CN109852597B

CN109852597B - Beta-galactosidase galRBM20_1 and preparation method and application thereof

Info

Publication number: CN109852597B
Application number: CN201910219359.0A
Authority: CN
Inventors: 许波; 张文洪; 黄遵锡; 杨云娟; 李俊俊; 唐湘华; 周峻沛
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2019-03-21
Filing date: 2019-03-21
Publication date: 2022-11-18
Anticipated expiration: 2039-03-21
Also published as: CN109852597A

Abstract

The invention discloses a beta-galactosidase galRBM20_1, a preparation method and application thereof, wherein the amino acid sequence of the beta-galactosidase is shown as SEQ ID NO. 1. The invention obtains a plurality of enzyme genes from the Yunnan golden monkey fecal microorganism and constructs a Yunnan golden monkey fecal microorganism metagenome Fosmid library. Beta-galactosidase gene galRBM20_1 is obtained by cloning from the Yunnan golden monkey fecal microorganism metagenome, and the research on the enzymology property and the salt tolerance of the gene is carried out after the heterologous expression of escherichia coli. The recombinase has high pH stability and thermal stability, and also has good salt tolerance. The invention provides a new source for obtaining the beta-galactosidase gene galRBM20_1, provides a large amount of beta-galactosidase gene galRBM20_1 for industrial production through high-efficiency recombinant expression, and can be widely applied to the dairy industry.

Description

Beta-galactosidase galRBM20_1 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to beta-galactosidase galRBM20_1, and a preparation method and application thereof.

Background

Beta-galactosidase (EC3.2.1.238) is known by the scientific name beta-D-galactoside galactohydrolase, commonly known as lactase, which catalyzes the hydrolysis of galactose glycosidic bonds, hydrolyzing lactose to glucose and galactose. Lactose is a specific carbohydrate in mammalian milk, and the vast majority of carbohydrates in milk is lactose, which is only hydrolyzed by beta-galactosidase to glucose and galactose and then absorbed by the small intestinal mucosal cells of the body. Most mammals have high β -galactosidase activity in vivo immediately after birth, but then gradually decline with age, eventually lactase deficiency and resulting in lactose intolerance. Asians and Africans are the people who are most prone to lactose intolerance in the world, and after the people ingest lactose, the lactose cannot be absorbed by small intestines and directly enters large intestines, so that the phenomenon that the large intestine peristalsis is accelerated, and borborygmus, diarrhea and other symptoms are caused is stimulated.

The beta-galactosidase is widely applied to the dairy industry. During the processing of milk and dairy products, safe beta-galactosidase is added to digest it into glucose and galactose, and such milk is called low-lactose milk. The low-lactose milk not only can meet the requirements of normal consumers, but also can meet the requirements of lactose intolerance people and congenital beta-galactosidase deficiency people on eating the dairy products and fully absorbing the nutrition of the dairy products. Currently, the beta-galactosidase obtained is mainly derived from a purely cultured strain of microorganism. The method for screening specific microorganisms from a special environment and separating to obtain pure culture is more traditional and has complicated steps. The method reduces the reliability of the experiment and greatly reduces the success rate of the experiment. Since only about 0.1-1% of microorganisms in nature can be obtained by the traditional pure culture separation method, the development and utilization of beta-galactosidase are greatly limited. The metagenomic technology avoids the problem of microorganism separation culture, realizes the obtainment of a large amount of uncultured microorganism gene resources from the environment, greatly expands the utilization space of microorganism enzymes and gene resources, and is an effective method for discovering a novel biocatalyst.

The research of the halophilic enzyme is an important part in an enzymology plate, and the beta-galactosidase has a wide source but has less practical application value. In the field of food industry, beta-galactosidase is widely used for hydrolysis of lactose, and improving the digestibility, sweetness, solubility, flavor and the like of dairy products. In addition, the beta-galactosidase is also applied to the fields of biotechnology, medicine and the like, and the beta-galactosidase with good properties can bring better economic benefit for actual production and life, so whether the salt tolerance is relevant to the application prospect. According to the reports of the literature, the beta-galactosidase with good salt tolerance is less, and the beta-galactosidase galRBM20_1 provided by the invention has good hydrolysis performance and also has strong salt tolerance.

