tyr Leu Asn Gly Glu Glu Ile Gly Tyr Trp Pro Tyr Gly Tyr

130 135 140

Gly Ser Phe Gln Leu Asp Ala Thr Arg Leu Leu Lys Pro Glu Gly Asn

145 150 155 160

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

165 170 175

Pro Gly Ala Gly Leu Tyr Arg Asn Val His Val Ile Val Ser Asp Glu

180 185 190

Ile Arg Ile Pro Leu Trp Gly Ile Arg Leu Thr Thr Pro Glu Ile Arg

195 200 205

Pro Asp His Ala Lys Val Arg Leu Gln Ala Asp Val Glu Ser Pro Ala

210 215 220

Gly Thr Asp Ser Arg Leu Val Leu Lys Thr Leu Leu Arg Asp Ala Gly

225 230 235 240

Gly Arg Val Val Ala Lys Ala Glu Thr Thr Leu Ala Glu Tyr Asp Ala

245 250 255

Gly Thr Phe Cys Gln Asp Leu Val Ile Asp Ala Pro Arg Leu Trp Ser

260 265 270

Pro Asp Thr Pro Asp Leu Tyr Glu Ala Glu Leu Arg Leu Tyr Ala Asp

275 280 285

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

290 295 300

Lys Ile Val Pro Asp Arg Gly Met Phe Leu Asn Gly Glu Pro Ile Lys

305 310 315 320

Phe Arg Gly Val Cys Leu His His Asp Leu Gly Pro Leu Gly Ala Ala

325 330 335

Val Asn Val Ser Ala Leu Arg Arg Gln Leu Ser Ile Leu Lys Glu Met

340 345 350

Gly Ala Asn Ala Val Arg Thr Ala His Asn Ile Pro Ala Pro Glu Leu

355 360 365

Val Glu Leu Cys Asp Arg Met Gly Leu Met Val Met Val Glu Thr Phe

370 375 380

Asp Glu Trp Arg Thr Pro Lys Met Lys Asn Gly Tyr His Leu Tyr Phe

385 390 395 400

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

405 410 415

Asn His Pro Ser Val Val Met Trp Cys Ile Gly Asn Glu Val Pro Asp

420 425 430

Gln Ser Ser Tyr Glu Gly Ala Lys Ile Ala Arg Trp Leu Gln Asp Ile

435 440 445

Cys His Arg Glu Asp Pro Thr Arg Leu Val Thr Met Gly Ile Asp Arg

450 455 460

Val Gln Asp Ala Ile Asp Thr His Phe Ala Ala Val Met Asp Val Val

465 470 475 480

Gly Phe Asn Tyr Arg Thr His Leu Tyr Thr Lys Ala Tyr His Glu Leu

485 490 495

Pro Gln Gln Ile Met Met Gly Ser Glu Thr Ala Ser Thr Phe Ser Ser

500 505 510

Arg Gly Thr Tyr His Phe Pro Val Glu Arg Thr Val Asn Lys Val Arg

515 520 525

Pro Asp Asn Gln Ser Ser Gly Tyr Asp Leu Asp Cys Gly Ser Trp Ser

530 535 540

Asn Leu Pro Glu Asp Asp Phe Val Leu His Asp Asp Tyr Asp Trp Cys

545 550 555 560

Ile Gly Glu Phe Val Trp Thr Gly Phe Asp Tyr Leu Gly Glu Pro Thr

565 570 575

Pro Tyr His Glu Ile Trp Pro Asn His Ser Ser Leu Phe Gly Ile Val

580 585 590

Asp Leu Ala Gly Leu Pro Lys Asp Arg Tyr Tyr Leu Tyr Arg Ser His

595 600 605

Trp Arg Pro Glu Glu Glu Thr Leu His Val Leu Pro His Trp Thr Trp

610 615 620

Pro Gly Arg Glu Gly Glu Val Thr Pro Val Phe Val Tyr Thr Asn Tyr

625 630 635 640

Pro Ser Ala Glu Leu Phe Val Asn Gly Arg Ser Gln Gly Arg Ile Ala

645 650 655

Lys Asp Thr Thr Met Thr Gln Ala Ala Thr Asp Ser Glu Glu Ala Ala

660 665 670

Arg Gly Leu Trp Arg Gln Arg Arg Tyr Arg Leu Met Trp Met Asp Val

675 680 685

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

690 695 700

Arg Pro Ala Ala Glu Thr Glu Val His Thr Ala Gly Glu Pro Cys Arg

705 710 715 720

Leu Glu Leu Ser Ala Asp Arg Gln Thr Leu Arg Ala Asp Gly Lys Asp

725 730 735

Leu Ser Phe Val Thr Val Arg Val Val Asp Arg Ala Gly Asn Leu Cys

740 745 750

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

755 760 765

Phe Arg Ala Ala Ala Asn Gly Asp Pro Thr Cys Leu Glu Pro Phe His

770 775 780

His Pro Arg Met Lys Ala Phe Lys Gly Gln Leu Val Ala Ile Val Arg

785 790 795 800

Ser Gly Glu Arg Pro Gly Lys Ile Gly Phe Glu Ala Ser Ala Glu Gly

805 810 815

Leu Arg Lys Ala Arg Leu Glu Ile Ser Val Lys

820 825

<210> 2

<211> 2484

<212> DNA

<213> beta-galactosidase coding Gene (GalNC 3-89)

