A lactase mutant and its application

Abstract

The present invention belongs to the technical field of enzyme engineering, and particularly relates to a lactase mutant and its application. Based on lactase molecules from two sources (BglD305 derived from Bacillus circulans B2301 and BglD derived from Bacillus circulans ATCC 31382), molecular evolution is carried out to obtain a new lactase enzyme molecule with high efficiency in synthesizing galactooligosaccharides and good expression performance; and a high-yield strain of lactase is constructed, which can efficiently synthesize lactase during deep fermentation and secrete the enzyme protein molecule into the culture medium, and directly prepare a high-activity enzyme preparation from the fermentation supernatant. The expression level of lactase can reach 2208 U / mL, which helps to reduce the fermentation manufacturing cost of lactase, simplify the fermentation manufacturing process and improve the quality of lactase enzyme preparation.

Description

[0001] This application is a divisional application of the invention patent application No. 202011051056.1. The filing date of 202011051056.1 is September 29, 2020, the application number is 202011051056.1, and the invention title is: A special enzyme for producing galactooligosaccharides and its preparation and application. Technical Field:

[0002] The present invention belongs to the technical field of enzyme engineering, and specifically relates to lactase for generating galactooligosaccharides and its preparation and application. Background Art:

[0003] Oligosaccharides, also known as oligosaccharides, refer to straight-chain or branched carbohydrates with a degree of polymerization of 2-10 formed by glycosidic bonds between monosaccharide molecules, and can be simply divided into two categories: functional oligosaccharides and ordinary oligosaccharides. Among them, functional oligosaccharides are those formed by polymerizing 2-10 identical or different monosaccharides through glycosidic bonds; they have the common sweet taste and sensory characteristics of sugars, can directly replace sucrose as a sweet ingredient in desserts, but are not degraded by human gastric acid and gastric enzymes, are not absorbed in the small intestine, and can reach the large intestine; they are oligosaccharides with physiological characteristics such as promoting the proliferation of Bifidobacterium in the human body. Among functional oligosaccharides, due to the configuration of the anomeric carbon atom (C1 or C2) of the monosaccharide, their glycosidic bonds are not easily hydrolyzed and digested by the hydrolases in the human gastrointestinal tract, and are also called non-digestible sugars.

[0004] Naturally occurring functional oligosaccharides, such as galactooligosaccharides (GOS) present in human milk, cow milk, goat milk, etc., are an important prebiotic and play an important role in human health. In the 1950s, there were already reports on the research of industrial technologies for catalyzing lactose to produce galactooligosaccharides using β-galactosidase, and GOS products were successfully launched on the international market in 1988.

[0005] The production of galactooligosaccharides mainly uses lactose as the raw material. Through the transglycosylation of lactase, oligosaccharides with a low degree of polymerization connected by one glucose or all galactose molecules are synthesized during the hydrolysis of lactose, which can be expressed as Gal-(Gal)n-Glc / Gal (n is 1-4). Lactase (a kind of β-galactosidase, EC3.2.1.23) applied in the production of galactooligosaccharides can generate GOS through its transgalactosylation, and it is an enzyme with great commercial value in the dairy industry. Commercially, strains such as Aspergillus niger (A. niger), Aspergillus oryzae (A. oryzae), Kluyveromyces lactis, Kluyveromyces fragilis, Cryptococcus laurentii, and Bacillus circulans are generally selected to prepare lactase products through the deep fermentation method. Although there are certain differences in the enzyme activity composition and transglycosylation activity due to different sources of lactase and production processes of GOS, the galactooligosaccharides catalytically synthesized are mainly connected by β-1,3, β-1,4, and β-1,6 glycosidic bonds, with β-1,4 glycosidic bonds being the main ones. In addition, some new heat-resistant lactases have been developed for the high-temperature resistance of the enzyme. For example, lactases from microorganisms such as Sulfolobus solfataricus, Saccharopolyspora rectivirgula, Pyrococcus furiosus, and Thermotoga maritima can catalytically synthesize GOS at high temperatures of 70°C - 80°C. Commercially available lactases generally can form 5% - 50% galactooligosaccharides with lactose as the raw material. The lactase produced by Bacillus circulans ATCC 31382 (such as ) is the lactase with the strongest ability to synthesize GOS so far. This enzyme exists in four different forms in enzyme preparation products (Song J, Abe K, Imanaka H, Imamura K, Minoda M, Yamaguchi S, and Nakanishi K, Biosci. Biotechnol. Biochem., 2011; 75, 268 - 278). Among them, β-Gal-C and β-Gal-D are considered the most valuable for GOS production. In addition, the lactase identified from Bacillus circulans B2301 can catalyze lactose to form 54.5% GOS under high-temperature conditions above 60 °C, which is the lactase with the best GOS synthesis performance among all reported lactases so far (Zhao Jihua et al., Food and Fermentation Industries, 2020). The complete open reading frame of the lactase-encoding gene of Bacillus circulans B2301 is 5,133 bp in size, encodes 1,710 amino acid residues, does not contain a typical bacterial signal peptide sequence, and has a maximum identity of 93.6% with the reported β-galactosidases; this enzyme exhibits the highest catalytic activity at 60 °C and pH 6.0 - 6.5, and Zn 2+ 、Fe 2 + 、Cu 2+ 、EDTA and SDS show different degrees of inhibitory effects on the recombinant enzyme, and the V max for the catalytic synthesis of galactooligosaccharides is 2.47 g / (L·h), and the K m is 14.37 g / L (Tian Kangming et al., Food and Fermentation Industries, 2020).

[0006] However, at present, the fermentation production of lactase by Bacillus circulans is very uneconomical, requiring a long fermentation time, usually 96 h - 200 h; the enzyme production level is also relatively low, usually only able to produce 2 - 5 U / mL of lactase (Zhao Jihua et al., Food and Fermentation Industries, 2020); the enzyme preparation technology is complex, and most of the enzyme activity of lactase exists inside the cells and needs to be released by complex cell disruption methods, and the quality of the enzyme product is thus greatly affected.