Disclosure of Invention

The invention aims to provide a beta-galactosidase galRBM20_1 with salt tolerance and a preparation method thereof. And after the gene sequence is compared by BLASTn, the similarity of the gene sequence and the beta-galactosidase gene sequence of the 35 th family recorded in NCBI is highest, and the gene sequence has the activity of transglycosylation and GOS production.

The invention is realized by the following technical scheme:

the amino acid sequence of the beta-galactosidase galRBM20_1 is shown in SEQ ID NO. 1.

The coding gene of the beta-galactosidase galRBM20_1 is shown as SEQ ID NO. 2.

A recombinant vector is a vector containing a nucleotide sequence shown in SEQ ID NO. 2.

Preferably, the vector is pEASY-E2.

A recombinant bacterium is a strain containing a nucleotide sequence shown in SEQ ID NO. 2.

Preferably, the strain is Escherichia coli BL-21 (DE 3).

The preparation method of the beta-galactosidase galRBM20_1 comprises the following steps:

1) Designing primers F1 and R1, and carrying out PCR amplification by taking a metagenome Fosmid library mixed plasmid of the Yunnan golden monkey fecal microorganism as a template to obtain a beta-galactosidase galRBM20_1 gene;

2) Designing primer primers F2 and R2, and carrying out PCR amplification by using a beta-galactosidase gene galRBM20_1 as a template to obtain a gene fragment galRBM20_1/Nde I/Xho I containing recognition sites of endonuclease Nde I and Xho I;

3) Cutting the expression vector pEASY-E2 by using restriction enzymes Nde I and Xho I, and connecting the cut expression vector pEASY-E2 with a gene fragment galRBM20_1/Nde I/Xho I;

4) Transforming the obtained expression plasmid into Escherichia coli BL-21 (DE 3) to obtain a positive clone of a recombinant Escherichia coli strain BL-21 (DE 3)/galRBM 20_1, which is named as BL-21 (DE 3)/galRBM 20_1;

5) The strain BL-21 (DE 3)/galRBM 20_1 is inoculated in LB liquid medium for culture and purified to obtain the beta-galactosidase galRBM20_1.

The nucleotide sequences of the primers F1 and R1 are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The nucleotide sequences of the primers F2 and R2 are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The primers F2 and R2 contain recognition sites of restriction enzymes Nde I and Xho I respectively.

The LB liquid culture medium contains 100mg/L Amp.

The beta-galactosidase galRBM20_1 disclosed by the invention is applied to lactose hydrolysis.

The enzymatic properties of the beta-galactosidase galRBM20_1 are as follows: the enzyme can better hydrolyze lactose into galactose and glucose, has the optimum action temperature of 45 ℃, has unchanged enzyme activity after being endured for one hour at 37 ℃ and 45 ℃, and has 50 percent of residual enzyme activity after being endured for 10min at 50 ℃. The optimum pH value is 5.0, and the activity of 70% or more can be still maintained between pH 4-7.

The invention has the beneficial effects that:

the invention obtains a plurality of enzyme genes from the Yunnan golden monkey fecal microorganism, constructs a Yunnan golden monkey fecal microorganism metagenome Fosmid library, and clones the Yunnan golden monkey fecal microorganism metagenome to obtain the beta-galactosidase gene galRBM20_1, and the recombinase has higher pH stability and thermal stability and good salt tolerance. The invention provides a new source for obtaining the beta-galactosidase gene galRBM20_1, provides a large amount of beta-galactosidase gene galRBM20_1 for industrial production through high-efficiency recombinant expression, and can be widely applied to the dairy industry.

Drawings

FIG. 1 shows PCR amplification of gene galRBM20_1;

FIG. 2 is an SDS-PAGE analysis of β -galactosidase galRBM20_1;

FIG. 3 shows the optimum temperature for β -galactosidase galRBM20_1;

FIG. 4 is the temperature stability of β -galactosidase galRBM20_1;

FIG. 5 is the optimum pH for β -galactosidase galRBM20_1;

FIG. 6 shows the pH stability of β -galactosidase galRBM20_1;

FIG. 7 is the effect of NaCl on the beta-galactosidase galRBM20_1;

FIG. 8 shows the NaCl stability of β -galactosidase galRBM20_1;

FIG. 9 is a TLC analysis of β -galactosidase galRBM20_1 degrading lactose;

FIG. 10 is a TLC analysis of the product after 24h reaction of beta-galactosidase galRBM 20-1 with 25% (w/v) lactose.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to 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 not all 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.

