SPECIAL ENZYME FOR THE PRODUCTION OF GALACTOOLIGOSACCHARIDS, PRODUCTION OF THEM AND USE OF THEM

Description

[0001] This application claims the priority of the Chinese patent application filed to the Chinese Patent Office on September 29, 2020, numbered CN2020110510561, and entitled "SPECIAL ENZYME FOR GALACTOOLIGOSACCHARIDE PRODUCTION AS WELL AS PREPARATION AND APPLICATION THEREOF.Technical Field

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

[0003] Oligosaccharides, also known as oligose, refers to linear or branched carbohydrates with a degree of polymerization of 2-10 connected by monosaccharide molecules through glycosidic bonds. They can be simply divided into functional oligosaccharides and ordinary oligosaccharides. Among them, functional oligosaccharides refer to the polymerization of 2-10 identical or different monosaccharides with glycosidic bonds; it has the sweet taste and sensory characteristics shared by sugars, and can directly replace sucrose as a sweet ingredient. It is not degraded by human gastric acid and gastric enzymes, not absorbed in the small intestine, and can reach the large intestine; and has physiological properties such as promoting the proliferation of probiotics in the human body. Among the functional oligosaccharides, the glycosidic bond is not easily hydrolyzed and digested by hydrolytic enzymes in the human intestines and stomach due to the anomeric carbon atom (C1 or C2) configuration of the monosaccharide, which is also called non-digestible sugar.

[0004] Naturally occurring functional oligosaccharides, such as galactooligosaccharides (GOS) present in human milk, cow milk, goat milk, etc., is an important prebiotic and plays an important role in human health. In the 1950s, there have been reports on the use of β-galactosidase to catalyze the industrialization of lactose to produce galactooligosaccharides. Internationally, GOS products were successfully marketed in 1988.

[0005] In the production of galactooligosaccharides, lactose is mainly used as the raw material, and oligosaccharides with a low degree of polymerization containing one glucose or all galactose molecules are synthesized during the hydrolysis of lactose by the transglycosylation of lactase, which can be expressed as Gal-(Gal) n -Glc / Gal (n is 1-4). The lactase (a type of β-galactosidase, EC3.2.1.23) used in the production of galactooligosaccharides can generate GOS through its transgalactosylation, which is a kind of enzyme very commercial value in the dairy industry. Commercially, Aspergillus niger (A. niger), Aspergillus oryzae (A. oryzae), Kluyveromyces lactis (K. lactis), K. fragilis (K. fragilis), Cryptococcus laurentii (C. laurentii), Bacillus circulans and other strains are generally chosen, through the submerged fermentation method to prepare lactase products. The source of lactase and the production process of GOS are different. Although there are certain differences in the composition of enzyme activity and transglycosylation activity, the catalyzed synthesis of galactooligosaccharides is mainly composed of β-1,3, β-1,4, and β-1,6 glycosidic bonds connection, of which β-1,4 glycosidic bond is the main one. In addition, in view of the thermotolerant of enzymes, some new types of high thermotolerant lactases have been researched and developed. Lactases derived from microorganisms such as Sulfolobus solfataricus, Saccharopolyspora rectivirgula, Pyrococcus furiosus and Thermotoga maritima can be used to catalyze synthesis of GOS at temperature of 70°C-80°C. The existing commercial lactases generally form 5%-50% galactooligosaccharides from lactose as a raw material. The lactase (such as Biolacta ®< ) produced by B. circulans ATCC 31382 is the lactase with the strongest ability to synthesize GOS so far. This enzyme has 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 which β-Gal-C and β-Gal-D are considered the most valuable for GOS production. In addition, the lactase identified from B. circulans B2301 can catalyze lactose to form 54.5% GOS at a high temperature above 60°C. It is the lactase with the best GOS synthesis performance among all reported lactases (Zhao Jihua, et al., Food and Fermentation Industries, 2020). The complete open reading frame size of the B. circulans B2301 lactase encoding gene is 5133 bp, encoding 1710 amino acid residues, with no typical bacterial signal peptide sequences, and the highest similarity with the previously reported β-galactosidase is 93.6%; this enzyme shows the highest catalytic activity at 60°C and pH 6.0-6.5. Zn 2+< , Fe 2+< , Cu 2+< , EDTA and SDS show different degrees of inhibition on the enzyme. The V max of catalyzing the synthesis of galactooligosaccharide is 2.47 g / (L·h), K m is 14.37g / L (Tian Kangming, et al., Food and Fermentation Industries, 2020).

[0006] However, the current fermentation of B. circulans to produce lactase is very uneconomical and requires a long fermentation time, usually 96 h-200 h; the level of enzyme production is also relatively low, usually only 2-5 U / mL of lactase can be produced (Zhao Jihua, et al., Food and Fermentation Industries, 2020). The process for the enzyme preparation is complex, most of the enzyme activity of lactase exists in the cells, and it needs to be released through complex cell disruption methods, and the quality of enzyme products is therefore greatly affected.

[0007] The coding gene of B. circulans lactase or its mutants was expressed in a variety of host cells to understand the expression level of lactase. For example, when expressed in Escherichia coli, the expression level of lactase is 1 to 3 U / mL, and it is hard to secrete lactase into the fermentation broth, which increases the difficulty of lactase preparations. When expressed in Pichia pastoris GS115, the expression level of shake flask fermentation can reach 70 U / mL, but it is also difficult to realize the secretory expression of lactase, and the separation and purification of the enzyme is also difficult.