[0007] The encoding genes of Bacillus circulans lactase or its mutants have been expressed in various host cells to understand the expression level of lactase. For example: when expressed in Escherichia coli, the expression level of lactase is 1 - 3 U / mL, and it is very difficult for this recombinant bacterium to secrete the synthesized lactase into the fermentation broth, increasing the difficulty of preparing lactase enzyme preparations. When expressed in Pichia pastoris GS115, the expression level in shake-flask fermentation can reach 70 U / mL, but it is also very difficult to achieve the secretory expression of lactase, and the separation and purification of the enzyme are extremely difficult. Summary of the Invention:

[0008] Based on obtaining excellent oligosaccharide-specific enzyme molecules, the object of the present invention is to obtain a recombinant bacterium with good large-scale fermentation production performance and ideal lactase synthesis and secretion ability, thereby significantly reducing the fermentation manufacturing cost of lactase, simplifying the fermentation manufacturing process of lactase, and significantly improving the quality of lactase.

[0009] To achieve the above object, one of the technical solutions of the present invention is to provide a variety of lactases, namely BglD305, BglD305-C, BglD305-D, BglD, BglD-C, BglD-D, BcBG168, BcBG168-C, BcBG168-D;

[0010] Among them, BglD305 is from Bacillus circulans B2301 (Zhao Jihua et al. Establishment and preliminary application of a rapid screening method for high transglycosylation activity lactase, Food and Fermentation Industries, 2020) screened and isolated by the inventors themselves, and the amino acid sequence is as shown in SEQ ID NO.2 in the sequence listing; BglD305-C and BglD305-D are truncated sequences of BglD305 respectively, and the amino acid sequences are as shown in SEQ ID NO.4 and SEQ ID NO.6 in the sequence listing;

[0011] Among them, BglD is from Bacillus circulans ATCC 31382, and the amino acid sequence is as shown in SEQ ID NO.8 in the sequence listing; BglD-C and BglD-D are truncated sequences of BglD respectively, and the amino acid sequences are as shown in SEQ ID NO.10 and SEQ ID NO.12 in the sequence listing;

[0012] Among them, BcBG168 is obtained by DNA shuffling and rearrangement of BglD305 and BglD, and BcBG168-C and BcBG168-D are obtained by further deleting part of the C-terminal amino acid sequence on the basis of BcBG168; the amino acid sequences of BcBG168, BcBG168-C and BcBG168-D are as shown in SEQ ID NO.14, 16 and 18 in the sequence listing.

[0013] The second technical solution provided by the present invention is a recombinant vector or recombinant strain containing the above lactase-encoding gene;

[0014] Preferably, the expression vectors used for the recombinant vectors include but are not limited to pHY-WZX, pBL-WZX, pHY300plk, pUB110, pE194, pHT1469 (MoBiTec), pWH1520 (Rygus and Hillen, 1991);

[0015] Preferably, the expression vectors used for the recombinant vectors include, but are not limited to, pHSE-001, pHSE-002, pHSE-003, pHSE-004, pHSE-005, pHSE-006, pHSE-007, pHSE-008, pHSE-009, pHSE-010, pHSE-011, pHSE-012, pHSE-013, pHSE-014, pHSE-015, pHSE-016, pHSE-017, pHSE-018;

[0016] More preferably, the expression vector used for the recombinant vector is the pHSE-008 plasmid, which is based on the backbone of the expression vector pHY-WZX and integrates the promoter P of the amylase from Bacillus licheniformis amyL (SEQ ID NO.20) and the signal peptide S of the alkaline protease aprE aprE (SEQ ID NO.23);

[0017] Preferably, the expression hosts used for the recombinant strains include, but are not limited to, Bacillus subtilis, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Bacillus licheniformis, etc.;

[0018] More preferably, the expression host used for the recombinant strain is the mutant strain BCBT0529, which is obtained by knocking out the aprE, vpr, wpr, lacR, lacA, lacA2, and yesZ genes on the genome of Bacillus licheniformis CBB3008 (accession number CCTCC NO.M208236). The GenBank accession numbers corresponding to the gene sequences are: MT885340, MT885341, MT885342, MT885336, MT885337, MT885338, MT885339;

[0019] Preferably, the present invention provides a recombinant strain - Bacillus licheniformis BCBTBc168D with high lactase productivity. The strain is obtained by integrating the BcBG168-D coding gene onto the pHSE-008 plasmid and expressing it in the host cell mutant strain BCBT0529. The expression level of lactase BcBG168-D prepared by fermenting the recombinant Bacillus licheniformis BCBTBc168D can reach 2208 U / mL.

[0020] The present invention also provides a method for fermenting and producing lactase using the above recombinant strain:

[0021] (1) Shake flask fermentation for lactase production: Inoculate the recombinant strain into a shake flask medium and culture it at 30 - 45 °C and 120 - 270 r / min for 2 - 3 days;

[0022] Shaking flask medium: yeast extract 0.5 - 1.5%, peptone 1.2 - 3.6%, glucose 8 - 20%; pH 7.0.

[0023] (2) Fermentation tank fermentation for producing lactase: Inoculate the strain into the fermentation tank medium at an inoculation amount of 5% - 10%; during the fermentation process, the fermentation temperature is 33 - 45°C, the dissolved oxygen is controlled at 0.1% - 20%, the pH is 6.0 - 7.8, and 30% - 60% (w / w) maltose syrup is added dropwise while maintaining the reducing sugar content at 0.1% - 5%; the fermentation lasts for 90 - 120 h, samples are taken regularly during the fermentation process for analysis, and the end point of fermentation is controlled such that the increase in fermentation enzyme activity is less than 5 - 20 U / (mL·h).

[0024] The composition of the fermentation tank medium is: maltose syrup 1% - 5%, cottonseed powder 0% - 5%, corn steep liquor 0% - 4%, soybean cake powder 0.5 - 5%, ammonium sulfate 0.1 - 5%, pH 6.0 - 8.0.

[0025] After fermentation, the enzyme activity of the fermentation broth of lactase by shaking flask fermentation can reach 25 - 54 U / mL; the enzyme activity of the fermentation broth of lactase by fermentation tank fermentation can reach 826 - 2208 U / mL.