EXAMPLE 1 cloning of the beta-galactosidase Gene galRBM20_1

Primers galRBM20_1F1 (5'-ATGGCCTGCCGGCGTAGGGGCGTT-3') and galRBM20_1R1 (5'-CCACTGGTTGTCCAGGTTTTTCGTA-3') are designed, and Touch-down PCR amplification is carried out on a beta-galactosidase gene galRBM20_1 by taking a yunnan monkey fecal microorganism metagenome Fosmid library mixed plasmid as a template.

And (3) PCR reaction system: 2 XGC buffer I25.0. Mu.L, 2.5mmol/L dNTP Mix 4.0. Mu.L, LA-Taq DNA Polymerase 1U, 10. Mu. Mol/L galRBM 20-1DNA F11.0. Mu.L, 10. Mu. Mol/L galRBM 20-1DNA R11.0. Mu.L, the Fosmi d mixed plasmid of metagenomic library 0.5. Mu.L, dd H ₂ O make up to 50.0. Mu.L.

The PCR reaction program is: 5min at 94 ℃; 30s at 94 ℃;63 ℃ for 30s (0.5 ℃ drop per cycle); 1min at 72 ℃ and 20 cycles; 30s at 94 ℃, 30s at 53 ℃, 1min at 72 ℃ and 10 cycles; 10min at 72 ℃.

The PCR reaction products were analyzed by electrophoresis on a 1% agarose gel (see FIG. 1). After obtaining a large amount of target genes by PCR, the PCR products were separated by coagulation electrophoresis, and the beta-galactosidase gene galRBM20_1 was recovered from the gel using a D NA recovery kit from Promega.

The positive clone of a recombinant escherichia coli strain BL-21 (DE 3)/galRBM 20_1 is obtained by transforming escherichia coli BL-21 (D E) with a prokaryotic expression plasmid pEASY-E2/galRBM20_1 obtained by connecting beta-galactosidase gene galRBM20_1 and a linearized vector pEASY-E2, is named as BL-21 (DE 3)/galRBM 20_1, and is sent to the Shanghai worker for sequencing.

Example 2 construction of E.coli recombinant expression System

Designing an expression primer according to the full-length sequence of the beta-galactosidase gene galRBM20_1 and the use instruction of the one-step rapid cloning kit: galRBM20_1Nde F2 (5'-TAAGAAGGAGATATACATATGGAATTGGCTGCCGGCGTAGGGGCG-3') and galRBM20_1Xho R2 (5'-GTGGTGGTGGTGGTGCTCGAGACAAATTGCAAAGGAATAT-3'), wherein galRBM20_1NdeF and galRBM20_1Xho contain recognition sites for restriction enzymes NdeI (CATATG) and Xho I (CTCGAG), respectively. The gene for β -galactosidase galRBM20_1 was used as a template, and galRBM20_1 was used.

NdeF and galRBM20_1XhoR primers are used for PCR to obtain a gene fragment galRBM20_ 1/NdeI/XhoI containing recognition sites of endonuclease NdeI and XhoI. The expression vector pEASY-E2 was linearized by cleavage with restriction enzymes Nde I and Xho I. A one-step rapid cloning kit is adopted to clone a gene fragment galRBM20_1/Nde I/Xho I onto a linearized expression vector pEASY-E2, a prokaryotic expression plasmid pEASY-E2/galRBM20_1 is obtained by connection, and a positive clone of a recombinant Escherichia coli strain BL-21 (DE 3)/galRBM 20_1 is obtained by transforming Escherichia coli BL-21 (DE 3), which is named as BL-21 (DE 3)/galRB M20_1.

