WO2021155674A1

WO2021155674A1 - Microbulbifer arenaceous β-galactosidase, encoding gene thereof and application thereof

Info

Publication number: WO2021155674A1
Application number: PCT/CN2020/122118
Authority: WO
Inventors: 江正强; 姚宇晨; 温永平; 杨绍青; 闫巧娟; 刘瑜
Original assignee: 中国农业大学
Priority date: 2020-02-07
Filing date: 2020-10-20
Publication date: 2021-08-12
Also published as: CN111154740B; CN111154740A

Abstract

Provided are a Microbulbifer arenaceous β-galactosidase, an encoding gene thereof and an application thereof. The Microbulbifer arenaceous β-galactosidase is a protein shown in sequence 2 in the sequence table. Said enzyme has excellent enzymatic properties, and the specific enzymatic activity thereof to the natural substrate lactose is 38.69 U/mg. At the same time, lactose can be hydrolyzed efficiently. At a refrigeration temperature of 4°C, 1 U/mL of enzyme is added; after reacting for 36 hours, more than 80% of the lactose in milk is hydrolyzed; after reacting for 48 hours, more than 90% of the lactose in the milk is hydrolyzed; and after reacting for 72 hours, the lactose in the milk is fully hydrolyzed. In addition, after reacting for 84 hours, more than 90% of the lactose in a 5% (w/v) whey solution is hydrolyzed. The described enzyme has important application value for the production of low-lactose or lactose-free dairy products in the food industry.

Description

A kind of sandy microcystis β-galactosidase and its coding gene and application

Technical field

The invention relates to a kind of microvesicular microvesicle beta-galactosidase and its coding gene and application.

Background technique

β-galactosidase (EC3.2.1.23, β-D-galactoside galactosyl hydrolase) belongs to the glycoside hydrolase family, which catalyzes the hydrolysis of the glycosidic bond between the non-reducing terminal β-D-galactoside And the function of transgalactoside. At present, it is mainly used in food, medicine, analysis and other fields.

Lactose is a disaccharide composed of a molecule of galactose and a molecule of glucose connected by β-1,4 glycosidic bonds. Lactose is mainly found in dairy products, and milk contains about 4.5% lactose. Adults cannot digest lactose due to lack of β-galactosidase activity in the intestine. Intestinal microorganisms can ferment lactose and cause a series of symptoms such as bloating, abdominal pain and diarrhea, which is called lactose intolerance. Using β-galactosidase to treat dairy products can hydrolyze lactose into galactose and glucose that the human body can absorb, thereby eliminating the symptoms of lactose intolerance.

β-galactosidase exists in animals, plants and microorganisms. Microbial β-galactosidase has the advantages of large enzyme production, diverse properties and low cost, and is widely used in dairy processing. Currently known β-galactosidase is mainly distributed in glycoside hydrolase GH1, GH2, GH35, GH42, GH59 and GH147 family based on amino acid sequence homology.

Milk in which 70%-80% of lactose is hydrolyzed can be called low-lactose milk, which can effectively solve the problem of lactose intolerance. Compared with medium-temperature and high-temperature β-galactosidase, low-temperature β-galactosidase can hydrolyze lactose at low temperatures, avoiding the deterioration of milk quality due to heating, and maximizing the preservation of milk flavor, heat-sensitive components, and reducing microorganisms Pollution risk, thereby saving resources and reducing production costs. At the same time, in the processing of dairy products, low-temperature hydrolysis of lactose can avoid crystallization during concentration, improve the quality of dairy products, increase the reaction rate and fermentation efficiency, and thus has certain advantages. At present, most commercial β-galactosidase enzymes used in dairy processing are medium temperature β-galactosidase, which has very low activity at refrigerated temperature. For example, DSM Neutral β-galactosidase (Maxilact LG2000) is the most suitable The temperature is 37°C, and the highest temperature of hydrolysis rate is 43°C, and the hydrolysis ability is poor below 37°C. Therefore, the discovery of a new type of low-temperature neutral β-galactosidase has very important practical significance.

Whey is a by-product of cheese production, which contains a lot of lactose (about 44-52g/L). After the lactose in whey is hydrolyzed into galactose and glucose by β-galactosidase, the sweetness is increased, and it can replace sucrose and corn syrup as a sweetener in the food and beverage industry. Therefore, the production of sweetener by hydrolyzing lactose in whey by β-galactosidase can realize high-value utilization of whey.

Microbulbiferarenaceous is a type of gram-negative bacteria, belonging to the γ-proteobacteria (γ-proteobacteria) Microbulbiferaceae family. There are no reports and patents of Microbulbifer β-galactosidase.

Invention Disclosure

The purpose of the present invention is to provide a kind of Microcystis serrata β-galactosidase and its coding gene and application.

In the first aspect, the present invention first provides proteins, which are the following A1) or A2) or A3) or A4) proteins:

A1) The amino acid sequence is the protein shown in sequence 2;

A2) The amino acid sequence is the protein shown in sequence 2 from positions 19 to 795 at the N-terminus;

A3) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of A1) or A2);

A4) A1) or A2) after substitution and/or deletion and/or addition of one or several amino acid residues to obtain a protein with the same function;

A5) A protein that has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the amino acid sequence defined by A1) or A2) and has the same function.