[0008] Jingyuan Song et, al, Duan Xuguo et al, and US 2017 / 049120 Al disclose a beta-galactosidase from Bacillus circulans having the UniProt accession number E5RWQ2 (corresponding to the GenBank accession number AB605256) (JINGYUAN SONG ET AL: "The Discoidin Domain of Bacillus circulans &bgr;-Galactosidase Plays an Essential Role in Repressing Galactooligosaccharide Production", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 77, no. 1, 23 January 2013 (2013-01-23), pages 73-79, DUAN XUGUO ET AL: "Optimal extracellular production of recombinant Bacillus circulans [beta]-galactosidase in Escherichia coliBL21 (DE3)", PROCESS BIOCHEMISTRY, ELSEVIER LTD, GB, vol. 53, 11 November 2016 (2016-11-11), pages 17-24). However, there is still a need for a lactase with an increased lactase activity.Summary

[0009] The purpose of the present invention is to obtain a novel strain with good performance of large-scale fermentation production and ideal lactase synthesis and secretion capabilities on the basis of obtaining excellent special enzyme molecules for galactooligosaccharide, which can significantly reduce manufacturing cost of the fermentation of lactase, simplify the fermentation manufacturing process of lactase, and significantly improved the quality of lactase preparations.

[0010] In order to achieve the above objectives, the technical solution of the present invention is to provide a lactase mutant, namely BcBG168-D; BcBG168 is obtained by DNA shuffling modification of BglD305 and BglD, BcBG168-C and BcBG168-D are obtained by further deleting the partial amino acid sequence of the C-terminal on the basis of BcBG168; The amino acid sequences of BcBG168, BcBG168-C and BcBG168-D are shown in SEQ ID NO. 14, 16 and 18, respectively.

[0011] The expression vector used in the recombinant vector is the pHSE-008 plasmid, which is based on the backbone of the expression vector pHY-WZX, and integrates the amylase promoter P amyL (SEQ ID NO. 20) derived from Bacillus licheniformis and signal peptide S aprE (SEQ ID NO.23) of the alkali protease aprE; The expression host used in the recombinant strain is the mutant strain BCBT0529, which is obtained by knocking out aprE, vpr, wpr, lacR, lacA, lacA2, yesZ genes (The GenBank accession numbers corresponding to its gene sequence are: MT885340, MT885341, MT885342, MT885336, MT885337, MT885338, MT885339, respectively) from the genome of B. licheniformis CBB3008 (numbered CCTCC NO.M208236).

[0012] The present invention provides a recombinant strain with high lactase production-B. licheniformis BCBTBc168D, which is obtained by integrating the BcBG168-D coding gene into the pHSE-008 plasmid and expressing it in the host cell mutant strain BCBT0529; The expression level of lactase BcBG168-D prepared by fermentation of B. licheniformis BCBTBc168D can reach 2208 U / mL.

[0013] The present invention also provides a method for fermentation and production of lactase using the above-mentioned recombinant strain: (1)Shake flask fermentation to produce lactase: inoculating recombinant strain to shake flask culture medium, culturing at 30-45°C and 120-270 r / min for 2-3 days; Shake flask culture medium: yeast extract 0.5-1.5%, peptone 1.2-3.6%, glucose 8-20%; pH7.0. (2)Fermentation tank fermentation to produce lactase: inoculating the strain to the fermentation tank culture medium according to the inoculation amount of 5%-10%; during the fermentation process, the fermentation temperature is 33-45°C, the dissolved oxygen is controlled to 0.1%-20%, and the pH is 6.0 -7.8, adding 30%-60%(w / w) maltose syrup and maintain the reducing sugar content at 0.1%-5%; fermentation lasts for 90-120 h, sampling and analyzing regularly during the fermentation process, the end of the fermentation is controlled as the increase value of fermentation enzyme activity less than 5-20 U / (mL·h).

[0014] The culture medium of the fermentation tank is composed of: 1%-5% of maltose syrup, 0%-5% of cottonseed powder, 0%-4% of corn syrup, 0.5-5% of soybean meal powder, 0.1-5% of ammonium sulfate, and pH 6.0-8.0.

[0015] After fermentation, the lactase activity of the fermentation broth in the shake flask fermentation can reach 25-54 U / mL; the lactase activity in the fermentation tank can reach 826-2208 U / mL.

[0016] Further, after the fermentation is completed, the strains are removed by simply filtration, and then filtered by an ultra-filtration system to obtain an enzyme solution.

[0017] The present invention also provides the application of the above-mentioned lactase in the production of galactooligosaccharides.Beneficial effects:

[0018] The special enzyme preparation for the production of galactooligosaccharides in the present invention is a lactase with extremely high activity of catalyzing lactose to produce galactooligosaccharides obtained through gene cloning and artificial evolution; the lactase high-yielding strain of the present invention is recombinant strain by microorganisms breeding, which can efficiently synthesize lactase during submerged fermentation and secrete enzyme molecules into the culture medium, directly prepare high-activity enzyme preparations from the fermentation broth, and apply them to the high-efficiency production of galactooligosaccharides.

[0019] With the high-efficiency preparation method of lactase of the present invention, the expression level of lactase can reach 2208 U / mL under the lactase high-producing strain and fermentation process provided by the present invention. The invention helps to reduce the fermentation manufacturing cost of lactase, simplify the fermentation manufacturing process and improve the quality of the lactase enzyme preparation.Brief Description of FIG.s

[0020] FIG. 1. is a validation map of protease-encoding gene knockout. Lane M is the 1kb molecular weight standard; Lane 1 is the mutant strain with the correct genome verification after the aprE gene is deleted, and the PCR amplification size is 1.5 kb; Lane 2 is the mutant strain with the correct genome verification after the protease wpr gene is deleted, and the PCR amplification size is 1.3 kb; Lane 3 is the mutant strain verified by the correct genome of the protease vpr deletion strain, and the PCR amplification size is 1.3 kb; FIG. 2. is a validation map of knockout of coding genes such as endogenous lactase. Lane M is the 1 kb molecular weight standard; Lane 1 is the electrophoretic pattern confirmed by PCR after the gene lacR is successfully knocked out, the size is 1.3 kb; Lane 2 is the electrophoretic pattern confirmed by PCR after the gene lacA is successfully knocked out, the size is 1.2 kb; Lane 3 is the electrophoretic pattern confirmed by PCR after the gene lacA2 is successfully knocked out, the size is 1.2 kb; Lane 4 is the electrophoretic pattern confirmed by the PCR after the gene yesZ is successfully knocked out, the size is 1.6 kb; FIG. 3. is a physical map of expression vector. a: optimized expression vector pHES-008; b: lactase expression plasmid pLEBG168; FIG. 4. is an enzyme production curve of lactase fermentation. FIG. 5. is a protein electrophoresis pattern of fermentation broth. Lane M is the protein molecular weight standard; Lane 1 is the result of direct electrophoresis of the fermentation broth, and the arrow mark is the lactase in the fermentation broth; FIG. 6. is a HPLC sugar spectrum of galactooligosaccharides produced by lactase from lactose DP2: galactobiose or lactose, DP3: glucosylgalactobiose or galactotriose; DP4: glucosylgalactotriose or galactotetraose; DP5: glucosylgalactotetraose or half lactopentaose. Detailed Description