Inoculating Escherichia coli strain BL-21 (DE 3)/galRBM 20_1 at 0.1% into LB (containing 100mg/L Amp) liquid culture medium, shake culturing at 37 deg.C and 180r/minNourishing for about 3-4h (OD) ₆₀₀ 0.6-1.0), IPTG (final concentration of 0.7 mmol/L) is added and the mixture is incubated at 20 ℃ for 10h at 160r/min with shaking to induce recombinant protein production. Centrifuging at 8000r/min for 6min, collecting thallus, resuspending thallus with Tris-HCl (PH 7.0) buffer solution, and ultrasonically breaking cells in ice water bath (300W, ultrasonic on for 5s, and ultrasonic off for 7 s). The cell liquid after ultrasonic disruption was centrifuged at 12000r/min for 10min at 4 ℃ in a refrigerated centrifuge, and the supernatant was collected by repeating the procedure 2-3 times (without contaminating cell debris). And (3) purifying the supernatant through an affinity chromatography column Ni-NTA Agarose to obtain the purified protein, namely the beta-galactosidase galRBM20_1 containing 6 XHis-tag. BL-21 (DE 3) transformed with the empty vector pEASY-E2 was used as a negative control to examine the activity of β -galactosidase galRB M20_1 and the purity of the purified enzyme was examined by SDS-PAGE (FIG. 2).

EXAMPLE 3 determination of optimum temperature and optimum pH

2-Nitrophenyl beta-D-galactopyranoside (oNPG) was dissolved in 0.1mol/L citric acid-disodium hydrogen phosphate buffer (pH 5.0) to prepare a substrate solution having a concentration of 2 mmol/L. Taking 450 mu L of substrate, preheating in 45 ℃ water bath for 5min, adding 50 mu L of purified and properly diluted enzyme solution, and reacting for 10min. Then, 1mL of 1mol/L NaCO was added ₃ The reaction was stopped and the absorbance at 405nm was measured.

Definition of enzyme activity unit: one enzyme activity unit (U) is the amount of enzyme required to hydrolyze the substrate oNPG at 45 ℃ at pH 5.0 per minute to 1. Mu. Mol oNP.

The enzyme activity of beta-galactosidase galRBM20_1 was measured in a buffer solution of pH 7.0 at different temperatures (0 ℃,10 ℃,20 ℃, 30 ℃, 40 ℃,45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 80 ℃).

Different buffers (pH 2.2-8.0; 0.1mol/L citric acid-disodium hydrogen phosphate buffer; pH 8.0-10.0: glycine-sodium hydroxide buffer) at pH 2.2-10.0 were prepared, respectively, the substrate oNPG was dissolved in the above prepared different pH buffers, and the β -galactosidase was diluted with the respective pH buffers. The enzyme activity at each pH was measured at 45 ℃.

Optimum temperature and temperature stability: the recombinant beta-galactosidase galRBM20_1 has the highest relative enzyme activity at 45 ℃ (FIG. 3), which is slightly higher than the optimum temperature of commercial beta-galactosidase (optimum temperature 40 ℃). The relative enzyme activity of the enzyme is above 60% at 37-50 ℃. After treatment at 37 deg.C, 45 deg.C and 50 deg.C for 0-1h, respectively, it is found that 100% of activity can still be retained at 37 deg.C and 45 deg.C, and after treatment at 50 deg.C for 10min, the residual enzyme activity is 50%, and after treatment for 20min, the residual enzyme activity is 20% (fig. 4).

Thermal stability and pH stability: the galRBM20_1 has the highest relative enzyme activity at pH 5.0, and can still maintain 70% or more of activity between pH4 and pH 7 (FIG. 5). The enzyme has good reaction potential in the pH value range (pH 6.5) of the natural cow milk. After being treated for 1 hour in the range of pH4-8, the enzyme activity is over 60 percent, wherein the enzyme activity is about 100 percent in the treatment range of pH 5-8 (figure 6).

Substrate specificity and kinetic parameters: beta-galactosidase galRBM20_1 had a higher specific activity on the substrate, ornpg, followed by pNPG and lactose, with no detectable activity on other nitrobenzene derivatives (data not shown). The kinetic parameters for hydrolysis of oNPG, pNPG and lactose are given below (Table 1).