The protein is derived from Microbulbiferarenaceous.

The protein can be artificially synthesized, or its coding gene can be synthesized first, and then obtained by biological expression.

Among the proteins, a protein-tag refers to a polypeptide or protein expressed by fusion with the target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag can be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.

The fusion protein may specifically be a protein shown in sequence 4 of the sequence listing.

The present invention also protects the gene encoding the protein.

The gene is shown in any one of the following B1)-B5):

B1) The DNA molecule shown in sequence 1 in the sequence listing;

B2) The coding sequence is the DNA molecule shown in sequence 1 in the sequence listing;

B3) The DNA molecule shown in sequence 1 from position 55 to position 2388 at the 5'end in the sequence listing;

B4) The coding sequence is the DNA molecule shown in sequence 1 from position 55 to position 2388 at the 5'end in the sequence table;

B5) DNA molecules that hybridize with DNA molecules defined by B1) or B2) or B3) or B4) under stringent conditions and encode the same functional protein.

The stringent conditions are hybridization in a solution of 2×SSC, 0.1% SDS at 68°C and washing the membrane twice, 5 min each time, and hybridization in a solution of 0.5×SSC, 0.1% SDS at 68°C And wash the membrane 2 times, 15min each time.

The gene encoding the fusion protein shown in sequence 4 is shown in sequence 3 in the sequence listing.

The present invention also protects a gene construct, which includes the aforementioned gene and a heterologous regulatory element connected to the gene.

Further, the heterologous regulatory element may be a promoter and/or an enhancer or the like.

The present invention also protects recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the aforementioned genes or gene constructs.

The recombinant expression vector can be specifically obtained by using the sequence 1 of the sequence table to replace the fragment between the NheI and XhoI restriction sites of the pET-28a(+) vector from the DNA fragment shown at positions 55-2388 at the 5'end. Recombinant expression vector.

The recombinant bacteria may specifically be recombinant bacteria obtained by introducing the aforementioned recombinant expression vector into Escherichia coli Rosetta (DE3).

In the second aspect, the present invention also protects the application of the above-mentioned protein as β-galactosidase.

In the third aspect, the present invention also protects the application of the above-mentioned genes or gene constructs or recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria in the preparation of β-galactosidase.

In the fourth aspect, the present invention also protects the application of the above-mentioned genes or gene constructs or recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria, which are at least one of the following (C1)-(C6):

(C1) Hydrolyzed lactose;

(C2) Hydrolysis of lactose in milk;

(C3) Hydrolyze lactose in whey;

(C4) Preparation of low-lactose or lactose-free products;

(C5) Preparation of low-lactose or lactose-free milk;

(C6) Use whey to produce sweeteners.

In the fifth aspect, the present invention also protects the method for preparing β-galactosidase, which includes introducing the above-mentioned gene into a recipient microorganism to obtain a recombinant microorganism expressing β-galactosidase, culturing the recombinant microorganism, and expressing β-galactosidase. -Galactosidase.

In the above method, the recipient microorganism is a prokaryotic microorganism. Specifically, the prokaryotic microorganism is Escherichia coli. More specifically, the Escherichia coli is Escherichia coli Rosetta (DE3).

In the above method, the gene can be introduced into the recipient microorganism through a recombinant expression vector containing the gene. The recombinant expression vector can be specifically obtained by using the sequence 1 of the sequence table to replace the fragment between the NheI and XhoI restriction sites of the pET-28a(+) vector from the DNA fragment shown at positions 55-2388 at the 5'end. Recombinant expression vector.

The present invention also protects the β-galactosidase prepared by the above method.

The optimal pH of the β-galactosidase is 6.5. After 30 minutes of incubation at pH 5.5-7.5, the residual enzyme activity is above 80%; the optimal temperature is 30°C, which is stable below 30°C.

In the sixth aspect, the present invention also protects the method for hydrolyzing lactose, which includes the following steps: using the above-mentioned protein or the β-galactosidase prepared by the above-mentioned method to hydrolyze the lactose in the sample.

In the above method, the sample may specifically be milk or whey.

In the above method, the concentration of the protein or β-galactosidase in the hydrolysis system may be 1 U/mL-5 U/mL. Specifically, it can be 1U/mL, 2U/mL, 3U/mL, 4U/mL, or 5U/mL.

In the above method, the temperature of the hydrolysis reaction may be a refrigeration temperature (specifically 4°C) or room temperature (specifically 20-25°C).

In the above method, the hydrolysis time can specifically be 0-84h. More specifically, it can be 4h, 8h, 24h, 36h, 48h, 72h or 84h.

In the seventh aspect, the present invention also claims a method for preparing low-lactose or lactose-free products, which uses the aforementioned protein or the aforementioned gene or gene construct or the aforementioned recombinant expression vector, expression cassette, and transgene Low-lactose or lactose-free products are prepared by cell lines or recombinant bacteria or β-galactosidase prepared by the method described above.

In the above method, the product may specifically be milk.

