Lactase and its application in in-situ conversion of fresh milk lactose

Abstract

The present invention belongs to the field of enzyme engineering and food engineering technology, and specifically relates to the directed evolution and site-directed mutagenesis of a lactase enzyme molecule and its application in fresh milk. The amino acid sequence of the lactase mutant obtained by the present invention is shown in SEQ ID NO.4. Through the directed artificial evolution of the lactase molecule, a new lactase molecule is obtained that can efficiently convert lactose in fresh milk and synthesize oligosaccharides at a relatively low temperature, and is rapidly inactivated at a subsequent elevated temperature (such as pasteurization). This facilitates the highly nutritious in situ bioconversion of lactose in fresh milk into prebiotic oligosaccharides, further improving the nutritional value of fresh milk and expanding the fresh milk consumer group.

Description

Technical field:

[0001] The invention belongs to the technical fields of enzyme engineering and food engineering, and particularly relates to the directed evolution and site-directed mutagenesis of a lactase enzyme molecule and its application in fresh milk. Background technology:

[0002] Dairy products refer to a variety of liquid or solid foods made primarily from milk through processes such as heating, drying, freezing, or fermentation. Dairy products provide comprehensive and balanced nutrients for human health and growth. They not only provide a variety of carbohydrates, but also high-quality protein, calcium, and vitamins. Dairy products have become an essential food in human life, consumed daily by 6 billion people worldwide, accounting for approximately 10% of their daily energy intake.

[0003] Lactose in dairy products can cause abdominal pain, bloating, flatulence, and diarrhea. 70% of adults worldwide experience varying degrees of lactose intolerance, significantly limiting their intake and consumption of dairy products. Therefore, removing lactose from dairy products has become a pressing issue for the dairy industry. Low-lactose products must contain less than 2g / 100g of lactose, while lactose-free products must contain less than 0.5g / 100g. There are two common methods for removing lactose from dairy products. One is membrane filtration, which uses microfiltration and ultrafiltration membranes to remove lactose from dairy products, resulting in low-lactose or lactose-free dairy products. The other is enzymatic conversion, which utilizes the hydrolytic activity of lactase (a type of β-galactosidase, EC 3.2.1.23) to hydrolyze lactose into glucose and galactose. Lactase, also known as lactase, can catalyze the transaside conversion of lactose to form galactooligosaccharides (GOS), effectively converting lactose into GOS with higher functional value. Lactases used for lactose hydrolysis are primarily derived from Kluyveromyces fragilis, Kluyveromyces fragilis, Aspergillus niger, and Aspergillus oryzae. Among them, lactase from Kluyveromyces sp. is a neutral lactase, which is mainly used for the hydrolysis of lactose in liquid dairy products; lactase from Aspergillus sp. is mostly acidic lactase, which is mainly used as a nutritional supplement and cannot be directly applied to the direct enzymatic hydrolysis of lactose in fresh milk.

[0004] Galacto-oligosaccharides (GOS) are functional oligosaccharides naturally found in human, cow, and goat milk. They have a mild sweetness and organoleptic properties, and can be used in foods as a partial replacement for fat and sugar to improve food texture. However, they are not digested by human intestinal enzymes and reach the large intestine, where they act as growth factors to promote the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus, enhance mineral absorption, lower blood lipids, cholesterol, and glycemic index, improve blood pressure, and have a beneficial impact on human health. Currently, lactases used to produce GOS are primarily derived from Bacillus circulans, although enzyme preparations derived from Aspergillus oryzae are also used, but their transglycosidation efficiency is relatively low. Therefore, Bacillus circulans lactase is currently the best enzyme preparation for the efficient GOS enzymatic preparation. For example, previous studies have found that the lactase identified from Bacillus circulans B2301 has the ability to catalyze lactose to form 54.5% GOS under high temperature conditions above 60°C, making it the lactase with the best GOS synthesis performance among all reported lactases (Zhao Jihua et al., Food and Fermentation Industries, 2020); the expression level of lactase BcBG168-D prepared by fermentation of high-lactase-producing Bacillus licheniformis BCBTBc168D also has good application value (Chinese invention patent, ZL202011051056.1).