[0021] In order to make the purpose, technical solutions and advantages of this patent clearer, the following will further describe this patent in detail with reference to specific embodiments. It should be understood that the specific embodiments described here are only used to explain the patent, and not used to limit the present invention.

[0022] The plasmid pHY-WZX used in the present invention is the prior art, and its construction method has been disclosed in Niu DD and 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 through the Biocatalysis and Biotransformation Laboratory of the College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology.

[0023] The B. licheniformis CBB3008 used in the present invention has been deposited in the China Center for Type Culture Collection (CCTCC), the preservation date is November 25, 2008, and the preservation number is CCTCC NO: M208236.

[0024] The present disclosure is based on two sources of lactase (BglD305 derived from B. circulans B2301 and BglD derived from B. circulans ATCC 31382) as the basis for molecular evolution, which are used to obtain new lactases (BglD305-C*, BglD305-D*, BglD-C*, BglD-D*, BcBG168*, BcBG168-C*, BcBG168-D), which have high efficiency in synthesis of galactooligosaccharides and good expression performance; (* not part of the present invention).

[0025] In the present invention, a host cell B. licheniformis CBB3008, has been deposited in the Chinese Type Culture Collection, and the preservation number is CCTCC NO: M208236, which is further genetically modified, multiple genes (alkaline protease coding gene aprE, minor serine protease coding gene vpr, cell wall protease coding gene wpr, regulatory protein coding gene lacR, β-galactosidase coding gene lacA, β-galactosidase coding gene lacA2, β-galactosidase encoding gene yesZ) affecting the expression of lactase are knocked out, to obtain a new host strain suitable for high-efficiency expression of lactase;

[0026] The present disclosure constructs and optimizes an expression vector suitable for secretion and expression of lactase, which is optimized on the basis of pHY-WZX. The expression vector contains a preferred promoter for guiding high expression of lactase and a signal peptide that efficiently mediates secretion and expression of lactase; In the present disclosure, the coding genes of B. circulans lactase or its mutants (corresponding to nucleotide sequence 1, 3, 5, 7, 9, 11, 13, 15, 17; the amino acid sequence 2, 4, 6, 8, 10, 12, 14, 16, 18) are cloned into the expression vector constructed in the present invention, genetically transformed into B. licheniformis host strains to obtain lactase-producing recombinant strain. The fermentation conditions and process are established and optimized. A new process of enzyme separation, purification and refining are developed to produce lactase products for the industrial manufacturing of GOS.

[0027] The method for constructing a new strain with high lactase yield of the present invention is to clone an expression vector by molecular cloning technology and obtain an expression plasmid for lactase, and transform it into a new host strain of B. licheniformis to obtain recombinants to realize lactase high-efficiency secretion and expression. The enzyme synthesis and secretion system is optimized to reach high-efficiency secretion of the synthesized lactase into the medium. And through the fermentation process, lactase is recovered and refined from the fermentation broth to obtain lactase products.

[0028] The main experimental methods used in the present invention are as follows:1. Gene cloning, molecular evolution and construction of expression plasmid

[0029] Conventional molecular cloning operations were carried out with reference methods (Sambrook et al. Molecular Cloning: A Laboratory Manual, 1989). The coding gene of B. circulans lactase or its mutants was used as the target gene in the present invention; the basic expression vector was pHY-WZX (Niu & Wang. J Ind Microbiol Biotechnol, 2007 ). The coding gene of lactase or its mutant is amplified by PCR and then cloned into an expression vector to obtain a series of recombinant plasmids expressing lactase.2. Chromosomal DNA extraction

[0030] The chromosomal DNA extraction method was carried out according to the literature (Zhuge Jian and Wang Zhengxiang. Manual of Industrial Microbiology Experiment Technology, China Light Industry Press, 1994).3. Plasmid DNA extraction

[0031] The plasmid DNA was extracted using a certain concentration of lysozyme to lyse the cell wall and using Sigma's plasmid small extraction kit.4. Gene amplification

[0032] DNA amplification was performed in 0.2 mL PCR thin-walled tubes. The PCR amplification conditions are: 1×(95°C, 5min); 30×(94°C, 10s; 58°C, 30s, 72°C, 30-300s); 1×(72°C 10min). Depending on the length of the amplification, the extension temperature and time of the PCR reaction were different. Unless otherwise specified, all PCR reactions were performed with Pfu DNA polymerase.5. Artificial evolution of enzyme molecules

[0033] The molecular evolution of lactase was carried out using DNA shuffling according to the literature method (Stemmer W P C, et al,. Rapid evolution of a protein in vitro by DNA shuffling [J]. Nature, 1994, 370(6488): 389-391). Firstly, the lactase gene was partially digested with DNase, and the 100-200 bp fragments were recovered by the density gradient method. After mixing, the gene amplification was performed for 15-25 cycles without primers, and then specific primers at both ends were added to amplify the full-length gene; PCR product purification kit (Sigma) was used for purification, the purified DNA was cloned into the expression vector pHY-WZX, CaCl 2 method was used (Zhuge Jian and Wang Zhengxiang. Industrial Microbiology Experimental Technology Manual, China Light Industry Press, 1994) to transform into E. coli JM109; the lactase activity was measured and compared.6. Overlap PCR