[0026] Furthermore, after fermentation, the thallus is removed by plate - frame filtration, and then the enzyme solution is obtained after filtration by an ultrafiltration system.

[0027] The present invention also provides the application of the above - mentioned lactase in the production of galactooligosaccharides.

[0028] Beneficial effects:

[0029] The special enzyme preparation for producing galactooligosaccharides in the present invention is a lactase obtained through gene cloning and artificial evolution, which has extremely high activity in catalyzing lactose to produce galactooligosaccharides; the high - yield lactase strain of the present invention is a recombinant bacterium obtained through microbial strain breeding, which can efficiently synthesize lactase during deep - layer fermentation and secrete the enzyme protein molecule into the culture medium, and directly prepare a high - activity enzyme preparation from the fermentation supernatant for the efficient production of galactooligosaccharides.

[0030] The high - efficiency preparation method of lactase of the present invention can reach an expression level of 2208 U / mL under the high - yield lactase strain and fermentation process provided by the present invention. The present invention helps to reduce the fermentation manufacturing cost of lactase, simplify the fermentation manufacturing process and improve the quality of lactase enzyme preparation. Description of the drawings:

[0031] Figure 1 . Verification map of protease - encoding gene knockout

[0032] Lane M is the 1 kb molecular weight standard; Lane 1 is the mutant strain with correct genomic verification after deletion of the aprE gene, and the PCR amplification size is 1.5 kb; Lane 2 is the mutant strain with correct genomic verification after deletion of the protease wpr gene, and the PCR amplification size is 1.3 kb; Lane 3 is the mutant strain with correct genomic verification after deletion of the protease vpr gene, and the PCR amplification size is 1.3 kb;

[0033] Figure 2 . Verification map of knockout of coding genes such as endogenous lactase

[0034] Lane M is the 1 kb molecular weight standard; Lane 1 is the electrophoresis map confirmed by PCR after successful knockout of the lacR gene, with a size of 1.3 kb; Lane 2 is the electrophoresis map confirmed by PCR after successful knockout of the lacA gene, with a size of 1.2 kb; Lane 3 is the electrophoresis map confirmed by PCR after successful knockout of the lacA2 gene, with a size of 1.2 kb; Lane 4 is the electrophoresis map confirmed by PCR after successful knockout of the yesZ gene, with a size of 1.6 kb;

[0035] Figure 3 . Physical map of the expression vector

[0036] a: Optimized expression vector pHES-008; b: Lactase expression plasmid pLEBG168;

[0037] Figure 4 . Curve graph of lactase production by fermentation

[0038] Figure 5 . Protein electrophoresis map of the fermentation broth

[0039] Lane M is the protein molecular weight standard; Lane 1 is the result map of direct electrophoresis of the fermentation broth, and the arrow-marked part is lactase in the fermentation broth;

[0040] Figure 6 . HPLC sugar spectrum of galactooligosaccharides produced after lactase acts on lactose

[0041] DP2: Galactobiose or lactose, DP3: Glucosylgalactobiose or galactotriose; DP4: Glucosylgalactotriose or galactotetraose; DP5: Glucosylgalactotetraose or galactopentaose. Specific implementation mode:

[0042] In order to make the purpose, technical solution and advantages of this patent clearer, the following further details this patent in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0043] The plasmid pHY-WZX used in the present invention is a prior art, and its construction method has been published 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 public can also obtain it from the Laboratory of Biocatalysis and Biotransformation, College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology.

[0044] Bacillus licheniformis CBB3008 used in the present invention has been deposited in the China Center for Type Culture Collection (abbreviated as CCTCC) on November 25, 2008, with the deposit number CCTCC NO: M208236.

[0045] Based on lactase molecules from two sources (BglD305 from Bacillus circulans B2301 and BglD from Bacillus circulans ATCC 31382), molecular evolution was carried out in the present invention to obtain new lactase enzyme molecules (BglD305-C, BglD305-D, BglD-C, BglD-D, BcBG168, BcBG168-C, BcBG168-D) with high efficiency in synthesizing galactooligosaccharides and good expression performance;

[0046] In the present invention, a host cell, classified and named Bacillus licheniformis CBB3008, which has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M208236, was further genetically modified by knocking out multiple genes affecting lactase expression (alkaline protease encoding gene aprE, minor serine protease encoding gene vpr, cell wall protease encoding gene wpr, regulatory protein encoding gene lacR, β-galactosidase encoding gene lacA, β-galactosidase encoding gene lacA2, β-galactosidase encoding gene yesZ) to obtain a new host strain suitable for high-efficiency expression of lactase;

[0047] In the present invention, an expression vector suitable for secretory expression of lactase was constructed and optimized. It was optimized and constructed based on pHY-WZX. The expression vector has a preferred promoter for guiding high expression of lactase and a signal peptide for efficiently mediating secretory expression of lactase;

[0048] The encoding genes (corresponding nucleotide sequences 1, 3, 5, 7, 9, 11, 13, 15, 17) of Bacillus circulans lactase or its mutants (amino acid sequences 2, 4, 6, 8, 10, 12, 14, 16, 18) of the present invention are cloned into the expression vector constructed by the present invention, and genetically transformed into the host strain of Bacillus licheniformis to obtain the recombinant bacteria producing lactase; the fermentation conditions and processes are established and optimized, and a new method for enzyme separation, purification and refinement is established to produce lactase products for the industrial manufacture of GOS.

[0049] The method for constructing a new strain with high lactase production of the present invention is to clone and obtain the expression plasmid of lactase in the expression vector through gene cloning technology, and transform it into the new host strain of Bacillus licheniformis to obtain recombinant bacteria, realizing the efficient secretion and expression of lactase; the lactase is efficiently synthesized through the optimized protein secretion system of the recombinant bacteria, and the synthesized lactase enzyme protein is efficiently secreted into the culture medium; through the fermentation production process, lactase is recovered and refined from the fermentation broth to obtain lactase products.