[0005] However, if Bacillus circulans lactase is directly applied to fresh milk, the lactose in the fresh milk is directly bioconverted, and the lactose therein is converted into GOS in situ, it is necessary to solve the problem of the efficiency of the transglycosides of the enzyme at a lower temperature and the convenience of inactivation of the enzyme after transglycosides. To this end, the present invention mutates the specific amino acid sites of Bacillus circulans B2301 lactase BcBG168-D (Wang Zhengxiang, Niu Dandan, Tian Kangming. Chinese invention patent, ZL202011051056.1) according to the fresh milk processing requirements and processing characteristics, in order to obtain a new enzyme molecule that can efficiently convert lactose into GOS at a lower temperature, and can simultaneously meet the requirements of being rapidly inactivated during subsequent heat treatment (such as pasteurization), so as to realize its application in low-lactose dairy products, which has important application significance for improving the nutritional value of fresh milk and improving the universality of consumers. Summary of the invention:

[0006] Through the directed artificial evolution of lactase molecules, a new lactase molecule was obtained that can efficiently convert lactose in fresh milk at a lower temperature and synthesize oligosaccharides at the same time, and is quickly inactivated at a subsequent elevated temperature (such as pasteurization). This is beneficial to the highly nutritious in situ bioconversion of lactose in fresh milk into prebiotic oligosaccharides, which can further enhance the nutritional value of fresh milk and expand the fresh milk consumer group.

[0007] To achieve the above objectives, the present invention is based on the lactase BcBG168-D encoding gene (nucleotide sequence shown in SEQ ID NO.1 in the sequence listing) or its amino acid sequence (shown in SEQ ID NO.2 in the sequence listing), and obtains a low-temperature high-activity mutant through site-directed mutagenesis and screening. Through the expression and preparation of the mutant, its enzymatic application performance is analyzed and evaluated, and its application value in fresh milk is evaluated.

[0008] One of the technical solutions provided by the present invention is a lactase mutant, which is obtained by subjecting the lactase shown in SEQ ID NO. 2 to double point mutations in amino acid residues T446 and R457 in the amino acid sequence;

[0009] Furthermore, the lactase mutant is obtained by subjecting the lactase shown in SEQ ID NO.2 to T446L and R457P mutations, and is named mutant T446L-R457P, and the amino acid sequence is shown in SEQ ID NO.4.

[0010] The second technical solution provided by the present invention is the gene encoding the above-mentioned lactase mutant T446L-R457P;

[0011] Furthermore, the nucleotide sequence of the gene encoding the mutant T446L-R457P is shown in SEQ ID NO.3.

[0012] The third technical solution provided by the present invention is a recombinant vector or recombinant strain comprising the gene encoding the lactase mutant T446L-R457P;

[0013] Furthermore, the expression vector used in the recombinant vector is pHY-WZX plasmid;

[0014] Furthermore, the host of the recombinant strain is Bacillus licheniformis BCBT0529 (published in Chinese invention patent ZL202011051056.1);

[0015] Furthermore, the recombinant strain is obtained by cloning the gene encoding the lactase mutant shown in SEQ ID NO. 3 into the expression vector pHY-WZX and performing recombinant expression in the Bacillus licheniformis BCBT0529 host cell.

[0016] The fourth technical solution provided by the present invention is the use of the above-mentioned recombinant vector or recombinant strain, especially the use in producing the lactase mutant T446L-R457P described in the first technical solution.

[0017] The fifth technical solution provided by the present invention is the use of the lactase mutant T446L-R457P described in the first technical solution, particularly in catalyzing lactose to produce galacto-oligosaccharides, and more particularly in producing low-lactose dairy products containing galacto-oligosaccharides;

[0018] Furthermore, the invention is used in the in-situ conversion of lactose in fresh milk into galacto-oligosaccharides.

