CN113637660A - Beta-galactosidase GalNC3-89 and preparation method and application thereof - Google Patents

Abstract

The invention discloses a beta-galactosidase GalNC3-89, a preparation method and an application thereof, wherein the amino acid sequence of the beta-galactosidase GalNC3-89 is shown as SEQ ID NO.1, and the amino acid sequence totally 827 amino acids have the theoretical molecular weight of 91.50kDa, and the coding gene is shown as SEQ ID NO. 2. The beta-galactosidase GalNC3-89 has good salt tolerance, temperature stability, 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 (EC3.2.1.23) 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 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 specifically adopts the following technical scheme:

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

The optimum action pH of the beta-galactosidase is 6.5, the beta-galactosidase is processed for 1h under the pH value of 5.0 to 9.0, and the residual enzyme activity is above 70 percent; the optimum action temperature is 40 ℃, the treatment is carried out for 1h at 37 ℃ and 40 ℃, and the enzyme activity is over 90 percent; the enzyme has better NaCl stability, and the enzyme activity is kept above 100% under 0.5-2.0mol/L NaCl; the enzyme activity was maintained at 55% -99% even at 2.5-5.0mol/L NaCl.

In another aspect of the invention, the 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 gene size is 2484 bp.

In another aspect of the invention, a recombinant expression vector comprising the beta-galactosidase GalNC3-89 encoding gene is provided, and the recombinant expression vector is pEASY-E2/GalNC 3-89.

In another aspect of the present invention, there is provided a recombinant strain comprising the gene encoding beta-galactosidase GalNC3-89, said strain including but not limited to escherichia coli, yeast, bacillus or lactobacillus, preferably recombinant strain BL21(DE3)/GalNC 3-89.

The invention clones a beta-galactosidase GalNC3-89 coding gene by a PCR method, connects the coding gene 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 β -galactosidase GalNC3-89, 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 3-89.

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

In another aspect of the invention, the application of the beta-galactosidase GalNC3-89 in food processing, bioethanol production and industrial dairy product preparation is provided.

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 used as a reporter gene; in the chemical process, monosaccharide after lactose hydrolysis can be utilized to produce bioethanol; in industrial dairy products, the problem of environmental pollution caused by excessive whey generated in the production process is solved; in addition, in the dairy industry, it is used to produce low lactose milk.

The invention has the beneficial effects that:

the optimum action pH of the beta-galactosidase is 6.5, the beta-galactosidase is processed for 1h under the pH of 5.0 to 9.0, and the residual enzyme activity is more than 70 percent; the optimum action temperature is 40 ℃, the treatment is carried out for 1h at 37 ℃ and 40 ℃, and the enzyme activity is over 90 percent; the enzyme has better NaCl stability, and the enzyme activity is kept above 100% under 0.5-2.0mol/L NaCl; the enzyme activity was maintained at 55% -99% even at 2.5-5.0mol/L NaCl. Km and Vmax of the enzyme are respectively 1.935mmol/L and 0.8948 mmol/min; na (Na)⁺、Fe³⁺、Pb²⁺Tween80 and TritonX-100 have activating effect on GalNC3-89, and respectively improve the enzyme activity by 10%, 14%, 28%, 31% and 9%; the other metal ions and chemical agents all inhibit their activity to varying degrees. The properties show that the beta-galactosidase prepared by the invention has good application potential in food industry, synthesis of Galactooligosaccharides (GOS), report genes and the like. Secondly, the beta-galactosidase can effectively utilize monosaccharide obtained 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 low-lactose milk produced by the method has good application prospect in the dairy product industry.

Drawings

FIG. 1 is an SDS-PAGE analysis of recombinant β -galactosidase GalNC3-89 expressed in E.coli provided by an embodiment of the invention, wherein M: low molecular weight protein Marker; 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 the transglycosidic product GOS of GalNC 3-89;

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 3-89.

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 GalNC3-89

The DNA sequence was expressed as GalNC3-89 DNA F: 5'-taagaaggagatatacatatggaattggcccggacggaattcaaactg-3' and GalNC3-89 DNA R: 5'-gtggtggtggtggtgctcgagtttcacggaaatttccaac-3' is an upstream primer and a downstream primer, and PCR amplification is carried out by using the Western black-crown ape stool microorganism metagenome DNA 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 target gene GalNC3-89 was obtained as a result of PCR.