In the eighth aspect, the present invention also claims a method for producing sweeteners using whey, which uses the aforementioned protein or the aforementioned gene or gene construct or the aforementioned recombinant expression vector, expression cassette, The transgenic cell line or recombinant bacteria or the β-galactosidase prepared by the method described above uses whey as a raw material to produce a sweetener.

Description of the drawings

Figure 1 is the electrophoresis diagram of the purification of the β-galactosidase from Microcystis serrata.

Figure 2 is a graph showing the optimal pH determination curve of Microcystis serrata β-galactosidase. Among them, citric acid-trisodium citrate pH 3.0-6.0(●), acetic acid-sodium acetate pH 3.6-5.6(■), MES pH 5.0-6.5(▼), MOPS pH 6.5-7.5(◆) disodium hydrogen phosphate- Sodium dihydrogen phosphate pH 6.0-8.0(▲), Tris-HCl pH 7.0-9.0

CAPS pH 10.0-11.0

Figure 3 is a graph showing the determination of pH stability of Microspora serrata β-galactosidase. Among them, citric acid-trisodium citrate pH 3.0-6.0(●), acetic acid-sodium acetate pH 3.6-5.6(■), MES pH 5.0-6.5(▼), MOPS pH 6.5-7.5(◆) disodium hydrogen phosphate- Sodium dihydrogen phosphate pH 6.0-8.0(▲), Tris-HCl pH 7.0-9.0

CAPS pH 10.0-11.0

Figure 4 is a graph showing the optimal temperature determination curve of Microcystis serrata β-galactosidase.

Fig. 5 is a graph showing the temperature stability determination curve of Microcystis serrata β-galactosidase.

Figure 6 is a graph showing the half-life determination curve of Microcystis serrata β-galactosidase. The treatment temperatures were 25°C (●), 30°C (■) and 35°C (▲).

Figure 7 is a diagram showing the hydrolysis of lactose by β-galactosidase from Microcystis serrata (A: TLC detection result; B: HPLC detection result). Fig. 7B shows the concentration of galactose (▲), glucose (●) and lactose (■).

Figure 8 The hydrolysis of lactose in milk by β-galactosidase plus enzyme (A: refrigerated temperature; B: room temperature; C: TLC test results; D: HPLC test results). Figure 8D shows the concentrations of galactose (▲), glucose (●) and lactose (■).

Figure 9 The hydrolysis of lactose in whey by β-galactosidase plus enzyme amount of Microvesicularis serrata (A: refrigerated temperature; B: room temperature; C: TLC test results; D: HPLC test results). Figure 9D shows the concentration of galactose (▲), glucose (●) and lactose (■).

The best way to implement the invention

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.

Sandy Microvesicle BH1: References: Junwen Ma, Qiaojuan Yan, Ping Yi, Shaoqing Yang, Haijie Liu, Zhengqiang Jiang. Biochemical characterization of a truncated β-agarase from Microbulbifer sp.suitable for efficient Bioproduction, tetra chemical production, tetra chemical ,87:119-127.https://doi.org/10.1016/j.procbio.2019.08.021.; the public can get it from China Agricultural University.

The method of determining the activity of β-galactosidase refers to the literature: Katrolia P, Yan Q, Jia H, et al. Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris[J].Journal Catalysis B Enzymatic, 2011,69(3-4):112-119. The specific measurement steps are as follows (the method is named as the general standard method):

(1) Add 75 μL of 15 mM o-nitro β-D galactopyranoside (oNPG) solution (solvent is water) to 150 μL phosphate buffer (pH 6.5, 50 mM), and preheat in a constant temperature water bath at 30°C for 3 minutes;

(2) Add 25μL of the sample solution to be tested (the enzyme solution to be tested or the diluted solution of the enzyme solution to be tested) into the above reaction system and place it in a constant temperature water bath at 30°C for 10 minutes;

(3) Add 750 μL of Na ₂ CO ₃ (2M) solution to the above reaction system to terminate the reaction, and use a spectrophotometer to measure the OD _410nm value (dilute the enzyme solution appropriately to _{control the OD 410nm} within the range of 0.200-0.800).

The definition of β-galactosidase enzyme activity unit (1U): Under the above experimental conditions, the amount of enzyme required to produce 1 μmol o-nitrophenol per minute. Enzyme activity calculation formula: H(U/mL)=Y×V ₁ ×n/(T×V ₂ ), where H represents enzyme activity (U/mL), Y represents the concentration of o-nitrophenol in the test solution (mmol/L), V ₁ represents the total volume of the reaction reagents (mL), n represents the dilution factor of the enzyme solution, T represents the reaction time (min), and V ₂ represents the volume of the diluted enzyme solution (mL).

Taking bovine serum albumin as the standard protein, using Lowry method (Lowry O H, Rosebrough N J, Farr A L, Randall R J, Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry,1951,193(1): 265 -275) Determine the protein content.

Specific enzyme activity is defined as the unit of enzyme activity per milligram of protein, expressed as U/mg.

The agarose Ni-IDA affinity column is a product from GE in the United States, and the product catalog number is 17-0575-01.