[0019] Beneficial effects:

[0020] The lactase mutant T446L-R457P of the present invention has an optimal operating temperature of 55°C, 5°C lower than that of the original enzyme. When applied to fresh milk, it can significantly reduce the residual lactose to 17.84g / L, meeting the relevant regulations for low-lactose products (lactose less than 2g / 100g). The total lactose bioconversion rate is 82.16%, a 10.94% increase over the original enzyme's 74.06%). It also produces approximately 31.17g / L of galacto-oligosaccharides, effectively converting lactose into prebiotic galacto-oligosaccharides during fresh milk processing. Subsequent high-temperature treatment (such as pasteurization) can inactivate the enzyme. T446L-R457P completely loses its enzyme activity after incubation at 62.8°C for 10 minutes, while the original enzyme still retains approximately 50% of its activity after 30 minutes. Description of the drawings:

[0021] Figure 1 Comparison of the spatial structures of the original enzyme BcBG168-D and the proposed mutant T446L-R457P

[0022] Among them, Figure A shows the hydrogen bond formation between the T446 and R457 sites in the original enzyme and the adjacent amino acid residues; Figure B shows the hydrogen bond formation between the L446 and P457 sites in the proposed mutant T446L-R457P and the adjacent amino acid residues; the red stick structure represents the amino acid residue at site 446, and the green stick structure represents the amino acid residue that forms a hydrogen bond with it; the blue stick structure represents the amino acid residue at site 457, and the brown stick structure represents the amino acid residue that forms a hydrogen bond with it; the yellow dotted line represents the hydrogen bond.

[0023] Figure 2 The optimal temperature for lactase

[0024] Among them, ◆: mutant T446L-R457P; ○: original lactase.

[0025] Figure 3 Heat inactivation of enzymes under pasteurization conditions

[0026] Among them, ◆: mutant T446L-R457P; ○: original lactase.

[0027] Figure 4The lactase mutant catalyzes the formation of galacto-oligosaccharides from lactose in milk

[0028] Among them, Figure A is the HPLC sugar spectrum of products with different polymerization degrees, the blue line (lower side) is the sugar spectrum of the original enzyme (BcBG168-D), and the orange line (upper side) is the sugar spectrum of the mutant T446L-R457P, DP2: disaccharide, DP3: glucosylgalactobiose or galactotriose; DP4: glucosylgalactotriose or galactotetraose; DP5: glucosylgalactotetraose or galactopentaose; Figure B is the HPLC sugar spectrum of DP2 in Figure A, 1: galactobiose, 2: lactose, 3: allolactose. Specific implementation method:

[0029] In order to make the purpose, technical solutions and advantages of this patent more clear, the following is a further detailed description of this patent in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not intended to limit the present invention.

[0030] The starting (original) lactase molecule used in the present invention is lactase BcBG168-D, the nucleotide sequence of its encoding gene and the corresponding amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0031] The coding gene for the lactase mutant of the present invention is a nucleotide sequence with a specific codon change obtained by chemical synthesis.

[0032] The method for targeted screening of lactase mutants of the present invention comprises cloning the encoding gene of the lactase mutant into the expression vector pHY-WZX (Niu DD, Wang ZX. Development of a pair of bifunctional expression vectors for Escherichia coli and Bacillus licheniformis. J Ind Microbiol Biotechnol (2007) 34: 357-362. DOI 10.1007 / s10295-0204-x.), genetically transforming the mutant into the Bacillus licheniformis strain BCBT0529 (Chinese invention patent, ZL202011051056.1) to obtain a lactase-producing recombinant bacterium; preparing an enzyme solution by shake flask fermentation and purifying the enzyme solution, comparing and analyzing the optimum temperature, and comparing the enzyme solution with the original lactase (BcBG168-D), thereby targeted screening for mutants with a lower optimum temperature.

[0033] The following definitions are used in the present invention:

[0034] 1. Nomenclature of amino acid and DNA sequences

[0035] The generally accepted IUPAC nomenclature for amino acid residues is used, either in three-letter code or single-letter form. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0036] 2. Identification of Lactase Mutants

[0037] The mutated amino acid in the lactase mutant is represented by "original amino acid + position + replaced amino acid". For example, Thr446Leu (T446L) indicates that the amino acid at position 446 is replaced by Leu from the original lactase; for example, Arg457Pro (R457P) indicates that the amino acid at position 457 is replaced by Pro from the original lactase. The position numbering corresponds to the amino acid sequence numbering of the original lactase in SEQ ID NO. 2.