TABLE 1 kinetic parameters of the hydrolysis of the substrates oNPG, pNPG and lactose by β -galactosidase galRBM 20-1

EXAMPLE 4 determination of the Effect of Metal ions and chemical reagents on recombinant β -galactosidase galRBM20_1

Mixing various metal ions and chemical agent (Na) ⁺ 、Li ⁺ 、Ca ²⁺ 、K ⁺ 、Fe ²⁺ 、Zn ²⁺ 、Mg ²⁺ 、Na ⁺ 、Mn ²⁺ 、Fe ³⁺ 、Pb ²⁺ 、i ²⁺ 、Co ²⁺ 、Ag ⁺ 、Cu ²⁺ 、Hg ²⁺ 、Al ³⁺ EDTA, SDS, CTAB, beta-Mercaptoethanol, triton X-100, tween-80) was added to the enzymatic reaction system so that the final concentration thereof was 1mmol/L (wherein the final concentration of Triton X-100 and Tween-80 was 0.5% (by volume)), and the concentration was measured at 45 ℃ at pH 5.0Beta-galactosidase activity.

Effects of metal ions and chemical agents: 1mmol/L Hg ²⁺ 、Ag ²⁺ SDS inhibit the activity of the beta-galactosidase galRBM20_1 completely, ag ²⁺ Has strong inhibiting effect on the activity of the enzyme, and Triton X-100 has strong activating effect on the enzyme (Table 2). Consistent with most reports, most divalent metal ions have an activating effect on the β -galactosidase, mg2+ is a physiological ion, and is associated with increased efficiency of β -galactosidase (increased Kcat), and thus increased affinity for the substrate (decreased Km).

TABLE 2 Effect of Metal ions and chemical reagents on recombinase Activity

Example 5 determination of the Effect and stability of NaCl on recombinant β -galactosidase galRBM20_1

NaCl solutions (0.5-5 mol/L) at various concentrations were prepared with a buffer of pH 5.0 (0.1 mol/L pH 5.0 citric acid-disodium hydrogen phosphate). At 45 ℃, adding substrates and enzyme solution into NaCl solutions with different concentrations to carry out enzymatic reaction, setting the activity of the reaction enzyme without NaCl in the reaction system as 100%, setting 1 control under each NaCl with different concentrations and 3 repeatedly measuring the influence of NaCl on beta-galactosidase galRBM20_1. Adding the enzyme solution into NaCl solutions with different concentrations to make the final concentration of NaCl be 0.5-5mon/L, placing in a constant-temperature water bath at 45 ℃ for 1h and 24h, and measuring the residual enzyme activity of the beta-galactosidase under the conditions of 45 ℃ and pH 5.0 (0.1 mol/L pH 5.0 citric acid-disodium hydrogen phosphate).

The research on the salt tolerance of the beta-galactosidase galRBM20_1 shows that when the concentration of NaCl in the reaction environment is 2mol/L, the enzyme has 130 percent of relative enzyme activity which is 40 percent higher than that of the beta-galactosidase in the microbial metagenome of the wheat straw degradation product. When the concentration of NaCl is 4mol/L, the enzyme still has more than 40% of enzyme activity, which is more than two times higher than salt-tolerant beta-galactosidase from Planococcus antarctica (Planococcus). The activity of the beta-galactosidase galRBM20_1 treated by 5mol/L NaCl for 1h is improved to 135 percent compared with the original enzyme activity (100 percent), and 45 percent of the enzyme activity still remains after treated by 1mol/L NaCl for 24 h. Compared with other microorganisms and metagenome-derived beta-galactosidase, the recombinant beta-galactosidase galRBM20_1 studied by the experiment has good salt tolerance and good application prospect in the aspects of the food industry, the report gene [ and the like ] in the field of biotechnology.

Effect of NaCl concentration: when the concentration of NaCl in the system is 1mol/L, the relative enzyme activity reaches 153%; when the concentration of NaCl is 0-2 mol/L, the relative enzyme activity is more than 100%; in the reaction system with 5M NaCl, the beta-galactosidase galRBM20_1 has the remaining 20% of enzyme activity (FIG. 7).

NaCl stability: after the beta-galactosidase galRBM20_1 is treated for 1h at 45 ℃ by NaCl with the concentration of 1-5 mol/L, the relative enzyme activity can be kept above 100%, when the action concentration of NaCl is 4mol/L, the relative enzyme activity reaches the highest value of 146%, and when the action concentration of NaCl is 5mol/L, the relative enzyme activity still reaches 135% (figure 8). After NaCl with the concentration of 1-5 mol/L is treated at 45 ℃ for 24h, the enzyme still has 45 percent of activity when the action concentration of the NaCl is 1mol/L (figure 8), and the results show that the enzyme has good NaCl stability.