[0034] Refer 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 steps were: using the primers of fragment F1 and fragment F2 (P1+P2; P3+P4, primers P2 and P3 were reverse complementary sequences) to mediate PCR amplification to obtain gene fragments; gel recovery and purification of amplified fragment F1 And F2; the purified two fragments F1 and F2 were diluted by an appropriate multiple, and mixed with a 1:1 molar ratio as a template, and primers P1+P4 were used to mediate a new PCR reaction to obtain a full-length sequence.7. B. licheniformis genetic transformation

[0035] Refer to the method introduced in the literature (Xu Min, Ma Junshuang, Wang Zhengxiang. The effect of high osmotic pressure on the electrical conversion rate of bacteria. Journal of Wuxi University of Light Industry, 2004(04):98-100). The main steps were as follows: fresh single colony was inoculated into liquid LB medium, and cultivated overnight at 37°C at 200 r / min, then 5% inoculation amount was transfered to new LB medium and continued to cultivate until the OD600 was 0.75-0.90. The cells were collected by centrifugation at 6000 r / min at 40°C for 10min after ice bath for 10 min. The cells were repeatedly washed 4 times with pre-cooled electroporation washing solution (0.5 mol / L sorbitol, 0.5 mol / L mannitol and 10% glycerol). The cell pellet was suspended in 1 mL of pre-cooled electroporation washing solution to complete the preparation of competent cells. 1 µL of plasmid DNA and about 100 µL of competent cells were taken to mix, and were immediately electroporated (1800v, 5 ms), then electroporation resuscitation solution (LB medium containing 0.65 mol / L sorbitol and 0.45 mol / L mannitol) was added, after recovery at 37°C, 160 r / min, the system was then spread on the corresponding resistant LB plate and cultivated at the appropriate temperature until a single colony grew. The correct transformants were verified by colony PCR verification, plasmid extraction, enzyme digestion, and fermentation verification methods.8. Deletion of specific genes in B. licheniformis

[0036] Reference method (Cai D, et al High-level expression of nattokinase in B. licheniformis by manipulating signal peptide and signal peptidase. J Appl Microbiol. 2016, 121: 704-712) was performed. The deletion of aprE gene in B. licheniformis CCTCC NO: M208236 was taken as an example, the general procedure was as follows: B. licheniformis genomic DNA was used as a template, apr-up1 (sequence 30) and apr-up2 (sequence 31) and primer apr-dn1 (sequence 32) and apr-dn2 (sequence 33) were primers to amplify the upper and lower homology arm fragments respectively to obtain the correct size PCR products and then gel recovery was used to purify them, and gel recovery product DNA was used as a template to overlap by PCR, the deletion mutation box ΔaprE was obtained. The mutant cassette was purified and digested with Xba I and cloned into the Sma I and Xba I locus of the plasmid pT2 ts< (pT2 ts< is based on T2(2)-ori (Chen Shouwen, etc., Chinese invention patent, ZL201310562150.7) as the starting plasmid, after reverse amplification of primers T2-1 (sequence 28), T2-2 (sequence 29) was performed, the PCR product was self-circularized and ligated to obtain the new plasmid pT2 ts< ), and was transformed into E. coli JM109 competent cells, and cultured on LB plate with 20 µg / mL kanamycin to obtain the correct deletion plasmid pT2-ΔaprE. According to the steps described in the genetic transformation method of B. licheniformis, the deletion plasmid pT2-ΔaprE was transformed into B. licheniformis host cells. After two homologous recombination, the primers apr-F: (sequence 34) and apr-R (sequence 35) were designed on both sides of the homology arm, and colony PCR was performed with these primers to verify the transformants (other genes for deletion, refer to the above method, design and replace primers according to the deleted gene sequence).9. Fermentation test

[0037] Shake flask fermentation to produce lactase: 30 mL fermentation medium (yeast extract 0.5~1.5%, peptone 1.2~3.6%, glucose 8~20%; pH 7.0) were added in a 250 mL Erlenmeyer flask, the recombinant strain was inoculated and incubated at 30~45°C, under 120-270 r / min for 2 to 3 days.

[0038] Fermentation tank to produce lactase: the composition of the fermentation medium was: 1% to 5% of maltose syrup, 0% to 5% of cottonseed meal, 0% to 4% of corn syrup, 0.5 to 5% of soybean meal, and 0.1 to 5% of ammonium sulfate, pH 6.0-8.0; fermentation was carried out in a 50 L-10 ton fermentation tank, with an inoculum amount of 5%-10%; during the fermentation process, the fermentation temperature was 33-45°C, the dissolved oxygen was controlled at 0.1%-20%, and the pH was 6.0-7.8. 30%-60% (w / w) maltose syrup was added and the reducing sugar content was maintained at 0.1%-5%; fermentation was lasted for 90-120 h, sampling and analysis were performed during the fermentation process, the end of the fermentation was controlled when the increased value of the enzyme activity is less than 5-20 U / (mL·h).10. Preparation method of lactase preparation

[0039] After the fermentation, the strains were removed by filtration, and then filtered by an ultrafiltration system to obtain the enzyme solution.11. Lactase activity determination

[0040] The enzyme activity determination of lactase is improved in accordance with the Chinese National Standard GB / T 33409-2016. The general process is that the reaction is carried out at pH 5.0 and 40°C with lactose as the substrate. A biosensor was used to determine the amount of glucose released.