[0038] The original lactase (BcBG168-D) involved in the present invention has an amino acid sequence as shown in SEQ ID NO.2: MKSTTSAAGKSVSYNDGERRVNFENWRFQRETNGSIAGAQNPGFDDSSWRKLNLPHEWSIDLDFNKNSLATHEGGYLDGGIGWNRKTFTIPESMKGKRFSLDFDGVYMNSTTYLNGEVLGTYPFGYNAFSYDISDKLYKDGRANVLVVKVNTNQPSGRWYSGSSIYRNVYLTVTDPIHVARYGTFVTTPNLEKSIKEDRDAVNIKTKISNAEAKQVKIAIKSTIYDGAGNTVQTVETEEKTAAAGTVTPFEQNTVIKQPKLWSIDKPYRYNLVTEVIVGGQTVDTYETKFGVRYFKFDENEGYSLNGEFMKLHGVSMHHDLAGGAALTNARGVERQMQIMKDVGVNAIRVTHNPASPELGLEFAANKLLIIEEAFDSWAQSKKPYDYGRFFNAWAEHDIKEMVDRGKNEPAIIMWSIGNEIYDTTNAAGVETARNLVGWVKEIDTTPRATIGEDKTRGDNVTPINSYIKEIFNIVDVVGLNYSENNYDGYHKQNPSWKLYGSETSSATRSRGVYTHPYQYNHDTKYADLQQSSYDNDYVGWGRTAQDAWKYDRDLKHIAGQFIWTGFDYIGEPTPYYNSYPAKSSYFGAVDTAGFPKDIFYYYQSQWKKEPMVHLLPHWNWKEGEKVRVLAYTNASKVELVLNGESLGNEKYDNKQTSWGAPYKETKDGKTYLEWAVPFKPGKLEAVAKDENGKVIARDQVVTAGEPASVRLTADRKVVKADGTDLSFITADIVDSKGIVVPDADHLITFNVTGQGELAGVDNGNASSVERYKDNKRKAFSGKALAIVQSSKLSGKITVHASVAGLSSDSTSVFTVTPAD。

[0039] The lactase mutant T446V-R457P of the present invention has an amino acid sequence as shown in SEQ ID NO.4:

[0040] The main experimental methods used in the present invention are as follows:

[0041] 1. Gene cloning, molecular evolution and construction of expression plasmids

[0042] Conventional molecular cloning procedures were performed according to the literature (Sambrook et al. Molecular Cloning: A Laboratory Manual, 1989). The gene encoding lactase or its mutant was used as the target gene in the present invention; the expression vector pHY-WZX is prior art (Wang Zhengxiang, Niu Dandan. Chinese Invention Patent No. ZL200510051648; its construction method is disclosed in Niu DD, Wang ZX. Development of a pair of bifunctional expression vectors for Escherichia coli and Bacillus licheniformis. J Ind Microbiol Biotechnol (2007) 34:357-362. DOI 10.1007 / s10295-0204-x.). The gene encoding lactase or its mutant was amplified by PCR and cloned into the expression vector pHY-WZX to obtain a recombinant plasmid expressing lactase.

[0043] 2. Extraction of plasmid DNA

[0044] Plasmid DNA was extracted using a plasmid extraction kit from Sigma after cell wall lysis with a certain concentration of lysozyme.

[0045] 3. Gene amplification

[0046] DNA amplification was performed in 0.2 mL thin-walled PCR tubes. PCR amplification conditions were: 1× (95°C for 5 min); 30× (94°C for 10 s, 58°C for 30 s, 72°C for 30–300 s); and 1× (72°C for 10 min). PCR extension temperature and time varied depending on the amplicon length. Unless otherwise noted, all PCR reactions were performed using Pfu DNA polymerase.