EXAMPLE 6 hydrolysis assay of recombinant β -galactosidase galRBM20_1

Adding purified beta-galactosidase into 1mL of 5% (w/v) lactose solution and milk respectively, reacting at 45 deg.C and pH 5.0, sampling at different time intervals (30 min, 2h, 8h, 12 h), boiling for 5min to inactivate denaturation, adding n-butanol: ethanol: water (5.

And (3) determination of hydrolysis performance: after the reaction of beta-galactosidase galRBM20 — 1 with 5% (w/v) lactose for 30min, part of the lactose was hydrolyzed. Lactose was almost completely hydrolyzed when the reaction was as long as 12h (fig. 9). Beta-galactosidase is mainly used in the dairy industry for the production of lactose-free milk. After analysis by thin layer chromatography, after 12h, the beta-galactosidase galRBM20_1 can completely hydrolyze 5% (w/v) of lactose, which indicates that the beta-galactosidase can effectively hydrolyze lactose, and therefore has great potential in the production of sugar-free milk.

Example 7 assay for the Performance of recombinant β -galactosidase galRBM20_1 transglycosylation GOS production

Adding the purified beta-galactosidase into 25% (w/w) lactose solution, reacting at 45 deg.C and pH 5.0 for 24h, boiling for 5min to inactivate enzyme denaturation, and performing TLC analysis with the inactivated enzyme solution as negative control.

Determination of transglycosylation GOS production performance: after 24h reaction of beta-galactosidase galRBM20_1 with 25% (w/v) lactose, the production of partial galactooligosaccharides (GO S) was analyzed by Thin Layer Chromatography (TLC) (FIG. 10), indicating that the enzyme has the activity of transglycosylation to produce GOS.

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> beta-galactosidase galRBM20_1, and preparation method and application thereof

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Met Ala Ala Gly Val Gly Ala Leu Ala Trp Ala Gly Pro Gln Ala Ile

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Glu Asn Val Gln Arg Pro Ala Pro Thr Arg Gly Val Pro Val Thr Phe

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Ser Phe Gly Gly Glu Gly Asn Gln Glu Phe Met Leu Asn Gly Lys Pro

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Phe Gln Ile Arg Gly Ala Glu Met His Pro Gln Arg Ile Pro Arg Glu

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Tyr Trp Arg His Arg Ile Gln Thr Ala Lys Ala Met Gly Leu Asn Thr

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Ile Ala Phe Tyr Val Phe Trp Asn Asp His Glu Gln Pro Asp Gly Ser

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Phe Asp Phe Lys Thr Asp Arg Arg Asn Leu Ser Glu Phe Leu Lys Ile

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Cys Gln Glu Glu Gly Met Trp Val Leu Phe Arg Pro Gly Pro Tyr Thr

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Cys Gly Glu Trp Asp Phe Gly Gly Leu Pro His Tyr Leu Leu Lys Asp

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

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Gln Thr Arg Tyr Leu Glu Ala Val Ala Arg Val Ala Glu Pro Phe Leu

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Ala Lys Asn Gly Gly Pro Ile Leu Met Thr Gln Leu Glu Asn Glu Tyr

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Gly Ser Phe Gln Arg Lys Asp Arg Lys Tyr Met Glu Trp Leu Gln Ala

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Phe Trp Thr Lys Lys Gly Phe Gly Pro Phe Tyr Thr Ser Asp Gly Ala

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Gly Glu His Phe Leu Lys Gly Val Thr Leu Pro Gly Val Ala Ile Gly

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Leu Asp Pro Gly Leu Asn Asp Gly Ala Trp Asn Val Ala Asn Lys Cys

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Asn Pro Gly Val Pro Val Phe Ser Ser Glu Thr Tyr Pro Gly Trp Leu

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Arg His Trp Gly Glu Gly Asn Trp Ala Pro Thr Pro Gln Ile Val Asn

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His Val Arg Trp Phe Met Asp Lys Gly Arg Ser Phe Ser Leu Phe Val

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

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Gly Pro Gly Asp Tyr Gln Pro Asp Leu Thr Ser Tyr Asp Tyr Gly Ser