An aqueous solution containing 5% (mass percentage) of whey powder, wherein the whey powder is produced by Bio-Nutrition International, a product of the United States, produced by spray drying the by-product whey after cheese production, and the lactose content is ≥78%.

pET-28a(+) vector: Novagen, catalog number: 69864-3CN.

pET-40b(+) vector: Novagen, catalog number: 70091-3CN.

pET-41a(+) vector: Novagen, catalog number: 70556-3CN.

pET-44a(+) vector: Novagen, catalog number: 71122-3CN.

Escherichia coli Rosetta (DE3): Bomed Gene Technology Co., Ltd.

Example 1. Cloning of Microcystis serrata β-galactosidase and its coding gene

A large number of sequence analysis and functional verification of Microcystis serrata BH1 were performed, and a β-galactosidase encoding gene was cloned from it, with a full length of 2388 bp, as shown in sequence 1 in the sequence list. The DNA molecule shown in sequence 1 of the sequence listing encodes the β-galactosidase shown in sequence 2, and is named MaBgal2A. MaBgal2A consists of 795 amino acids, of which amino acids from the 1st to 18th positions of the N-terminal are predicted to be the signal peptide sequence.

Example 2. Construction of engineered bacteria expressing Microcystis serrata β-galactosidase

1. Construction of the recombinant expression vector of Microcystis serrata β-galactosidase

1. Use sequence 1 in the sequence table to replace the fragment between the NheI and XhoI restriction sites of the pET-28a(+) vector from the DNA fragment shown at positions 55-2388 at the 5'end to obtain the recombinant expression vector pET-28a (+)-MaBgal2A (verified by sequencing).

The inserted DNA molecule is fused with part of the nucleotides on the vector backbone to form the fusion gene shown in Sequence 3 of the Sequence Listing, which encodes the fusion protein shown in Sequence 4 of the Sequence Listing. In sequence 4 of the sequence listing, positions 5-10 are 6×His tags, and positions 24-800 are MaBgal2A without signal peptide.

2. Use sequence 1 in the sequence table to replace the fragment between the BamHI and XhoI restriction sites of the pET-40b(+) vector from the DNA fragment shown at positions 55-2388 at the 5'end to obtain the recombinant expression vector pET-40b (+)-MaBgal2A (verified by sequencing).

The inserted DNA molecule is fused with part of the nucleotides on the vector backbone to form a fusion gene, which encodes the fusion protein. The fusion protein includes DsbC protein and MaBgal2A without signal peptide. DsbC protein can promote the folding of proteins containing disulfide bonds.

3. The DNA fragment shown at positions 55-2388 from the 5'end of sequence 1 in the sequence list was used to replace the fragment between the BamHI and XhoI restriction sites of the pET-41a(+) vector to obtain the recombinant expression vector pET-41a (+)-MaBgal2A (verified by sequencing).

The inserted DNA molecule is fused with part of the nucleotides on the vector backbone to form a fusion gene, which encodes the fusion protein. The fusion protein includes GST protein and MaBgal2A without signal peptide. GST protein can promote the soluble expression of the target protein and avoid the formation of inclusion bodies.

4. The DNA fragment shown at positions 55-2388 from the 5'end of sequence 1 in the sequence list is used to replace the fragment between the BamHI and XhoI restriction sites of the pET-44a(+) vector to obtain the recombinant expression vector pET-44a (+)-MaBgal2A (verified by sequencing).

The inserted DNA molecule is fused with part of the nucleotides on the vector backbone to form a fusion gene, which encodes the fusion protein. The fusion protein includes NusA protein and MaBgal2A without signal peptide. NusA protein can promote the soluble expression of the target protein and avoid the formation of inclusion bodies.

2. Construction of engineered bacteria expressing Microvesicularis serrata β-galactosidase

The 4 kinds of recombinant expression vectors constructed in step 1 were introduced into Escherichia coli Rosetta (DE3) (Bomed Gene Technology Co., Ltd.) to obtain 4 kinds of recombinant bacteria.

Example 3. Preparation, purification and enzymatic properties of Microcystis serrata β-galactosidase

1. Preparation of recombinant protein MaBgal2A

1. The 4 recombinant bacteria prepared in Example 2 were respectively inoculated into LB liquid medium containing 50μg/mL kanamycin, cultured with shaking at 37°C and 200rpm until the OD _600nm of the bacterial solution reached between 0.6-0.8, and then cultivated Isopropyl-β-D-thiogalactoside (IPTG) was added to the system. The concentration of IPTG in the culture system was 1mmol/L. The culture system was induced overnight at 30℃ and 200rpm, and then the culture system was centrifuged at 11510g to collect the bacteria. Precipitate, resuspend in buffer A and sonicate it (250W, 20min), centrifuge at 11510g for 10min after sonication, and collect the supernatant to obtain the crude enzyme solution. After fragmentation, the target protein is soluble in E. coli containing various expression vectors. Therefore, the recombinant bacteria containing the recombinant plasmid pET-28a(+)-MaBgal2A is preferred for subsequent work.

Buffer A is Tris-HCl buffer (pH 8.0) containing NaCl (300mM) and imidazole (20mM).