[0047] 4. Overlap PCR

[0048] Refer to the literature (Krishnan, et al. Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones. Nucleic Acids Research, 1991, 22:6177-82). The general steps are as follows: PCR amplification using primers for fragments up and dn (P1+PA; PB+P2, with primers PA and PB being reverse complementary sequences) to obtain gene fragments; gel recovery and purification of the amplified fragments up and dn; dilution of the purified fragments up and dn by an appropriate multiple, mixing them at a 1:1 molar ratio as templates, and performing a new PCR reaction using primers P1 and P2 to obtain the full-length sequence.

[0049] 5. Genetic transformation of Bacillus licheniformis

[0050] The main steps are as follows: inoculate a fresh single colony into liquid LB medium, culture at 37°C 200r / min overnight, transfer 5% of the bacterial solution into new LB medium and continue to culture until OD 600 The pH value is 0.75-0.90. After ice bathing the cells for 10 minutes, centrifuge at 4°C and 6000 rpm for 10 minutes to collect the cells. Wash the cells four times with pre-cooled electroporation solution (0.5 mol / L sorbitol, 0.5 mol / L mannitol, and 10% glycerol). Resuspend the cell pellet in 1 mL of pre-cooled electroporation solution to complete the preparation of competent cells. Take 1 μL of plasmid DNA and approximately 100 μL of competent cells, mix them, and immediately electroporate (1800 v, 5 ms). Then add electroporation recovery solution (LB medium containing 0.65 mol / L sorbitol and 0.45 mol / L mannitol). After recovery at 37°C and 160 rpm, spread on the corresponding resistance LB plate and culture at 37°C until a single colony grows. Correct transformants are verified by colony PCR, plasmid extraction and enzyme digestion, and fermentation verification function.

[0051] 6. Preparation of lactase and its mutants

[0052] Lactase production by shake flask fermentation: 50 mL of fermentation medium (0.5-1.5% soybean meal, 0.5-1.2% cottonseed meal, 1-8% glucose, 0.1-0.5% ammonium sulfate; pH 7.0) is inoculated into a 250 mL Erlenmeyer flask, and the recombinant bacteria are incubated at 30-45°C and 120-270 rpm for 3-5 days. After fermentation, the supernatant is centrifuged at 6000 rpm for 10 minutes, and the supernatant is collected and precipitated with 50-60% saturated ammonium sulfate. The enzyme protein is then purified using a Sephadex G-200 column, and the active enzyme fraction is lyophilized for later use.

[0053] 7. Lactase activity assay

[0054] The enzymatic activity of lactase is defined as the amount of enzyme required to decompose lactose into 1 micromole of glucose per minute in 1 mL of liquid enzyme (or 1 g of solid enzyme powder) at a certain pH and temperature (pH 5.0 and temperature 40°C unless otherwise specified). This is defined as one enzyme activity unit (U) and is expressed in U / mL or U / g.

[0055] Lactase activity assay: Lactose is used as the substrate, and 1 mL of the reaction system is used for enzyme activity assay reactions at pH 5.0 and 40°C. 900 μL of 10% (w / v) lactose solution is placed in a centrifuge tube and preheated at 40°C for 5 minutes. 100 μL of the diluted enzyme solution to be tested is added. The tube is immediately timed, shaken, and reacted for 20 minutes. The reaction is terminated by boiling in water for 5 minutes, cooled on ice, and the glucose content is determined using a biosensor.

[0056] X=P*10*10*n / (180*t)

[0057] in:

[0058] X: enzyme activity of the original enzyme, U / mL;

[0059] P: biosensor display data, mg / 100mL;

[0060] 10: Coefficient for converting biosensor readings into μg / mL;

[0061] 10: The volume of the reaction system / 0.1 mL of absorbed enzyme sample, calculated as 1 mL;

[0062] n: enzyme sample dilution multiple;

[0063] 180: molecular weight of glucose;

[0064] t: reaction time, min.