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Pro Val Asp Glu Gln Gly Arg Met Asp Gln Tyr Tyr Ala Gln Met Arg

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Glu Ile Ile Leu Ser Lys Leu Pro Ala Gly Ala Ala Val Pro Glu Pro

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Pro Ala Asp Ile Pro Ala Met Glu Ile Pro Glu Phe Thr Pro Lys Met

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His Ala Gly Leu Trp Glu Asn Met Pro Lys Pro Leu Arg Asn Lys Phe

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Pro Gln Ala Pro Tyr Phe Glu Gln Trp Gly Gln Asn Gln Gly Met Ala

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Val Tyr Ser Thr Ala Val Pro Ala Gly Pro Ala Glu Lys Leu Glu Phe

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Thr Asn Val His Asp Tyr Ala His Val Tyr Leu Asn Gly Glu Pro Val

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Gly Thr Val Asp Arg Arg Leu Gly Gln Lys Ser Val Ser Leu Pro Glu

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gagtttatgc tgaatggtaa gcctttccag atccgcggtg cggaaatgca ccctcagcgg 180

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attgcgtttt atgttttctg gaatgatcat gaacagccgg acggtagctt cgattttaag 300

acggatcgcc gtaatttgag cgagttcctc aaaatctgtc aggaagaagg aatgtgggtt 360

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gggccgatcc tgatgactca gctggaaaat gaatacggaa gtttccagcg caaggatcgt 600

aagtatatgg aatggttgca ggctttctgg acgaagaagg gctttgggcc gttctataca 660

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gctcctaccc cacaaattgt gaaccatgtc cgttggttta tggataaggg gcgttctttc 900

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ggtccggggg attatcaacc ggacttgaca agttatgatt atggttctcc tgtggatgaa 1020

cagggacgca tggatcaata ttatgcccaa atgcgggaaa ttatcttgtc taaactccct 1080

gccggagcag ccgttccgga accgccggca gatattccgg cgatggaaat cccggaattt 1140

acgcctaaaa tgcatgccgg tctttgggaa aatatgccca agcccctacg taataagttc 1200

ccgcaagctc cttattttga acagtggggg cagaatcagg gaatggctgt ttacagcacc 1260

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gtgtatttga atggtgaacc ggtgggtacc gtggatcgcc gactggggca gaagagtgtg 1380

tcattgcctg aacgagccaa ggccgggaag ttggacatcc ttgtggaggc catgggccat 1440

atcaacttcc atatcagcat ggaaagtgac cgtaagggga tttatggacc tgtgaagttg 1500

ggctcccgtg agctgaaggg atggatggtc agaccgcttc cgctgaaggc ttcttccatt 1560

gttcaggctc ctaagggtaa gggcgtttcc aataagaggg aaggcgctca tttccgcgct 1620

gtcgtgaata ttgaaaatcc gcaggatacg ttcttggata tggctcgtta caccaagggg 1680

tatgtgtggg taaacggtat caatgtgggt cgttactgga atgtggggcc gcagttgaga 1740

ctgtacgttc cggctccctt cctgaagaag ggcgagaatg tgattgatgt gctggatttg 1800

cacatgacct ctcccaagcc cattcgtggg atgaaggaac gtaataagga accgggtaag 1860

attaatacga aaaacctgga caaccagtgg taa 1893

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcctgcc ggcgtagggg cgtt 24

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccactggttg tccaggtttt tcgta 25

<210> 5

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taagaaggag atatacatat ggaattggct gccggcgtag gggcg 45

<210> 6

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtggtggtgg tggtgctcga gacaaattgc aaaggaatat 40

Claims

1. The beta-galactosidase galRBM20_1 is characterized in that the amino acid sequence of the beta-galactosidase galRBM20_1 is shown as SEQ ID NO. 1.

2. The beta-galactosidase galRBM20_1 coding gene according to claim 1, wherein the coding gene is represented by SEQ ID No. 2.

3. A recombinant vector is characterized in that the recombinant vector is a vector containing a nucleotide sequence shown in SEQ ID NO. 2.

4. The recombinant strain is characterized by being a strain containing a nucleotide sequence shown in SEQ ID NO. 2.

5. Use of the β -galactosidase galRBM20_1 of claim 1 for lactose hydrolysis.