2. Take the crude enzyme solution obtained in step 1 and use an agarose Ni-NTA affinity chromatography column (1×5cm) to purify the recombinant protein. The specific steps are as follows: (1) Equilibrate the chromatography with buffer A at a flow rate of 1 mL/min Column 5-10 column volumes; (2) Load the crude enzyme solution at a flow rate of 0.5 mL/min; (3) Use buffer A and buffer B to elute at a flow rate of 1 mL/min to OD _280nm <0.05; ( 4) Elute with buffer C and collect _{the post-column solution with OD 280nm} >0.1; (5) Dialysis and concentration to obtain a purified product (recombinant protein MaBgal2A).

Buffer B is Tris-HCl buffer (pH 8.0) containing NaCl (300mM) and imidazole (50mM);

Buffer C is a Tris-HCl buffer (pH 8.0) containing NaCl (300mM) and imidazole (100mM).

The products in the above steps were subjected to SDS-PAGE electrophoresis, and the results are shown in Figure 1. In Figure 1, lane M is the molecular weight standard, lane 1 is the recombinant bacteria crude enzyme solution, and lane 2 is the recombinant protein MaBgal2A. The results in Figure 1 show that the size of the recombinant protein MaBgal2A is 88.3kDa, which is consistent with the expected size.

2. Enzyme activity determination of recombinant protein MaBgal2A

The crude enzyme solution obtained in step 1 and the recombinant bacteria crude enzyme solution obtained in step 2 were purified by Ni-IDA affinity chromatography, and the recombinant protein MaBgal2A was used as the enzyme solution to be tested, and the inactivated recombinant protein MaBgal2A was heated in a boiling water bath for 10 minutes. As a control, the enzyme activity of β-galactosidase was tested according to the general standard method.

The results are shown in Table 1.

Table 1 Purification table of β-galactosidase

The purification multiple is the ratio of the specific enzyme activity of each purification step to the specific enzyme activity of the crude enzyme solution;

The recovery rate is the ratio of the total enzyme activity of each purification step to the total enzyme activity of the crude enzyme solution.

3. Determination of the optimum pH and pH stability of the recombinant protein MaBgal2A

1. Determination of the optimum pH: at 30°C, determine the enzyme activity of the recombinant protein MaBgal2A prepared in step one in different buffer systems.

Choose the pH range and system of the buffer as follows (50mM):

(1) Citric acid-trisodium citrate (pH: 3.0-6.0);

(2) Acetic acid-sodium acetate (pH: 3.6-5.6);

(3) MES (pH: 5.0-6.5);

(4) Disodium hydrogen phosphate-sodium dihydrogen phosphate (pH: 6.0-8.0);

(5) MOPS (pH: 6.5-7.5);

(6) Tris-HCl (pH: 7.0-9.0);

(7) CAPS (pH: 10.0-11.0).

The substrate oNPG (15mM) was prepared with the above buffer, and the activity was measured according to the standard enzyme activity method. The highest point of the enzyme activity was set as 100%, and the relative enzyme activity of MaBgal2A was calculated when reacting in different pH buffers.

2. Determination of pH stability: Dilute the recombinant protein MaBgal2A prepared in step 1 with the above-mentioned different buffers, and place it in a water bath at 25°C for 30 minutes, and then quickly place it in ice water to cool for 30 minutes to determine the residual enzyme activity. The enzyme activity of the untreated recombinant protein MaBgal2A was taken as 100%, and the relative enzyme activity of MaBgal2A after different pH treatments was calculated.

The results are shown in Figure 2 and Figure 3. The results show that the optimal reaction pH of MaBgal2A is 6.5 (disodium hydrogen phosphate-sodium dihydrogen phosphate) (as shown in Figure 2), which has good pH stability, and 80% remains after 30 minutes of incubation in the pH range of 5.5-7.5 The above enzyme activity (Figure 3).

4. Determination of the optimum temperature of recombinant protein MaBgal2A

Use the recombinant protein MaBgal2A prepared in step 1 as the enzyme solution to be tested in the optimal pH buffer (50mM sodium hydrogen phosphate-sodium hydrogen phosphate buffer, pH 6.5) for enzyme activity determination (replace the temperature with 0-80 ℃), the relative enzyme activity is calculated by taking the highest enzyme activity as 100%.

The result is shown in Figure 4. The results show that the optimum temperature of MaBgal2A is 30°C.

5. Determination of temperature stability of recombinant protein MaBgal2A

The recombinant protein MaBgal2A prepared in step 1 was pretreated and the enzyme activity was determined. The pretreatment method is as follows: the recombinant protein MaBgal2A is kept at different temperatures (0-80°C) for 30 minutes, and then quickly placed in ice water to cool for 30 minutes. Measure the residual enzyme activity at the optimum pH buffer (50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 6.5) and the optimum temperature (30°C), and take the enzyme activity of the untreated recombinant protein MaBgal2A as 100%, calculate the relative enzyme activity of MaBgal2A after different temperature treatments.

The result is shown in Figure 5. The results show that MaBgal2A has good stability below 30°C.