[0041] The enzyme activity of lactase is 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 is defined as one unit (U), expressed in U / mL or U / g.12. Synthesis and product analysis of galactooligosaccharides

[0042] Using 300 g / L-800 g / L lactose as the substrate, adding 5 U / g-20 U / g lactase, the reaction is carried out at 50°C-70°C, and sampling is done regularly. For the analysis of the formation and content of reaction raw materials and galactooligosaccharides, the characteristics and formation of enzymatic products were analyzed by HPLC. The chromatographic conditions were: the mobile phase was 65% acetonitrile, the flow rate was 1.0 mL / min; the TSK-GEL G3000PWXL-CP (7.8mm×300mm, 7µm) chromatographic column, the column temperature was 25°C; the evaporative light scattering detector, the drift tube temperature was 90 °C, the carrier gas flow rate was 2.2 mL / min.13. Other analysis methods

[0043] Protease activity determination was carried out in accordance with the Chinese National Standard method (GB / T 23527-2009); Gene and amino acid sequence comparison, DNAMAN software was used; The nucleotide sequence determination was carried out by the Sanger method; Sequence protein content was carried out according to literature method (Bradford. Anal Chem, 1976); Glucose content was determined by enzyme electrode method (SBA-90, Shandong); The cell density was measured with a spectrophotometer (UV-2000, USA) at 600nm; Protein electrophoresis was carried out according to the literature method (Zhuge Jian and Wang Zhengxiang. Manual of Industrial Microbiology Experiment Technology, China Light Industry Press, 1994).

[0044] The following will further explain the present invention through specific embodiments.Example 1: Molecular evolution of lactase

[0045] The BglD305 and BglD coding genes shown in SEQ ID NO. 1 and SEQ ID NO.7 in the sequence table were used as a template, DNA shuffling was used to carry out molecular evolution. After the enzyme activity screening, the lactase enzyme molecule BcBG168 (nucleotide sequence SEQ ID NO.13) with significantly increased enzyme activity level was obtained, and its amino acid sequence (amino acid sequence SEQ ID NO. 14) was obtained.

[0046] By truncating the coding genes of BglD305, BglD and BcBG168 with varying degrees, and efficiently expressing the modified sequences and the original sequence, the corresponding gene sequences were amplified by PCR amplification technology and cloned into the expression vector pHY-WZX to obtain lactase expression plasmid 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, pHY-Bgl-12. The above recombinant plasmids were transformed into B. licheniformis CCTCC NO: M208236 by the above-mentioned B. licheniformis genetic transformation method, and 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, CBB-Bgl-12 were obtained, shake flask fermentation and analysis of enzyme production (enzyme activity determination on the supernatant of the fermentation broth) were further carried out, the main content and enzyme production results are shown in Table 1.

[0047] Under the same conditions, the expressed enzyme activity of BcBG168 was 103.2% of BglD305 and 109.8% of BglD, respectively.

[0048] The C-terminal truncated lactase of lactase showed an upward trend under the same expression conditions. Compared with the original gene sequence, the enzyme activity increased by 40%, 78%; 35%, 69% and 31%, 70%. Table 1 Expression efficiency of lactase from different sources and C-terminal truncationStrainsLactase expression and its sequenceEnzyme activity (U / mL)CBB-Bgl-1BglD305*nucleotide sequence 1 (amino acid sequence 2)3.21±0.15CBB-Bgl-2BglD305-C*nucleotide sequence 3 (amino acid sequence 4)4.52±0.17CBB-Bgl-3BglD305-D*nucleotide sequence 5 (amino acid sequence 6)5.74±0.21CBB-Bgl-7BglD*nucleotide sequence 7 (amino acid sequence 8)3.56±0.11CBB-Bgl-8BglD-C*nucleotide sequence 9 (amino acid sequence 10)4.82±0.14CBB-Bgl-9Bg1D-D*nucleotide sequence 11 (amino acid sequence 12)6.03±0.20CBB-Bgl-10BcBG168*nucleotide sequence 13 (amino acid sequence 14)3.82±0.16CBB-Bgl-11BcBG168-C *nucleotide sequence 15 (amino acid sequence 16)5.02±0.17CBB-Bgl-12BcBG168-Dnucleotide sequence 17 (amino acid sequence 18)6.48±0.22*comparative example Example 2: Genetic modification of expression host cells

[0049] Deletion of aprE gene in B. licheniformis CCTCC NO: M208236. B. licheniformis CCTCC NO: M208236 genomic DNA was used as a template, apr-up1 (sequence 30) and apr-up2 (sequence 31) and primers apr-dn1 (sequence 32) and apr-dn2 (sequence 33) were used as primers, the upper and lower homology arm fragments were respectively amplified, the sizes were 667 bp and 495 bp, respectively. After obtaining the PCR products of the correct size, they were purified by gel recovery, and overlap PCR was performed using the gel recovered product DNA as a template to obtain a deletion mutation box ΔaprE with a size of ~1.2 kb. The mutant box was purified and digested with Xba I, cloned into the Sma I and Xba I sites of plasmid pT2 ts< , transformed into E. coli JM109 competent cells, and cultured on LB plates containing 20 µg / mL kanamycin to obtain the correct deletion plasmid pT2-ΔaprE. Following the steps described in the "Genetic transformation of B. licheniformis" method, the deletion plasmid pT2-ΔaprE was transformed into the B. licheniformis CCTCC NO: M208236. After two homologous recombination, the primers apr-F (sequence 34) and apr-R (sequence 35) were designed on both sides of the homology arm, and colony PCR was performed with these primers to verify that the correct transformant BCBT01 was obtained, the size of the PCR product for correct transformant is ~1.5kb (FIG. 1, lane 1).

[0050] B. licheniformis CCTCC NO: M208236 β-galactosidase encoding gene lacA deletion. The method similar to the above aprE gene deletion was used. B. licheniformis CCTCC NO: M208236 genomic DNA was used as template, lacA-up1 and lacA-up2 (sequence 36 and sequence 37) and primers lacA-dn1 and lacA-dn2 (sequence 38 and sequence 39) were used as primers to respectively amplify the upper and lower homology arm fragments, 486 bp and 500 bp in size, respectively. After obtaining the PCR products of the correct size, they were purified by gel recovery, and overlap PCR was performed using the gel recovered product DNA as a template to obtain a deletion mutation box ΔlacA with a size of 936 bp. The mutant box was purified and digested with Xba I, cloned into the Sma I and Xba I sites of plasmid pT2 ts< , transformed into E. coli JM109 competent cells, and cultured on LB plates containing 20 µg / mL kanamycin to obtain the correct deletion plasmid pT2-ΔLacA. The deletion plasmid pT2-ΔLacA was transformed into B. licheniformis according to the steps described in the "Genetic Transformation of B. licheniformis" method. After two homologous recombination, using the primers lacA-F and lacA-R (sequence 40 and 41) on both sides of the homology arm to verify the correct transformant by colony PCR. The PCR product size of the correct transformant was ~1.2 kb (FIG. 2, lane 2).