[0050] The main experimental methods adopted in the present invention are as follows:

[0051] 1. Gene cloning, molecular evolution and construction of expression plasmid

[0052] Conventional molecular cloning operations are carried out according to the reference methods (Sambrook et al. Molecular Cloning: A Laboratory Manual, 1989). The encoding genes of Bacillus circulans lactase or its mutants are used as the target genes in the present invention; the basic expression vector is pHY-WZX (Wang Zhengxiang, Niu Dandan. Chinese Invention Patent, ZL200510051648; Niu & Wang. J Ind Microbiol Biotechnol, 2007). After the encoding genes of lactase or its mutants are amplified by PCR, they are cloned into the expression vector to obtain a series of recombinant plasmids expressing lactase.

[0053] 2. Extraction of chromosomal DNA

[0054] The method for extracting chromosomal DNA is carried out according to the literature (Zhuge Jian, Wang Zhengxiang. Industrial Microbiology Experiment Technology Manual, China Light Industry Press, 1994).

[0055] 3. Extraction of plasmid DNA

[0056] The extraction of plasmid DNA is carried out using the plasmid mini kit of Sigma Company after lysing the cell wall with a certain concentration of lysozyme.

[0057] 4. Gene amplification

[0058] DNA amplification was carried out in 0.2 mL PCR thin-walled tubes. The PCR amplification conditions were as follows: 1×(95°C for 5 min); 30×(94°C for 10 s, 58°C for 30 s, 72°C for 30 - 300 s); 1×(72°C for 10 min). Depending on different amplification lengths, the extension temperature and time of the PCR reaction were different. Unless otherwise specified, all PCR reactions were carried out using Pfu DNA polymerase.

[0059] 5. Molecular evolution of enzyme molecules

[0060] The molecular evolution of lactase was carried out by DNA shuffling according to the literature method (Stemmer W P C. Rapid evolution of a protein in vitro by DNA shuffling[J]. Nature, 1994, 370(6488): 389 - 391.). First, the lactase gene was partially digested with DNase, and fragments of 100 - 200 bp were recovered by density gradient method. After mixing, gene amplification was carried out for 15 - 25 cycles without adding primers, and then specific primers at both ends were added to amplify the full-length gene; the PCR products were purified using a PCR product purification kit (Sigma), and the purified DNA was cloned into the expression vector pHY-WZX and transformed into Escherichia coli JM109 by the CaCl2 method (Zhuge Jian, Wang Zhengxiang. Handbook of Industrial Microbiology Experimental Techniques, China Light Industry Press, 1994); the lactase activity was measured and compared.

[0061] 6. Overlap PCR

[0062] It was carried out with reference to the literature (Krishnan B R, 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 procedure was as follows: The amplification of PCR was mediated by the primers of fragment F1 and fragment F2 (P1 + P2; P3 + P4, and primers P2 and P3 were reverse complementary sequences) to obtain gene fragments; the amplified fragments F1 and F2 were recovered and purified by gel; the two purified fragments F1 and F2 were diluted by an appropriate multiple and mixed at a 1:1 molar ratio as the template, and a new PCR reaction was mediated by primers P1 + P4 to obtain the full-length sequence.

[0063] 7. Genetic transformation of Bacillus licheniformis

[0064] The method described in the reference (Xu Min, Ma Junshuang, Wang Zhengxiang. Effect of high osmotic pressure on the electrotransformation efficiency of bacteria. Journal of Wuxi University of Light Industry, 2004(04):98-100) was used. The main steps are as follows: Inoculate fresh single colonies into liquid LB medium and culture overnight at 37°C with 200 r / min. Transfer 5% of the bacterial solution to a new LB medium and continue to culture until the OD600 reaches 0.75 - 0.90. After the bacteria are ice-bathed for 10 min, centrifuge at 4°C and 6000 r / min for 10 min to collect the bacteria. Wash the cells 4 times repeatedly with pre-cooled electrotransformation washing solution (0.5 mol / L sorbitol, 0.5 mol / L mannitol and 10% glycerol). Suspend the cell pellet in 1 mL of pre-cooled electrotransformation washing solution to complete the preparation of competent cells. Mix 1 μL of plasmid DNA with about 100 μL of competent cells, immediately perform electroporation (1800 v, 5 ms), then add electrotransformation recovery solution (LB medium containing 0.65 mol / L sorbitol and 0.45 mol / L mannitol), resuscitate at 37°C with 160 r / min, and then spread on an LB plate with corresponding resistance and culture at an appropriate temperature until single colonies grow. The correct transformants were verified by methods such as colony PCR verification, plasmid extraction and enzyme digestion, and fermentation verification of function.

[0065] 8. Deletion of specific genes in Bacillus licheniformis

[0066] The method in the reference (Cai D, et al High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase. J Appl Microbiol. 2016, 121:704-712) was used. Taking the deletion of the aprE gene in Bacillus licheniformis CCTCC NO: M208236 as an example, the general steps are as follows: Using the genomic DNA of Bacillus licheniformis as a template, and using apr-up1 (sequence 30), apr-up2 (sequence 31), primer apr-dn1 (sequence 32) and apr-dn2 (sequence 33) as primers, amplify the upper and lower homologous arm fragments respectively. After obtaining the PCR products of the correct size, purify them by gel extraction respectively. Using the DNA of the gel extraction product as a template, perform overlap PCR to obtain the deletion mutant cassette, △aprE. After purifying this mutant cassette and digesting it with Xba I, clone it into plasmid pT2 ts (pT2 ts is based on T2(2)-ori (Chen Shouwen et al., Chinese invention patent, ZL201310562150.7) as the starting plasmid. After reverse amplification using primers T2-1 (sequence 28) and T2-2 (sequence 29), the PCR product is circularized and ligated by itself to obtain the new plasmid pT2ts ) The SmaI and XbaI sites were used to transform competent Escherichia coli JM109 cells, which were cultured on an LB plate containing 20 μg / mL kanamycin to obtain the correct deletion plasmid pT2-ΔaprE. According to the steps described in the genetic transformation method of Bacillus licheniformis, the deletion plasmid pT2-ΔaprE was transformed into the Bacillus licheniformis host cell. After two rounds of homologous recombination, primers apr-F: (Sequence 34) and apr-R (Sequence 35) were designed on both sides of the homologous arms, and colony PCR was performed with this pair of primers to verify the transformants (for the deletion of other genes, the above method can be referred to, and the primers can be designed and replaced according to the deleted gene sequence).