EXAMPLE 2 preparation of beta-galactosidase GalNC3-89

The beta-galactosidase gene GalNC3-89 prepared in example 1 was ligated with the plasmid pEASY-E2 to obtain a recombinant expression vector pEASY-E2/GalNC3-89, and E.coli BL21(DE3) was transformed to obtain a recombinant E.coli strain BL21(DE3)/GalNC 3-89. Escherichia coli strain BL21(DE3)/GalNC3-89 containing recombinant expression vector pEASY-E2/GalNC3-89 was inoculated in an amount of 0.1% V/V to LB (containing 100. mu.g/mL Amp) culture solution and cultured at 37 ℃ and 180rpm for 12-16 hours. Then, the activated bacterial suspension was inoculated into fresh LB (containing 100. mu.g/mL Amp) culture medium in an amount of 1% (V/V), and shake-cultured at 37 ℃ and 180rpm37 ℃ 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. Suspending the bacteria in a suitable amount of sterile water, and disrupting the cells under high pressure(35 KPSI). And (3) freezing and centrifuging the crushed cell sap at 4 ℃ and 12000r/min for 10min, taking the supernatant, and purifying the target protein by using Nickel-NTA Agarose to obtain the salt-tolerant beta-galactosidase GalNC 3-89.

The purified protein GalNC3-89 was analyzed by 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 the present example, 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 beta-galactosidase GalNC3-89

Enzyme activity assay methods refer to zhangwenhong (zhangchenghong, 2019): recombinant beta-galactosidase enzyme activity was determined as p-nitrophenyl-beta-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 the optimum pH and pH stability of the beta-galactosidase GalNC3-89

Determination of the optimum pH of the enzyme: the enzyme activity in a buffer of pH3.0 to 12.0(pH3.0 to 7.0: 0.1mol/L citric acid-disodium hydrogenphosphate buffer; pH8.0 to 12.0: 0.2mol/L glycine-sodium hydroxide buffer) was measured at 37 ℃ with the purified beta-galactosidase GalNC3-89 purified in example 2.

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%.

Results referring to fig. 2 and 3, fig. 2 is the optimum pH of the salt-tolerant β -galactosidase provided by the embodiment of the present invention, and fig. 3 is the pH stability of the β -galactosidase provided by the embodiment of the present invention. As can be seen from FIGS. 2 and 3, the optimum pH of beta-galactosidase provided by the present invention is 6.5; treating at pH 5.0 and pH 9.0 for 1 hr to obtain residual enzyme activity of above 70%.

2) Optimum temperature and temperature stability determination of 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 for 1 hour at 37 ℃ and 40 ℃ and the enzyme reaction was carried out at pH6.5 and 40 ℃ every 10 minutes, using untreated enzyme solution as a control.

The results are shown in FIGS. 4 and 5. FIG. 4 shows the optimum temperature of beta-galactosidase provided by the embodiments of the present invention, and FIG. 5 shows the temperature stability of beta-galactosidase provided by the embodiments of the present invention. The results show that: the optimum temperature of the salt-tolerant beta-galactosidase is 40 ℃, and the salt-tolerant beta-galactosidase keeps stable at the conditions of 37 ℃ and 40 ℃.

3) Effect of NaCl on beta-galactosidase and NaCl tolerance assay

NaCl effect assay of the enzyme: the enzymatic reaction is carried out at 40 ℃ 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 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 40 ℃ for 1h, and then measuring the residual enzyme activity under the conditions of 40 ℃ 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 effect of beta-galactosidase provided by the embodiments of the invention, and FIG. 7 is the NaCl tolerance of salt-tolerant beta-galactosidase provided by the embodiments of the invention. The results show that: when the reaction system contains 3.5mol/L NaCl, the enzyme activity reaches half-life. After the temperature is respectively kept for 1h at 40 ℃ under 0.5-5.0mol/L NaCl, the enzyme activity of 117 percent of the GalNC3-89 is kept under 0.5mol/L NaCl; the activity of more than 100 percent is kept under 1.0-2.0mol/L NaCl; the concentration is maintained between 55 and 99 percent under 2.5 to 5.0mol/L NaCl.

4) Determination of kinetic parameters of recombinant beta-galactosidase

Kinetic parameters were measured at pH6.5, temperature 40 ℃ and first order reaction time with pNPGal at different concentrations as substrate (0.1-0.9mmol/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⁺、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 results are shown in Table 1, with reference to the enzyme activity without addition of metal ions and chemicals.

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

As can be seen from Table 1, Na⁺、Fe³⁺、Pb²⁺Tween80 and Triton X-100 have activating effect on Gal NC3-89, and respectively improve the enzyme activity by 10%, 14%, 28%, 31% and 9%;the other metal ions and chemical agents all inhibit their activity to varying degrees.

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 deg.C and pH6.5, boiling for 5min immediately to terminate the reaction, centrifuging at 12000r/min for 10min, and collecting 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> beta-galactosidase GalNC3-89, and preparation method and application thereof

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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 (6)

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 beta-galactosidase GalNC3-89 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 method of producing the β -galactosidase GalNC3-89 of claim 1, comprising the steps of:

1) taking Western black-crown ape excrement microorganism metagenome DNA as a template, and designing primers F and R shown as SEQ ID NO. 3-4 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 3-89.

6. Use of the beta-galactosidase GalNC3-89 of claim 1 in food processing, production of bioethanol and preparation of industrial dairy products.

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