[0051] Using the above-mentioned similar method, the vpr, wpr, lacR, lacA2 and yesZ genes in the genome of B. licheniformis CCTCC NO: M208236 were deleted in different combinations, where vpr corresponds to the homology primers and the verification primers are sequences 42-47; wpr corresponds to the homology primers and the verification primers are sequences 48-53; lacR corresponds to the homology primers and verification primers are the sequences 54-59; lacA2 corresponds to the homology primers and verification primers are the sequences 60-65; yesZ corresponds to the homology primers and verification primers are sequence 66-71, and different defective mutants were obtained. Among them, mutant BCBT03-15, renamed BCBT0529, its genetic background was (CBB3008, ΔaprE, Δvpr, Δwpr, ΔlacR, ΔlacA, ΔlacA2, ΔyesZ ).Example 3 The effect of knocking out some genes on host cell expression(1) The effect of partial proteases knockout on extracellular protease activity

[0052] The different mutant strains obtained in Example 2 were subjected to a shake flask fermentation test, and their extracellular protease activity was analyzed. As shown in Table 2, after deleting the alkaline protease encoding gene aprE, the total enzyme activity of proteolytic enzymes in the medium was reduced by 80%. After further deleting the two proteases encoding gene vpr and wpr, the total enzyme activity of proteolytic enzymes in the medium drops to 10% of the wild type. Table 2 Determination of alkaline protease activity of mutant strainsStrainsGenotypeExtracellular protease activity (U / mL)CCTCC NO: M208236Original strain560±20BCBT01ΔaprE112±12BCBT02ΔaprEΔvpr82±9BCBT03ΔaprEΔvprΔwpr58±7 (2) The effect of partial proteases knockout on the expression of lactase

[0053] The expression plasmid pHY-bgl-12 carried in the recombinant CBB-Bgl-12 with the highest enzyme-producing activity obtained in Example 1 was transformed into the host with the proteolytic enzyme gene deleted obtained in Example 2 to obtain the corresponding recombinants, with CCTCC NO: M208236 as a control. The results of recombinants in shake flask fermentation are shown in Table 3. After deleting the alkaline protease gene aprE, the lactase 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 further increased (12.12 U / mL), which was 60% higher than the original strain. Table 3 The expression level of lactase after deleting the proteasesHostStrainsLactase activity (U / mL)CCTCC NO: M208236 / 0.15±0.03CCTCC NO: M208236CBB-Bgl-127.57±0.24BCBT01BCBT01-Bgl-1210.68±0.44BCBT02BCBT02-Bgl-1211.72±0.18BCBT03BCBT03-Bgl-1212.12±0.28 (3) The effect of specific gene knockout on the expression of lactase

[0054] Table 4 shows the changes in lactase activity of the host cell after their endogenous lactase-related genes are mutated under shaking flask fermentation conditions. It can be seen that after the deletion of the alkaline protease encoding gene aprE and the lactose operon repressor protein encoding gene lacR, the lactase activity in the fermentation broth has increased. After further deletion of the related endogenous lactase structural genes, the lactase activity in the fermentation broth is as low as can not be measured according to existing methods. Table 4 Lactase enzyme activity of endogenous lactase gene mutant strainStrainsGenesLactase activity (U / mL)CCTCC NO: M20823 6Parent strain0.15±0.03BCBT03ΔaprEΔvprΔwpr0.14±0 .05BCBT03 -1ΔaprE ΔvprΔwprΔlacR2.86±0 .09BCBT03 -2ΔaprE ΔvprΔwprΔlacAn.d.*BCBT03 -3ΔaprEΔvprΔwprΔlacA20.12±0 .02BCBT03 -4ΔaprE ΔvprΔwprΔyesZ0.13±0 .02ΔaprEΔvprdΔwprΔlacRΔlacABCBT03 -5n.d.*BCBT03 -6ΔaprEΔvprΔwprΔlacRΔlacA22.61±0 .02BCBT03 -7ΔaprE ΔvprΔwprΔlacR ΔyesZ2.60±0 .04BCBT03 -8ΔaprE ΔvprΔwprΔlacA ΔlacA2n.d.*BCBT03-9ΔaprE Δvpr ΔwprΔlacAΔyesZn.d.*BCBT03-10ΔaprE ΔvprdwprΔlacA2ΔyesZ2.72±0 .03BCBT03 -11ΔaprE ΔvprΔwprΔlacRΔlacAΔlacA2n.d.*BCBT03 -12ΔaprE ΔvprΔwprΔlacRΔlacAΔyesZn.d.*BCBT03 -13ΔaprEΔvprΔwprΔlacRΔlacA2ΔyesZ2.58±0 .04BCBT03 -14ΔaprE ΔvprΔwprΔlacAΔlacA2ΔyesZn.d.*BCBT03 -15ΔaprEn.d*ΔvprΔwprΔlacRΔlacA2ΔvesZΔlacA*n.d.: Enzyme activity is not detected Example 4: Efficient expression of lactase