[0067] 9. Fermentation test

[0068] Flask fermentation for lactase production: In a 250 mL Erlenmeyer flask, 30 mL of fermentation medium (yeast extract 0.5 - 1.5%, peptone 1.2 - 3.6%, glucose 8 - 20%; pH 7.0) was filled, and the recombinant strain was inoculated and cultured at 30 - 45 °C and 120 - 270 r / min for 2 - 3 days.

[0069] Fermenter fermentation for lactase production: The composition of the fermentation medium was: malt syrup 1% - 5%, cottonseed meal 0% - 5%, corn steep liquor 0% - 4%, soybean cake powder 0.5 - 5%, ammonium sulfate 0.1 - 5%, pH 6.0 - 8.0; the fermentation was carried out in a 50 L - 10 t fermenter, and the inoculation amount was 5% - 10%; during the fermentation process, the fermentation temperature was 33 - 45 °C, the dissolved oxygen was controlled at 0.1% - 20%, the pH was 6.0 - 7.8, 30% - 60% (w / w) malt syrup was fed and the reducing sugar content was maintained at 0.1% - 5%; the fermentation continued until 90 - 120 h, samples were taken regularly during the fermentation process for analysis, and the end point of fermentation was controlled so that the increase in fermentation enzyme activity was less than 5 - 20 U / (mL·h).

[0070] 10. Preparation method of lactase enzyme preparation

[0071] After the fermentation was completed, the cells were removed by plate and frame filtration, and then the enzyme solution was obtained after filtration through an ultrafiltration system.

[0072] 11. Determination of lactase enzyme activity

[0073] The determination of lactase enzyme activity was improved according to GB / T 33409-2016. Generally, the reaction was carried out at pH 5.0 and 40 °C with lactose as the substrate. A biosensor was used to measure the release amount of glucose.

[0074] The enzyme activity of lactase was defined as: the amount of enzyme required to decompose lactose to produce 1 micromole of glucose per minute at pH 5.0 and 40 °C, which was defined as one enzyme activity unit (U), expressed as U / mL or U / g.

[0075] 12. Synthesis and Product Analysis of Galactooligosaccharides

[0076] Using lactose at 300 g / L - 800 g / L as the substrate, adding lactase at 5 U / g - 20 U / g, the reaction is carried out at 50°C - 70°C, and samples are taken at regular intervals. For the analysis of the reaction raw materials and the formation and content of galactooligosaccharides, HPLC is used to analyze the characteristics and generation of the enzymatic products. The chromatographic conditions are as follows: the mobile phase is 65% acetonitrile, the flow rate is 1.0 mL / min; a TSK-GEL G3000PWXL-CP (7.8 mm × 300 mm, 7 μm) chromatographic column, the column temperature is 25°C; an evaporative light scattering detector, the drift tube temperature is 90°C, and the carrier gas flow rate is 2.2 mL / min.

[0077] 13. Other Analytical Methods

[0078] The determination of protease activity is carried out according to the national standard method (GB / T 23527 - 2009);

[0079] The alignment of gene and amino acid sequences is carried out using DNAMAN software;

[0080] The determination of nucleotide sequences is carried out using the Sanger method;

[0081] The determination of the content of sequence proteins is carried out according to the literature method (Bradford. Anal Chem, 1976);

[0082] The glucose content is determined by the enzyme electrode method (SBA-90, Shandong);

[0083] The cell density is determined using a spectrophotometer (UV-2000, USA) at 600 nm;

[0084] Protein electrophoresis is carried out according to the literature method (Zhuge Jian, Wang Zhengxiang. Industrial Microbiology Experimental Technology Handbook, China Light Industry Press, 1994).

[0085] The present invention will be further explained and illustrated through specific examples below.

[0086] Example 1: Molecular Evolution of Lactase

[0087] Using the BglD305 and BglD coding genes shown in Sequence Listing SEQ ID NO.1 and SEQ ID NO.7 as templates, DNA shuffling method is used to carry out their molecular evolution. After screening for enzyme activity, a lactase enzyme molecule BcBG168 (nucleotide sequence SEQ ID NO.13) with a significantly improved enzyme activity level is obtained, and its amino acid sequence (amino acid sequence SEQ ID NO.14).

[0088] By truncating the coding genes of BglD305, BglD, and BcBG168 to different extents, highly expressing the modified sequences and the original sequences, amplifying the corresponding gene sequences by PCR amplification technology, and cloning them into the expression vector pHY-WZX, lactase expression plasmids pHY-Bgl-1, pHY-Bgl-2, pHY-Bgl-3, pHY-Bgl-4, pHY-Bgl-5, pHY-Bgl-6, pHY-Bgl-7, pHY-Bgl-8, pHY-Bgl-9, pHY-Bgl-10, pHY-Bgl-11, and pHY-Bgl-12 were obtained. The above recombinant plasmids were respectively transformed into Bacillus licheniformis CCTCC NO: M208236 by the above-mentioned genetic transformation method of Bacillus licheniformis to obtain the corresponding transformants CBB-Bgl-1, CBB-Bgl-2, CBB-Bgl-3, CBB-Bgl-4, CBB-Bgl-5, CBB-Bgl-6, CBB-Bgl-7, CBB-Bgl-8, CBB-Bgl-9, CBB-Bgl-10, CBB-Bgl-11, and CBB-Bgl-12. Further, flask fermentation was carried out and the enzyme production (the enzyme activity of the fermentation broth supernatant was measured) was analyzed. The main content and the enzyme production results are shown in Table 1.

[0089] Under the same conditions, the expressed enzyme activities of BcBG168 were 103.2% of BglD305 and 109.8% of BglD, respectively.

[0090] The lactase with the C-terminal truncated lactase showed an upward trend under the same expression conditions, and the enzyme activities were increased by 40%, 78%; 35%, 69% and 31%, 70% compared with the original gene sequence.