6. Determination of the half-life of recombinant protein MaBgal2A

Use phosphate buffer (50mM, pH 6.5) to properly dilute the recombinant protein MaBgal2A prepared in step 1 to an enzyme protein concentration of 1mg/mL. Incubate at 25°C, 30°C, and 35°C, and place samples on ice. Cool for 0.5h in a water bath and determine the residual enzyme activity. For 25℃, after incubation for 0min, 15min, 30min, 45min, 60min, 90min, 120min, 150min, 180min, and 240min, samples will be taken to determine the residual enzyme activity; for 30℃, insulation for 0min, 15min, 30min, 45min, 60min, 90min and 120min Afterwards, samples were taken to determine the residual enzyme activity; for 35°C, samples were taken to determine the residual enzyme activity after incubation for 0min, 15min and 30min. Using the untreated enzyme solution as a control, calculate the percentage of the residual enzyme activity after treatment at different temperatures to the enzyme activity of the blank control. The time required for the enzyme activity to decay to 50% at different temperatures is the half-life at that temperature. .

The result is shown in Figure 6. The results showed that the half-life of MaBgal2A at 25℃, 30℃ and 35℃ were 4032min, 252min and 62min, respectively.

7. Substrate specificity of recombinant protein MaBgal2A

Different nitrophenyl glycosides [o-nitrophenyl-β-D-galactopyranoside

(oNP-β-Galactopyranoside, CAS number: 369-07-3), p-nitrophenyl-β-D-galactopyranoside (pNP-β-Galactopyranoside, CAS number: 3150-24-1), p-nitrobenzene PNP-β-Glucopyranoside (pNP-β-Glucopyranoside, CAS number: 2492-87-7), pNP-β-D-mannopyranoside (pNP-β-D-mannopyranoside, CAS number: 252 -633-9),p-nitrophenyl-β-xylopyranoside (pNP-β-Xylopyranoside, CAS number: 2001-96-9),p-nitrophenyl-α-galactopyranoside (pNP-α -Galactopyranoside, CAS number: 7493-95-0), p-nitrophenyl-α-glucopyranoside (pNP-α-Glucopyranoside, CAS number: 3767-28-0)] and lactose as the substrate to determine the enzyme activity. The above-mentioned substrates were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; the product number of lactose was V900080.

The method for determining the enzyme activity of pNP-glycoside substrates was carried out in accordance with the above-mentioned general standard method. Using lactose as the substrate, the method for determining the enzyme activity refers to the GOP-POD method: mix a 10mg/mL lactose solution (prepared with 50mM phosphate buffer, pH 6.5) with an enzyme solution of an appropriate dilution, and incubate at 30°C for 10 minutes. The enzyme activity is finally determined by the concentration of glucose released in the solution, and the glucose concentration is determined with a glucose determination kit (glucose oxidase method, Shanghai Rongsheng Biopharmaceutical Co., Ltd., catalog number 361510).

When oNPG is used as the substrate, the β-galactosidase enzyme activity is 100%, and the specific and relative enzyme activities of β-galactosidase to different substrates are calculated respectively. Definition of enzyme activity: the amount of enzyme required to produce 1μmol of nitrobenzene (pNP or oNP) or glucose per minute according to the above measurement standards. Specific enzyme activity is defined as the unit of enzyme activity per milligram of protein, expressed as U/mg.

The results are shown in Table 2.

Table 2 The substrate specificity of MaBgal2A

The results showed that the enzyme hydrolyzed artificially synthesized substrates more strictly, and could only hydrolyze two artificial β-galactoside substrates. The relative enzymatic activity for pNPG was 60.4% of oNPG. At the same time, the enzyme has strong activity on the natural substrate lactose, and the relative enzyme activity is about four times that of oNPG as the substrate.

8. Hydrolysis characteristics of recombinant protein MaBgal2A

Prepare 5% (w/v) lactose solution in phosphate buffer (50mM, pH 6.5) as a substrate, add recombinant protein MaBgal2A5U/mL (enzyme activity measured by the general standard method) to the substrate, and mix well Incubate the reaction in a water bath at 30°C, and boil the sample for 5 minutes after sampling at regular intervals to inactivate the enzyme for testing. The hydrolyzed sample was spread twice on the TLC analysis plate, and then developed at 100°C after being completely soaked and blown dry with the color developing solution. The ratio of spreading agent is n-butanol:ethanol:water=5:3:2(v/v/v), the developer is 5%(v/v) sulfuric acid methanol solution, the standard control is glucose, galactose And lactose concentration of 1% (mass percentage, w/v) mixed solution.

HPLC detection conditions: chromatographic column is BP-800Pb++ (Benson Polymeric, Reno, NE, USA), injection volume is 10μL, column temperature is 80℃, flow rate is 0.6mL/min, mobile phase is ultrapure water; column B is Waters Xbridge Amide (250×4.6mm) amino column, the injection volume is 10μL, the column temperature is 45°C, the flow rate is 0.8mL/min, and the mobile phase is 75% acetonitrile in water. Calculated as follows:

Lactose hydrolysis rate (%, w/w) = [initial lactose content (%, w/v)—lactose content at the time of sampling (%, w/v)]/initial lactose content (%, w/v)

The result is shown in Figure 7. The results of TLC and HPLC showed that under the conditions of pH 6.5 and 30°C, MaBgal2A could completely hydrolyze lactose to produce glucose and galactose after 12 hours (Figure 7).