[0065] 8. Conversion and product analysis of lactose in milk

[0066] To simulate the efficiency of lactase in converting lactose, 5U / g to 30U / g of lactase was added to fresh milk. The reaction was carried out at 30°C to 60°C, and samples were taken at regular intervals.

[0067] The reaction raw materials and the formation and content of oligogalactose were analyzed by HPLC. The characteristics and generation of the enzymatic products were analyzed by HPLC. The detection conditions of the double column method were as follows: the degree of polymerization of the product was analyzed, the mobile phase was high-purity water, the flow rate was 0.5 mL / min; Sugar-Pak TMⅠ Column (6.5×300 mm), column temperature 80°C, differential refractive index detector; for analysis of disaccharides, the mobile phase was 70% acetonitrile by volume, at a flow rate of 1.0 mL / min, a YMC-Pack NH2 (250×4.6 mm, 7 μm) column, column temperature 35°C, differential refractive index detector.

[0068] 9. Other analysis methods

[0069] Gene and amino acid sequence alignments were performed using DNAMAN software;

[0070] The enzyme protein content was determined according to the literature method (Bradford. Anal Chem, 1976);

[0071] Glucose content was determined by enzyme electrode method (SBA-90, Shandong);

[0072] The cell density was measured using a spectrophotometer (UV-2000, USA) at 600 nm;

[0073] The present invention will be further explained below through specific examples.

[0074] Example 1: Acquisition of new lactase enzyme molecules

[0075] The present invention is based on the structural characteristics of lactase BcBG168-D. The T446 and R457 sites are located in the loop region of lactase. The loop region is a random coiled form and usually has great flexibility. Mutations usually affect the steric stability of the enzyme, thereby affecting the heat resistance of lactase. Figure 1 Figure A shows that T446 in lactase BcBG168-D forms two hydrogen bonds with the spatially adjacent D444, and R457 forms three hydrogen bonds with the adjacent D454; Figure 1 Figure B shows that in the proposed mutant T446L-R457P, L446 forms one hydrogen bond with the adjacent D444, while P457 does not form any hydrogen bonds with any adjacent amino acid residues. Compared to BcBG168-D, the number of hydrogen bonds and amino acid residues formed at the L446 and P457 sites in the mutant T446L-R457P is reduced, which would reduce the stability of the local structure of lactase and alter the intermolecular forces. Based on this, the present invention combined T446L and R457P to obtain a new enzyme molecule, T446L-R457P, whose full-length amino acid sequence is shown in SEQ ID NO. 4 and the encoding gene is shown in SEQ ID NO. 3.

[0076] Example 2: Preparation of mutants

[0077] The full-length sequence of the gene encoding the mutant T446L-R457P proposed in Example 1 (SEQ ID NO. 3) was commissioned to Sangon Biotechnology Co., Ltd. for synthesis and cloned into the expression plasmid pHY-WZX to obtain the lactase expression plasmid pHY-T446L-R457P. According to the Bacillus licheniformis genetic transformation method described above, the recombinant plasmid pHY-T446L-R457P was transformed into Bacillus licheniformis BCBT0529 to obtain the corresponding recombinant bacterium BCB-T446L-R457P.

[0078] Above-mentioned recombinant bacteria is inoculated in the 250mL triangular flask that 50mL fermentation medium (soybean meal 1.5%, cottonseed meal 1.0%, glucose 8%, ammonium sulfate 0.3%, pH 7.0) is housed, inoculum size 10%, 37 ℃, 220r / min following shaking table culture 5 days.After fermentation finishes, 6000r / min centrifugal 10min gets supernatant and is crude enzyme liquid.The crude enzyme liquid that obtains carries out graded precipitation through 50%~60% saturated ammonium sulfate, enzyme protein further uses SephadexG-200 chromatographic column to purify by protein chromatography purification system AKTA system, and enzyme activity component is used after the sterilization of 0.22 micron sterile filter membrane.

[0079] At the same time, the original lactase BcBG168-D was prepared using the same method as above.