[0055] The plasmid pHY-bgl-12 was transformed into the strains with different endogenous lactase genes deletion obtained in Example 2, and the recombinants were constructed and subjected to shake flask fermentation. The lactase activity in the fermentation broth was determined. The results are shown in Table 5. After the endogenous lactase-related genes of the host cell were deleted, it was found that the expression level of lactase of the present invention was greatly increased. Among them, BCBT03-15 had the highest enzyme activity when BCBT0529 was used as the host cell, and was 4.47 times of the enzyme activity when strain CCTCC NO: M208236 was used as the host cell. Table 5 The expression level of lactase after deletion of endogenous lactaseHostStrainsLactase activity (U / mL)CCTCC NO: M208236CBB-Bgl-127.57±0.24BCBT03-1BCBT03-1-Bgl-1213.63±0.38BCBT03-2BCBT03-2-Bgl-1228.25±2.42BCBT03-3BCBT03-3-Bgl-127.87±0.34BCBT03-4BCBT03-4-Bgl-127.02±0.46BCBT03-5BCBT03-5-Bgl-1230.56±1.46BCBT03-6BCBT03-6-Bgl-1212.27±0.32BCBT03-7BCBT 03-7-Bgl-1212.89±0.53BCBT03-8BCBT03-8-Bgl-1232.25±1.79BCBT03-9BCBT03-9-Bgl-1231.37±1.43BCBT03-10BCBT03-10-Bgl-1211.76±0.29BCBT03-11BCBT03-11-Bgl-1232.68±1.54BCBT03-12BCBT03-12-Bgl-1232.83±2.96BCBT03-13BCBT03-13-Bgl-1215.65±0.78BCBT03-14BCBT03-14-Bgl-1232.92±1.87BCBT03-15 (BCBT0529)BCBT03-15-Bgl-1233.87±2.65*n.d.: Enzyme activity is not detected.

[0056] On the basis of determining the optimal host cell, the expression element was further optimized, and the expression vector was modified by the combination of different promoters and different signal peptides to increase the expression level of lactase.

[0057] Based on the plasmid pHY-WZX as the backbone of the expression vector, three different constitutive promoters were selected, which were P cry (sequence 19, Bacillus thuringiensis insecticidal protein gene promoter), P amyL (sequence 20, Bacillus licheniformis amylase gene promoter), P 43 (sequence 21, Bacillus subtilis cytidine deaminase gene promoter) and 6 different signal peptides were selected, which were S amyL (sequence 22, Bacillus licheniformis amylase gene signal peptide), S aPrE (Sequence 23, Bacillus licheniformis alkaline protease signal peptide), S amyQ (sequence 24, Bacillus amyloliquefaciens amylase gene signal peptide), S amyE (sequence 25, Bacillus subtilis amylase gene signal peptide), S nprE (sequence 26, Bacillus licheniformis neutral protease gene signal peptide), S chi (sequence 27, Bacillus licheniformis chitinase gene signal peptide), replacing the original promoter and signal peptide on pHY-WZX with different combinations to construct 18 species new expression vectors pHSE-001-018 (see Table 6 for details). The BcBG168-D coding gene was cloned into the above 18 species new expression vectors and transformed into B. licheniformis BCBT0529 to obtain a series of recombinants, which were subjected to shake flask fermentation and determined enzyme activity. The results are summarized in Table 6. The tested 18 combinations of promoters and signal peptides can all mediate the secretion and expression of lactase BcBG168-D in B. licheniformis. Among them, the expression plasmid pHSE-008 (FIG. 3a), which is composed of a combination of the amylase promoter P amyL derived from theB. licheniformis and the signal peptide of the alkaline protease aprE, can mediate the highest enzyme expression, and the obtained lactase expression plasmid is pLEBG168 (The BcBG168-D coding gene was cloned into the expression plasmid pHSE-008, FIG. 3b); the obtained strain BCBTBc168D (The BcBG168-D coding gene sequence was cloned into the expression plasmid pHSE-008 and then transformed into Bacillus licheniformis host cell BCBT0529) expresses lactase activity of 53.79 U / mL in shake flask fermentation, more than 20 times of the enzyme production level of the wild strain, and more than 7 times that of the host cell before genetic modification and signal peptide optimization. Table 6 The effect of different promoter and signal peptide combinations on the expression of lactase BcBG168-DHostExpression plasmidExpression element assemblyLactase activityPromoterSignal peptide(U / mL)B2301 / / / 2.5 U / mLCCTCC NO: M208236pHY-WZXP amyL S amyL 7.57±0.24BCBT0529pHSE-001*P cry S amyL 23.35±1.82pHSE-002*S aprE 26.93±2.15pHSE-003*S amyQ 20.36±1.27pHSE-004*S amyE 23.78±1.93pHSE-005*S vprE 17.85±1.62pHSE-006*S chi 19.46±1.35pHSE-007(pHY-WZX )*P amyL S amyL 33.26±2.12pHSE-008S aprE 53.79±3.24pHSE-009*S amyQ 28.36±2.17pHSE-010*S amyE 25.58±1.97pHSE-011*S vprE 20.67±1.23pHSE-012*S chi 16.39±1.17pHSE-013*P 43 S amyL 30.68±2.17pHSE-014*S aprE 38.56±2.78pHSE-015*S amyQ 29.32±1.18pHSE-016*S amyE 23.93±1.69pHSE-017*S vprE 12.15±0.57pHSE-018*S chi 19.56±1.18*comparative examples Example 5: Lactase fermentation production process under 50 L fermentation system

[0058] The lactase high-producing strain BCBTBc168D was cultured at 37°C for 20-40 h, and 2-3 single colonies were picked and inoculated into 2 bottles of 5 L Erlenmeyer flasks containing 1000 mL LB liquid medium at 37°C, 230 r / min, and cultured on a shaker for 16 h, as seed liquid. Seed liquid was inoculated into a 50 L automatic fermentation tank containing 30 L fermentation medium (maltose syrup 4%, cottonseed powder 2.5%, soybean meal powder 3.5%, ammonium sulfate 0.5%, pH 6.5) according to the inoculum amount of 5%. The fermentation volume was 30 L. During the process, the dissolved oxygen was maintained at 0.1%-20% by adjusting the rotation speed and aeration, the fermentation temperature was 40-42°C, and the pH was controlled to 6.5±0.5, and 60% (w / w) maltose syrup was added as a carbon source, and the reducing sugar content was maintained at 0.5-5%. Regular sampling and analysis of the amount of residual sugar and enzyme activity were carried out, fermentation was performed to 120 hours, when the enzyme activity increase rate was less than 5 U / (mL·h), the fermentation was stopped for preparing the enzyme preparation.