Example 4 Application of Microcystis serrata β-galactosidase in the hydrolysis of lactose in milk

1. Configure the following reaction system: add recombinant MaBgal2A protein (100U/mL, enzyme activity measured by a general standard method) to commercially available milk (purchased from Inner Mongolia Yili Industrial Group Co., Ltd., ultra-high temperature instantaneously sterilized pure milk, 100 U/mL) Libao packaging, each bag (200mL) hydrolyzes the lactose in it, and the total volume of the reaction system is 10mL. Set the final enzyme concentration in different reactants to 1U/mL, 2U/mL, 3U/mL, 4U/mL, and 5U/mL. The volume of commercially available milk in the corresponding hydrolysis system is 9.5mL, and the volume of enzyme protein addition is respectively It is 0.1mL, 0.2mL, 0.3mL, 0.4mL and 0.5mL. If the volume is less than 10mL after mixing, make up the volume with distilled water.

2. The reaction system configured in step 1 is reacted at refrigerated temperature (4°C) and different room temperature (variation range 20-25°C). After regular sampling, the sample is boiled for 5 minutes, and the enzyme is inactivated for testing. Taking the lactose concentration as an index, HPLC was used to quantitatively analyze the remaining lactose concentration in the hydrolysate. TLC, HPLC detection conditions: the same as in Example 3, step 8.

The effect of different β-galactosidase additions on the hydrolysis of lactose in commercially available milk is shown in Figure 8. HPLC results showed that the concentration of lactose in the commercially available milk used in the experiment was 4.5% (w/v), and as the amount of enzyme added increased, the time required to hydrolyze the same content of lactose gradually shortened. At room temperature, the lactose in milk can be hydrolyzed completely in 36 hours when the enzyme amount is 1U/mL. Under refrigeration temperature, the amount of enzyme added is 1U/mL. After 36 hours of reaction, more than 80% of lactose in milk is hydrolyzed. After 48 hours of reaction, more than 90% of lactose in milk is hydrolyzed. After 72 hours of reaction, all lactose in milk is hydrolyzed.

Example 5 Application of Microcystis serrata β-galactosidase in hydrolyzing lactose in whey

1. Configure the following reaction system: add recombinant MaBgal2A protein (100U/mL, enzyme activity measured by the general standard method) to an aqueous solution containing 5% (mass percentage) of whey powder to hydrolyze the lactose in it. The total reaction system is The volume is 10 mL. Set the final enzyme concentration in different reactants to 1U/mL, 2U/mL, 3U/mL, 4U/mL, and 5U/mL, the volume of the whey solution in the corresponding hydrolysis system is 9.5mL, and the volume of enzyme protein addition is respectively It is 0.1/mL, 0.2/mL, 0.3/mL, 0.4/mL and 0.5mL. If the volume is less than 10mL after mixing, make up the volume with distilled water.

The effect of different β-galactosidase additions on the lactose in the hydrolyzed whey solution is shown in Figure 9. The results showed that as the amount of enzyme added increased, the hydrolysis time gradually shortened. When the amount of enzyme added is 1U/mL, the lactose in the 5% (w/v) whey solution can be completely hydrolyzed at room temperature for 84 hours. At the refrigeration temperature, more than 90% of lactose in the 5% (w/v) whey solution was hydrolyzed after 84 hours of reaction.

The content not described in detail in this specification belongs to the prior art known to those skilled in the art.

Industrial application

The present invention uses genetic engineering technology to clone a GH2 family β-galactosidase gene from Microbulbiferarenaceous BH1, and express it heterologously in Escherichia coli. The microvesicle sandy bacteria β-galactosidase (MaBgal2A) provided by the present invention has excellent enzymatic properties, and its optimum pH is 6.5, and the residual enzyme activity is above 80% when it is incubated at pH 5.5-7.5 for 30 minutes; It is 30°C and remains stable below 30°C. The specific enzymatic activity of MaBgal2A to the natural substrate lactose is 38.69U/mg. MaBgal2A can efficiently hydrolyze lactose. Add enzyme at 1U/mL at a refrigeration temperature of 4℃. After 36h of reaction, more than 80% of lactose in milk is hydrolyzed. After 48h of reaction, more than 90% of lactose in milk is hydrolyzed. After 72h of reaction, Fully hydrolyze the lactose in milk. In addition, after 84 hours of reaction, more than 90% of lactose in the 5% (w/v) whey solution was hydrolyzed. The protein provided by the invention has important application value for the production of low-lactose or lactose-free dairy products in the food industry.