[0091] Table 1 Expression efficiency of lactases from different sources and with C-terminal truncation

[0092]

[0093] Example 2: Genetic modification of the expression host cell

[0094] Deletion of the aprE gene in Bacillus licheniformis CCTCC NO: M208236. Using the genomic DNA of Bacillus licheniformis CCTCC NO: M208236 as a template, and using apr-up1 (sequence 30) and apr-up2 (sequence 31) and primers apr-dn1 (sequence 32) and apr-dn2 (sequence 33) as primers, two homologous arm fragments were amplified respectively, with sizes of 667 bp and 495 bp. After obtaining the PCR products of the correct sizes, they were purified by gel extraction respectively. Using the gel-extracted product DNA as a template for overlap PCR, a deletion mutant cassette △aprE with a size of ~1.2 kb was obtained. After purifying the mutant cassette and digesting it with Xba I, it was cloned into the Sma I and Xba I sites of plasmid pT2 ts , and transformed into competent Escherichia coli JM109 cells, and cultured on an LB plate containing 20 μg / mL kanamycin to obtain the correct deletion plasmid pT2-△aprE. According to the steps described in the method of "Genetic transformation of Bacillus licheniformis", the deletion plasmid pT2-△aprE was transformed into the Bacillus licheniformis host cell CCTCC NO: M208236. After two homologous recombinations, primers apr-F (sequence 34) and apr-R (sequence 35) were designed on both sides of the homologous arms, and colony PCR was performed with this pair of primers to verify the correct transformant BCBT01. The size of the PCR product of the correct transformant was ~1.5 kb( Figure 1 , lane 1).

[0095] Deletion of the β-galactosidase-encoding gene lacA in Bacillus licheniformis CCTCC NO: M208236. It was carried out by a method similar to the deletion of the aprE gene described above. Using the genomic DNA of Bacillus licheniformis CCTCC NO: M208236 as a template, and using lacA-up1 and lacA-up2 (sequence 36 and sequence 37) and primers lacA-dn1 and lacA-dn2 (sequence 38 and sequence 39) as primers, two homologous arm fragments were amplified respectively, with sizes of 486 bp and 500 bp. After obtaining the PCR products of the correct sizes, they were purified by gel extraction respectively. Using the gel-extracted product DNA as a template for overlap PCR, a deletion mutant cassette △lacA with a size of 936 bp was obtained. After purifying the mutant cassette and digesting it with Xba I, it was cloned into plasmid pT2 tsThe SmaI and XbaI sites were used to transform competent Escherichia coli JM109 cells, which were cultured on LB plates containing 20 μg / mL kanamycin to obtain the correct deletion plasmid pT2-ΔLacA. According to the steps described in the method of "Genetic transformation of Bacillus licheniformis", the deletion plasmid pT2-ΔLacA was transformed into Bacillus licheniformis. After two rounds of homologous recombination, colony PCR was performed using the primers lacA-F and lacA-R (sequences 40 and 41) on both sides of the homologous arms to verify the correct transformants. The size of the PCR product of the correct transformants was ~1.2 kb( Figure 2 , lane 2).

[0096] Using a similar method as above, different combinations of genes vpr, wpr, lacR, lacA2, and yesZ in the genome of Bacillus licheniformis CCTCC NO: M208236 were deleted. Among them, the homologous arm primers and verification primers corresponding to vpr were sequences 42-47; the homologous arm primers and verification primers corresponding to wpr were sequences 48-53; the homologous arm primers and verification primers corresponding to lacR were sequences 54-59; the homologous arm primers and verification primers corresponding to lacA2 were sequences 60-65; the homologous arm primers and verification primers corresponding to yesZ were sequences 66-71, and mutant strains with different deficiencies were obtained. Among them, mutant strain BCBT03-15 was renamed BCBT0529, and its genetic background was (CBB3008, ΔaprE, Δvpr, Δwpr, ΔlacR, ΔlacA, ΔlacA2, ΔyesZ).

[0097] Example 3 Effects of knocking out some genes on the expression of host cells

[0098] (1) Effects of knocking out some proteases on the extracellular protease activity

[0099] Flask fermentation experiments were carried out on the different mutant strains obtained in Example 2, and their extracellular protease activities were analyzed. As shown in Table 2, after deleting the alkaline protease-encoding gene aprE, the total enzyme activity of proteases in the medium decreased by 80%. After further deleting the two protease genes vpr and wpr, the total enzyme activity of proteases in the medium decreased to 10% of the wild type.

[0100] Table 2 Determination of alkaline protease activity of mutant strains

[0101]

[0102] (2) Effects of knocking out some proteases on the expression of lactase

[0103] The expression plasmid pHY-bgl-12 carried by the recombinant bacterium CBB-Bgl-12 with the highest enzyme production activity obtained in Example 1 was transformed into the host bacterium with the proteolytic enzyme gene deleted obtained in Example 2 to obtain the corresponding recombinant bacterium. Using CCTCC NO: M208236 as a control, the shake flask fermentation results of the recombinant bacterium are shown in Table 3. After deleting the alkaline protease gene aprE, the lactase enzyme activity in the fermentation broth increased significantly, reaching 10.68 U / mL, an increase of 41.08%; after deleting the other two proteolytic enzyme genes vpr and wpr, the lactase activity in the fermentation broth was further increased, and finally reached 12.12 U / mL, a 60% increase compared to the starting strain.

[0104] Table 3 Expression levels of lactase after protease deletion

[0105]

[0106] (3) Effect of partial gene knockout on lactase expression

[0107] After the endogenous lactase-related genes of the host bacterium mutated, the changes in lactase enzyme activity under shake flask fermentation conditions are shown in Table 4. It can be seen that after deleting the alkaline protease gene aprE and the lactose operon repressor gene lacR, the lactase activity in the fermentation broth increased. After further deleting the relevant endogenous lactase structural genes, the lactase activity in the fermentation broth was so low that it could not be detected by the existing method.