[0059] The typical production process curve of lactase is shown in FIG. 4. The enzyme protein in the fermentation broth was the most important protein molecules (FIG. 5), and the highest lactase activity in fermentation broth reached 1131 U / mL (at 108 h), which was approximately 21 times of shake flask fermentation.

[0060] Similarly, BglD305-D and BglD-D were expressed in B. licheniformis BCBT0529 using pHSE-008 as the expression vector under the mediated combination of the Bacillus licheniformis amylase promoter P amyL and the signal peptide S aprE of the alkaline protease AprE, the lactase high-producing strains BCBT305D and BCBTatccD obtained respectively, under the above fermentation conditions, the lactase production levels reached 820 U / mL and 870 U / mL, respectively.Example 6: Fermentation production and preparation of lactase

[0061] According to the process of the 50 L fermentor in Example 5, the BCBTBc168D strain was used to prepare lactase under a 10-ton fermentation system after adjusting the operation process accordingly. After the fermentation, the lactase activity in the fermentation broth reached 2208 U / mL.

[0062] After the fermentation is finished, biological flocculant (polyacrylamide: basic aluminum chloride = 8:1) was added to the fermentation broth according to 1.0%, 2% diatomite TS-20# was added after the flocculation was completed, and then plate and frame filtration was carried out for sterilization. The membrane material with a molecular weight cut-off of 30 kDa was selected for ultrafiltration and concentration, and the operating pressure was 0.05 MPa at 40°C for 2h.

[0063] After ultrafiltration, 1% sodium benzoate, 1% potassium sorbate, 2% sodium chloride, 10% sorbitol, and 10% glycerin were added as a stabilizer for the liquid dosage form to obtain a liquid dosage form product.

[0064] The 10% glycerin in the above liquid dosage form stabilizer was replaced with 3% lactose and 2% sodium sulfate, and other components remain unchanged, and a solid dosage form product was prepared by spray drying.

[0065] The above percentages (%) are all w / v.Example 7: Application of lactase in the preparation of galactooligosaccharides

[0066] A lactose solution with a concentration of 600 g / L was used as a substrate, and the lactase BcBG168-D prepared by the invention was used to catalyze the preparation of galactooligosaccharides, and the total volume of the reaction system was about 30 L. The enzyme was added according to the substrate concentration of 20 U / g, the pH was adjusted to 6.0, the reacction system was stirred and reacted for 10 h in the reactor at 65°C at a stirring speed of 50 r / min.

[0067] The content of galactooligosaccharides in the reaction product reached more than 50%. FIG. 6 is a typical result of the sugar profile analysis of the galactooligosaccharides produced above by the HPLC detection method.

[0068] The above-mentioned embodiments only express several implementation modes of the present invention, and the description is relatively specific and detailed, but it should not be understood 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 the patent, the above-mentioned embodiments can be modified, combined, and improved, which belong to the scope of protection of the patent. Therefore, the scope of protection of this patent should be subject to the claims.

Claims

1. A recombinant strain containing the lactase encoding gene having the sequence of SEQ ID NO. 17, wherein, the expression vector used is pHSE-008, the expression host used by the recombinant strain is the mutant strain BCBT0529, which is obtained by knocking out aprE, vpr, wpr, lacR, lacA, lacA2, and yesZ genes from the genome of B. licheniformis CBB3008, numbered CCTCC NO.M208236; the GenBank accession numbers of aprE, vpr, wpr, lacR, lacA, lacA2, yesZ genes are: MT885340, MT885341, MT885342, MT885336, MT885337, MT885338, MT885339, respectively, wherein, the recombinant strain is specifically B. licheniformis BCBTBc168D, obtained by inserting the BcBG168-D coding gene shown in SEQ ID NO. 17 into the pHSE-008 plasmid, and expressed in the host cell mutant strain BCBT0529, wherein the expression vector pHSE-008 is obtained by integrating the amylase promoter PamyL derived from Bacillus licheniformis and signal peptide of alkaline protease aprE; the PamyL is shown in SEQ ID NO.20, and the signal peptide of the alkaline protease aprE is shown in SEQ ID NO.23 .

2. A method for fermentation and production of lactase by recombinant strain according to claim 1, <b>characterized in that: shaking flask fermentation to produce lactase: inoculating the recombinant strain to the shake flask culture medium, cultivating for 2 to 3 days at 30-45°C and 120-270 r / min; shaking flask culture medium composition: yeast extract 0.5-1.5%, peptone 1.2-3.6%, glucose 8-20%; pH 7.0.

3. The method for fermenting lactase by recombinant strain according to claim 1, characterized in that the fermentation tank is used to produce lactase: inoculating the strain to the fermentation tank culture medium according to the inoculum amount of 5%-10%; the fermentation temperature is 33-45°C during the fermentation, controlling dissolved oxygen at 0.1%-20%, pH at 6.0-7.8, adding 30%-60% maltose syrup and maintaining reducing sugar content at 0.1%-5%; fermentation lasts for 90-120 h, an end of fermentation is controlled that the increased value of fermentation enzyme activity is less than 5-20 U / (mL·h); the culture medium of the fermentation tank is composed of: 1% to 5% of maltose syrup, 0% to 5% of cottonseed powder, 0% to 5% of corn syrup, 0.5 to 5% of soybean meal powder, 0.1 to 5% of ammonium sulfate, and pH 6.0 to 8.0.

4. The method for producing lactase by recombinant strain fermentation according to claim 2 or 3, characterized in that, after the fermentation is finished, the strains are removed by plate and frame filtration, and then the enzyme solution is obtained after filtration through an ultrafiltration system.

5. The use of the recombinant strain of claim 1 in the production of lactase.