Claims

Protein is the protein of A1) or A2) or A3) or A4) as follows:

A1) The amino acid sequence is the protein shown in sequence 2;

A2) The amino acid sequence is the protein shown in sequence 2 from positions 19 to 795 at the N-terminus;

A3) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of A1) or A2);

A4) A1) or A2) after substitution and/or deletion and/or addition of one or several amino acid residues to obtain a protein with the same function;

A5) The amino acid sequence defined by A1) or A2) has more than 99%, 95%, 90%, 85% or 80% homology and is derived from Microbulbiferarenaceous A protein with the same function.
The protein according to claim 1, wherein the fusion protein is the protein shown in sequence 4 of the sequence listing.
A gene encoding the protein of claim 1 or 2.
The gene according to claim 3, wherein the gene is any one of the following B1)-B5):

B1) The DNA molecule shown in sequence 1 in the sequence listing;

B2) The coding sequence is the DNA molecule shown in sequence 1 in the sequence listing;

B3) The DNA molecule shown in sequence 1 from position 55 to position 2388 at the 5'end in the sequence listing;

B4) The coding sequence is the DNA molecule shown in sequence 1 from position 55 to position 2388 at the 5'end in the sequence table;

B5) DNA molecules that hybridize with DNA molecules defined by B1) or B2) or B3) or B4) under stringent conditions and encode the same functional protein.
The gene according to claim 3, wherein the gene encoding the fusion protein shown in sequence 4 is shown in sequence 3 in the sequence listing.
A gene construct comprising the gene described in any one of claims 3-5 and a heterologous regulatory element connected to the gene.
The gene construct according to claim 6, wherein the heterologous regulatory element is a promoter and/or an enhancer.
A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria containing the gene of any one of claims 3-5 or the gene construct of claim 6 or 7.
The recombinant expression vector according to claim 8, characterized in that: the recombinant expression vector is a DNA fragment shown in sequence 1 from the 5'end position 55-2388 in the sequence listing instead of pET-28a(+) vector Recombinant expression vector obtained from the fragment between NheI and XhoI restriction sites.
The recombinant bacterium according to claim 8, wherein the recombinant bacterium is a recombinant bacterium obtained by introducing the recombinant expression vector of claim 8 or 9 into Escherichia coli Rosetta (DE3).
Use of the protein of claim 1 or 2 as a β-galactosidase.
The gene of any one of claims 3-5 or the gene construct of claim 6 or 7 or the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria of any one of claims 8-10 is in preparation Application of β-galactosidase.
The protein of claim 1 or 2 or the gene of any of claims 3-5 or the gene construct of claim 6 or 7 or the recombinant expression vector of any of claims 8-10, expression The application of the cassette, transgenic cell line or recombinant bacteria is at least one of the following (C1)-(C6):

(C1) Hydrolyzed lactose;

(C2) Hydrolysis of lactose in milk;

(C3) Hydrolyze lactose in whey;

(C4) Preparation of low-lactose or lactose-free products;

(C5) Preparation of low-lactose or lactose-free milk;

(C6) Use whey to produce sweeteners.
The method for preparing β-galactosidase comprises introducing the gene of any one of claims 3-5 into a recipient microorganism to obtain a recombinant microorganism expressing β-galactosidase, culturing the recombinant microorganism, and expressing Obtain β-galactosidase.
The method according to claim 14, wherein the recipient microorganism is a prokaryotic microorganism.
The method according to claim 15, wherein the prokaryotic microorganism is Escherichia coli.
The method of claim 16, wherein the Escherichia coli is Escherichia coli Rosetta (DE3).
The method according to claim 14, wherein the gene is introduced into the recipient microorganism through a recombinant expression vector containing the gene.
The method according to claim 18, characterized in that: the recombinant expression vector is a DNA fragment shown from 5'th position 55-2388 of sequence 1 in the sequence listing instead of NheI and pET-28a(+) vector Recombinant expression vector obtained by cutting the fragments between XhoI restriction sites.
The β-galactosidase prepared by the method of any one of claims 14-19.
The method for hydrolyzing lactose comprises the following steps: using the protein of claim 1 or 2 or the β-galactosidase prepared by the method of any one of claims 14-19 to hydrolyze the lactose in the sample.
The method according to claim 21, wherein the sample is milk or whey.
The method according to claim 21 or 22, wherein the concentration of the protein or the β-galactosidase in the system for performing the hydrolysis is 1 U/mL-5 U/mL.
The method according to claim 23, wherein the concentration of the protein or the β-galactosidase in the system for performing the hydrolysis is 1 U/mL, 2 U/mL, 3 U/mL, 4 U/mL Or 5U/mL.
The method according to any one of claims 21-24, wherein the temperature for carrying out the hydrolysis reaction is 4°C or 20-25°C.
The method according to any one of claims 21-25, wherein the time for carrying out the hydrolysis is 0-84h.
The method according to claim 26, wherein the hydrolysis time is 4h, 8h, 24h, 36h, 48h, 72h or 84h.
The method for preparing a low-lactose or lactose-free product is to use the protein of claim 1 or 2 or the gene of any one of claims 3-5 or the gene construct of claim 6 or 7 or claim 8- The recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria described in any one of 10 or the β-galactosidase prepared by the method described in any one of claims 14-19 to prepare low-lactose or lactose-free products.
The method according to claim 28, wherein the product is milk.
The method of using whey to produce sweeteners is to use the protein of claim 1 or 2 or the gene of any one of claims 3-5 or the gene construct of claim 6 or 7 or claim 8- The recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria of any one of 10 or the β-galactosidase prepared by the method of any one of claims 14-19 uses whey as a raw material to produce a sweetener .