<400> 2

atgaaacgat tatcttttac cgcgactctc ttattgaccg ctttttccgc attcgccgcc 60

cggacggaat tcaaactgga aaagggctgg cgatttaccc gcgaagatca tgcggaggcc 120

gttcgtccgg atttcgacga ttcggcctgg cagcgcgtga cggttccgca cgactgggcg 180

atttacgggc ctttcgacat cggcaacgac cctcagttcg tggccatcga gcaggacggc 240

gagaccgttc cgtcgctcaa agccggacgt accgggggac tgcccgtcgt cgggcccggt 300

tggtacagga ttcgtttcga cgtgccggat tttgccgccg ggaaacgggc cgatattctt 360

ttcgacggag ccatgagcaa tgcccgggtc tatctcaacg gagaggagat cggttactgg 420

ccctatggct acggcagttt ccagctggat gcgacccggc tgctgaagcc ggagggcaac 480

gtgctggccg tgcggttgga gaactatccc gaatcctccc ggtggtatcc gggggccgga 540

ttgtaccgga acgtgcatgt gatcgtttcg gatgaaattc gtatcccgct gtggggcatt 600

cgtctcacga cgcccgagat ccgtccggac catgcgaagg tgcggttgca ggcggatgtc 660

gaatcgccgg cggggaccga ttcacggctg gtgttgaaaa cgcttcttcg ggatgccgga 720

ggacgggtcg ttgcgaaggc tgaaacgacg cttgccgaat acgacgccgg gaccttctgt 780

caggatctgg tgatcgatgc cccgcggctt tggtcgcccg atacgcccga cctgtatgaa 840

gcggagctcc ggctatatgc cgacggggag cttcgggata cccgttcggt gccgttcggt 900

gtccgggagc tgaaaatcgt tcccgaccga gggatgttcc tcaacggcga accgatcaag 960

ttcaggggtg tttgcctgca tcacgatctc gggccgctgg gcgcggcggt caacgtcagt 1020

gcgctgcgcc ggcagttgtc gatcctcaag gagatggggg ccaatgccgt ccgcacggcg 1080

cataatatcc ccgctccgga gctggtcgaa ctgtgcgacc ggatggggct gatggtgatg 1140

gtggagacct tcgacgagtg gcgcaccccc aagatgaaga acggttatca cctctatttc 1200

gacgaatggg ccgagcgcga tctggtcaat acggtccggc gtttccgcaa tcatccgtcg 1260

gtggtgatgt ggtgcatcgg caacgaggtt cccgaccaaa gcagttacga aggggcgaag 1320

atcgcccggt ggttgcagga tatctgtcat cgggaggacc cgacgcgcct cgttaccatg 1380

gggatcgacc gggtgcagga tgctatcgac acccatttcg cggccgtcat ggacgtggtg 1440

ggcttcaact accgcaccca tctctatacg aaggcgtatc acgagctgcc ccagcagatt 1500

atgatggggt ccgagaccgc ttccacgttc agttcgcggg ggacctatca tttcccggtg 1560

gaacgcaccg tgaacaaggt ccgtccggat aaccagtcgt cgggctacga cctggactgc 1620

ggcagttggt ccaacctgcc cgaagatgat ttcgtgctgc acgacgatta cgactggtgc 1680

atcggcgagt tcgtatggac cggattcgat tatctggggg agcccacgcc ttaccatgag 1740

atctggccca accacagttc gctgttcggg atcgtggatc tggccgggtt gcccaaagac 1800

cgctattacc tctatcggag ccattggcgg cccgaggagg agaccttgca tgtcctgccg 1860

cattggacct ggcccggtcg tgaaggcgag gtgacccctg tgttcgtcta tacgaactac 1920

ccttctgccg agctgttcgt gaacggcagg agccagggcc gcattgccaa agatacgacg 1980

atgacacagg ctgcgaccga cagcgaagag gccgcccggg gactttggcg ccagcgccgt 2040

taccgtctga tgtggatgga tgtgaaatat gaacccggga cgttgagggt ggtggcttac 2100

gaccggaacg gccggccggc tgccgagacc gaggtgcaca cggcgggcga accctgccgg 2160

ctggagcttt cggccgacag gcagactctt cgtgccgacg gcaaggacct ttcgtttgtc 2220

acggtgcggg tcgtggacag agcgggcaac ctctgcccgg acgccgctcc ggaggtctcg 2280

ttccgcgtca ccggggccgg agggttccgg gcggccgcga acggggaccc gacctgtctg 2340

gaaccgttcc accatccgcg gatgaaggct ttcaagggac agctcgtggc gattgtccga 2400

tcgggggaga gacccgggaa gatcggattc gaggcttcgg cggagggact gcgcaaggcg 2460

cggttggaaa tttccgtgaa ataa 2484

<210> 3

<211> 48

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

taagaaggag atatacatat ggaattggcc cggacggaat tcaaactg 48

<210> 4

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gtggtggtgg tggtgctcga gtttcacgga aatttccaac 40

Claims

1. The beta-galactosidase GalNC3-89 is characterized in that the amino acid sequence of the beta-galactosidase GalNC3-89 is shown as SEQ ID NO. 1.

2. The coding gene of the beta-galactosidase GalNC3-89 of claim 1, wherein the coding gene is shown in 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. Use of the beta-galactosidase GalNC3-89 according to claim 1 for food processing, bioethanol production.