[0108] Table 4 Lactase enzyme activity of endogenous lactase gene mutant strains

[0109]

[0110]

[0111] *n.d.: Enzyme activity not detected

[0112] Example 4: High-level expression of lactase

[0113] The plasmid pHY-bgl-12 was transferred into the strains with different endogenous lactase genes deleted obtained in Example 2 to construct recombinant bacteria and perform shake flask fermentation. The lactase activity in the fermentation broth was measured, and the results are shown in Table 5. After deleting the endogenous lactase-related genes in the host cell, it was found that the expression level of the lactase of the present invention was greatly improved. Among them, when BCBT03-15, that is, BCBT0529 was used as the host cell, the enzyme activity reached the highest, which was 4.47 times that of CCTCC NO: M208236 used as the host cell.

[0114] Table 5 Expression levels of lactase after endogenous lactase deletion

[0115]

[0116]

[0117] * n.d.: Enzyme activity not detected.

[0118] On the basis of determining the optimal host cell, the expression elements were further optimized. The expression vector was modified by combining different promoters and different signal peptides to improve the expression level of lactase.

[0119] Using the plasmid pHY-WZX as the backbone of the basic expression vector, 3 different constitutive promoters were selected, which were P cry (Sequence 19, promoter of Bacillus thuringiensis insecticidal protein gene), P amyL (Sequence 20, promoter of Bacillus licheniformis amylase gene), P 43 (Sequence 21, promoter of Bacillus subtilis cytidine deaminase gene) and 6 different signal peptides, which were S amyL (Sequence 22, signal peptide of Bacillus licheniformis amylase gene), S aprE (Sequence 23, signal peptide of Bacillus licheniformis alkaline protease), S amyQ (Sequence 24, signal peptide of Bacillus amyloliquefaciens amylase gene), S amyE (Sequence 25, signal peptide of Bacillus subtilis amylase gene), S nprE (Sequence 26, signal peptide of Bacillus licheniformis neutral protease), S chi (Sequence 27, signal peptide of Bacillus licheniformis chitinase gene). The original promoter and signal peptide on pHY-WZX were replaced in different combinations to construct 18 new expression vectors pHSE-001 - 018 (see Table 6 for details). After cloning the BcBG168-D coding gene sequence and transforming the Bacillus licheniformis host cell BCBT0529, a series of recombinant bacteria were obtained. Flask fermentation was carried out and the enzyme activity was measured. The results are summarized in Table 6. All 18 combinations of promoters and signal peptides tested could mediate the secretion and expression of lactase BcBG168-D in Bacillus licheniformis. Among them, the expression plasmid pHSE-008 ( amyL ) composed of the combination of the promoter P Figure 3 a) of Bacillus licheniformis amylase and the signal peptide of alkaline protease aprE could mediate the highest enzyme expression. The obtained lactase expression plasmid was pLEBG168 (the BcBG168-D coding gene was cloned into the expression plasmid pHSE-008, Figure 3b); The obtained strain BCBTBc168D (transformed the Bacillus licheniformis host cell BCBT0529 after cloning the BcBG168-D coding gene sequence into the expression plasmid pHSE-008) expressed a lactase enzyme activity of 53.79 U / mL during shake flask fermentation, which was more than 20 times the enzyme production level of the wild strain and more than 7 times that before genetic modification of the host cell and signal peptide optimization.

[0120] Table 6 Effects of different promoter-signal peptide combinations on the expression of lactase BcBG168-D

[0121]

[0122]

[0123] Example 5: Lactase fermentation production process in a 50 L fermentation system

[0124] Cultivate the high lactase-producing strain BCBTBc168D at 37 °C for 20 - 40 h. Pick 2 - 3 single colonies and inoculate them into two 5 L Erlenmeyer flasks containing 1000 mL of LB liquid medium. Incubate them on a shaker at 37 °C and 230 r / min for 16 h as the seed liquid. Inoculate it into a 50 L fully automatic fermenter containing 30 L of fermentation medium (malt syrup 4%, cottonseed powder 2.5%, soybean cake powder 3.5%, ammonium sulfate 0.5%, pH 6.5) at an inoculation amount of 5%. The working fermentation volume is 30 L. During the process, maintain the dissolved oxygen at 0.1% - 20% by adjusting the rotation speed and ventilation volume, the fermentation temperature at 40 - 42 °C, and control the pH at 6.5 ± 0.5. Feed 60% (w / w) malt syrup as the carbon source and maintain the reducing sugar content at 0.5 - 5%. Regularly take samples to analyze the residual sugar content and enzyme activity. Stop fermentation when the enzyme activity increase rate is lower than 5 U / (mL·h) after 120 hours of fermentation. Discharge from the fermenter and prepare the enzyme preparation.

[0125] The typical enzyme production progress curve of lactase is shown in Figure 4 , and the enzyme protein in the fermentation broth is the main protein molecule ( Figure 5 ), and the lactase enzyme activity in the supernatant of the fermentation broth reached a maximum of 1131 U / mL (at 108 h), which was about 21 times that of the shake flask fermentation level.

[0126] Similarly, BglD305-D and BglD-D were cloned into the Bacillus licheniformis amylase promoter P amyL and the signal peptide S of alkaline protease AprE aprEUnder the mediation of the combination, using pHSE-008 as the expression vector, the lactase high-yield strains BCBT305D and BCBTatccD were expressed in Bacillus licheniformis BCBT0529, and the lactase production levels of the two strains under the above fermentation conditions reached 820 U / mL and 870 U / mL respectively.

[0127] Example 6: Fermentation production and preparation of lactase

[0128] According to the process of the 50 L fermenter in Example 5, after adjusting the operation process accordingly, the BCBTBc168D strain was used to prepare lactase in a 10-ton fermentation system. After fermentation, the lactase activity in the fermentation broth reached 2208 U / mL.

[0129] After fermentation, a bioflocculant (polyacrylamide: basic aluminum chloride = 8:1) was added to the fermentation broth at 1.0%. After flocculation, 2% of diatomaceous earth TS-20# was added, and then plate and frame filtration was carried out for sterilization. A membrane material with a molecular weight cut-off of 30 kDa was selected for ultrafiltration concentration. At 40 °C, the operating pressure was 0.05 MPa, and the operation lasted for 2 h.