[0080] Example 3: Enzymatic properties of lactase mutants

[0081] (1) Optimum working temperature

[0082] The effect of temperature on enzyme activity and stability was determined using the lactase mutant T446L-R457P prepared and purified in Example 2 and the original lactase BcBG168-D. The enzyme solution was appropriately diluted and 10% lactose was used as the substrate. The enzyme activity was measured at 20-70°C at 5°C intervals according to the lactase activity assay method. The highest enzyme activity was taken as 100%, and the relative enzyme activity at other temperatures was calculated to determine the optimal operating temperature of the enzyme. The optimal operating temperature is Figure 2 As shown, the optimum temperature of T446L-R457P is 55℃, and it exhibits high enzyme activity between 40-60℃, and also has good enzyme activity at 20-37℃. The optimum temperature of original lactase is 60℃.

[0083] (2) Enzyme inactivation rate under pasteurization conditions

[0084] Enzyme activity inactivation characteristics under pasteurization conditions. After incubating the enzyme solution at 62.8°C for 10, 20, and 30 minutes, the enzyme activity was determined according to the lactase activity assay method. The relative activity of the enzyme was calculated with the activity of the enzyme without temperature treatment as 100%. The enzyme inactivation of the mutant T446L-R457P under pasteurization conditions was as follows: Figure 3 After incubation at 62.8°C for 10 min, T446L-R457P completely lost its enzyme activity, while the original enzyme still retained about 50% of its activity after 30 min.

[0085] Example 4: The mutant can catalyze the conversion of lactose in milk into galacto-oligosaccharides

[0086] The lactase mutant T446L-R457P prepared in the present invention and the original lactase BcBG168-D were added directly to cow's milk at a substrate concentration of 20 U / g (based on lactose dry matter). The reaction volume was approximately 50 mL. The reaction was carried out at 40°C for 18 hours, and samples were collected and analyzed by HPLC using a dual-column chromatography column.

[0087] Figure 4 Table 2 shows the sugar profile analysis of the low-lactose milk product produced using dual-column HPLC. Figure A shows the sugar profiles of products with different degrees of polymerization, and Figure B shows the sugar profile of the disaccharide (DP2). The sugar contents of the reaction products were calculated using the area normalization method. The results are shown in Table 2. After lactase T446L-R457P catalyzed the conversion of milk, the residual lactose content was 17.84 g / L (approximately 1.8 g / 100 g), and the amount of GOS produced was 31.17 g / L. The lactose conversion rate was 82.16%, a 10.9% increase over the original enzyme. The original enzyme had a residual lactose content of 25.94 g / L (approximately 2.6 g / 100 g). After T446L-R457P in situ enzyme treatment, the lactose content of the milk met the requirements for low-lactose dairy products (lactose < 2 g / 100 g). After the reaction, the samples treated with T446L-R457P were heat-treated by pasteurization (62.8°C, 10 min), and samples were taken to analyze the residual enzyme activity of lactase T446L-R457P. The results showed that no enzyme activity was detected.

[0088] Table 2 Content of sugar components in lactose converted from bovine milk by lactase mutants

[0089]

[0090] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that, without departing from the concept of this patent, a person skilled in the art would be able to make various variations, combinations, and improvements to the above-described embodiments, all of which fall within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be determined by the claims.

Claims

1. A lactase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.4 in the sequence listing.

2. A gene encoding the lactase mutant according to claim 1.

3. A recombinant vector or recombinant strain comprising the encoding gene according to claim 2.

4. The recombinant vector according to claim 3, wherein The expression vector used was pHY-WZX plasmid.

5. The recombinant strain according to claim 3, characterized in that The host used was Bacillus licheniformis BCBT0529.

6. Use of the recombinant vector or recombinant strain according to claim 3 in producing the lactase mutant according to claim 1.

7. The use of the lactase mutant according to claim 1, characterized in that: It is used in catalyzing lactose to produce galacto-oligosaccharides.

8. The use according to claim 7, characterized in that It is used in the production of low-lactose dairy products containing galacto-oligosaccharides.

9. The use according to claim 7, characterized in that The invention is used for converting lactose in fresh milk into galacto-oligosaccharide in situ.

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