[0130] After ultrafiltration, 1% sodium benzoate, 1% potassium sorbate, 2% sodium chloride, 10% sorbitol, and 10% glycerol were added as stabilizers for the liquid dosage form to obtain a liquid dosage form product.

[0131] 10% glycerol in the above liquid dosage form stabilizer was replaced with 3% lactose and 2% sodium sulfate, and other components remained unchanged. A solid dosage form product was prepared by spray drying.

[0132] The above percentages (%) are all w / v.

[0133] Example 7: Application of lactase in the preparation of galactooligosaccharides

[0134] Using a lactose solution with a concentration of 600 g / L as the substrate, the lactase BcBG168-D prepared by the present invention was used to catalyze the preparation of galactooligosaccharides, and the reaction system had a total volume of about 30 L. The enzyme was added at a concentration of 20 U / g substrate, the pH was adjusted to 6.0, and the reaction was carried out in a reaction kettle at 65 °C with a stirring speed of 50 r / min for 10 h.

[0135] The content of galactooligosaccharides in the reaction product reached more than 50%. Figure 6 This is a typical result of the sugar spectrum analysis of the above-produced galactooligosaccharide product by HPLC detection method.

[0136] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this patent, several modifications, combinations, and improvements can be made to the above-mentioned embodiments, and these all fall within the protection scope of this patent. Therefore, the protection scope of this patent shall be subject to the claims.

Claims

1. A lactase, characterized in that, The amino acid sequence of the lactase is shown in Sequence Listing SEQ ID NO.6: MKSTTSAAGKSVSYNDGERRVNFENWRFQRETNGSIAGAQNPGFDDSSWRKLNLPHEWSIDLDFNKNSLATHEGGYLDGGIGWNRKTFTIPESMKGKRFSLDIDGVYMYSTTYLNAFVLGTYPFGYNGESYDISDKLYKDGRANVLVVKVNTNQPSGRWYSGSSIYRNVYLTVTDPIHVARYGTFVTTPNLEKSIKEDRDAVNIKTKISNAEAKQVKIADKSTIYDGAGNTVQTVETEEKTAAGATVTPFEQNTVIKQPKLWSIDKPYRYNLVTEVIVGGQTVDTYETKFGVRYFKFDENEGYSLNGEFMKLHGVSMHHDLAGGAALTNARGVERQMQIMKDVGVNAIRVTHNPASPELGLEFAANKLLIIEEAFDSWAQSKKPYDYGRFFNAWAEHDIKEMVDRGKNEPAIIMWSIGNEIYDTTNAAGVETARNLVGWVKEIDTTPRATIGEDKTRGDNVTPINSYIKEIFNIVDVVGLNYSENNYDGYHKQNPSWKLYGSETSSATRSRGVYTQPYQYNHDTKYADLQQSSYDNDYVGWGRTAQDAWKYDRDLKHIAGQFIWTGFDYIGEPTPYYNSYPAKSSYFGAVDTAGFPKDIFYYYQSQWKKEPMVHLLPHWNWKEGEKVRVLAYTNASKVELVLNGESLGNEKYDNKQTSWGAPYKETKDGKTYLEWAVKPLKPGKFDEAVAENGKVIARDQVVTAGEPASVRLTADRKVVKADGTDHSFITADIVDSKGIVVPDADHLITFNVTGQGELAGVDNGNASSVERYKDNKRKAFSGKALAIVQSSKLSGKITVHASVAGLSSDST。 2. The coding gene of lactase according to claim 1, characterized in that, The lactase-encoding gene is shown in Sequence Listing SEQ ID NO.

5.

3. A recombinant vector or recombinant strain containing the lactase-encoding gene according to claim 2.

4. The recombinant strain according to claim 3, characterized in that, The expression host used in the recombinant strain is Bacillus licheniformis CCTCC NO: M208236 or mutant strain BCBT0529, and the mutant strain BCBT0529 is obtained by knocking out aprE , vpr , wpr , lacR, lacA, lacA2 , yesZ genes from the genome of Bacillus licheniformis CBB3008, numbered CCTCC NO. M208236; aprE , vpr , wpr , lacR, lacA, lacA2 , yesZ The GenBank accession numbers of the genes are: MT885340, MT885341, MT885342, MT885336, MT885337, MT885338, MT885339.

5. The method for fermenting and producing lactase by the recombinant bacterium according to claim 3, characterized in that, Flask fermentation for lactase production: Inoculate the recombinant bacteria into the flask medium and culture at 30 - 45 o C, 120 - 270 r / min for 2 - 3 days; Flask culture medium composition: yeast extract 0.5 - 1.5%, peptone 1.2 - 3.6%, glucose 8 - 20%; pH 7.

0.

6. The method for fermenting and producing lactase by the recombinant bacterium according to claim 3, characterized in that, Fermentation tank fermentation for producing lactase: Inoculate the strain into the fermentation tank medium at an inoculation amount of 5% - 10%; during the fermentation process, use a fermentation temperature of 33 - 45°C, control the dissolved oxygen at 0.1% - 20%, the pH at 6.0 - 7.8, feed 30% - 60% malt syrup and maintain the reducing sugar content at 0.1% - 5%; the fermentation lasts for 90 - 120 h, and the end point of fermentation is controlled such that the increase in fermentation enzyme activity is less than 5 - 20 U / (mL·h). Fermentation tank culture medium composition: malt syrup 1% - 5%, cottonseed meal 0% - 5%, corn steep liquor 0% - 4%, soybean cake powder 0.5 - 5%, ammonium sulfate 0.1 - 5%, pH 6.0 - 8.

0.

7. The method for fermenting the recombinant bacterium according to claim 5 or 6 to produce lactase, characterized in that, After fermentation, remove the cells by plate and frame filtration, and then obtain the enzyme solution after filtration through an ultrafiltration system.

8. Use of the recombinant vector or recombinant strain according to claim 3 in the production of lactase.

9. Use of the lactase according to claim 1 or 2 in the production of galactooligosaccharides.

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