WO2021179652A1 - 一种低聚半乳糖生产专用酶及其制备与应用 - Google Patents

一种低聚半乳糖生产专用酶及其制备与应用 Download PDF

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WO2021179652A1
WO2021179652A1 PCT/CN2020/127693 CN2020127693W WO2021179652A1 WO 2021179652 A1 WO2021179652 A1 WO 2021179652A1 CN 2020127693 W CN2020127693 W CN 2020127693W WO 2021179652 A1 WO2021179652 A1 WO 2021179652A1
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lactase
phse
fermentation
recombinant
enzyme
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王正祥
牛丹丹
田康明
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天津科技大学
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Definitions

  • the invention belongs to the technical field of enzyme engineering, and specifically relates to a lactase producing galactooligosaccharide and its preparation and application.
  • Oligosaccharides also known as oligosaccharides, refer 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. Big category. Among them, functional oligosaccharides (functional oligosaccharides) refer to the polymerization of 2-10 identical or different monosaccharides with glycosidic bonds; they have the sweet taste and sensory characteristics shared by sugars, and can directly replace sucrose as sweet food ingredients.
  • oligosaccharides with physiological properties such as promoting the proliferation of bifidobacteria in the human body.
  • the glycosidic bond is not easy to be hydrolyzed and digested by hydrolytic enzymes in the human intestines and stomach due to the configuration of the anomeric carbon atom (C1 or C2) of the monosaccharide, which is also called non-digestible sugar.
  • Naturally occurring functional oligosaccharides such as galactooligosaccharides (GOS) present in human milk, cow milk, goat milk, etc.
  • GOS galactooligosaccharides
  • ⁇ -galactosidase to catalyze the industrialization of lactose to produce galacto-oligosaccharides.
  • Internationally, GOS products were successfully marketed in 1988.
  • lactose is mainly used as the raw material.
  • the transglycosylation of lactase is used to synthesize oligosaccharides with a degree of low polymerization containing one glucose or all galactose molecules connected during the hydrolysis of lactose, which can be expressed as Gal- (Gal)n-Glc/Gal (n is 1-4).
  • the lactase (a type of ⁇ -galactosidase, EC 3.2.1.23) used in the production of galactooligosaccharides can generate GOS through its transgalactosyl function, and is an enzyme with very commercial value in the dairy industry. .
  • lactase Commercially generally select Aspergillus niger (A.niger), Aspergillus oryzae (A.oryzae), Kluyveromyces lactis (Kluyveromyces lactis), K. fragilis (K.fragilis), Cryptococcus laurentii (Cryptococcus laurentii) ) And Bacillus circulans and other strains, through the submerged fermentation method to prepare lactase products.
  • the source of lactase is different from the production process of GOS.
  • Lactase derived from microorganisms such as Sulfolobus solfataricus, Saccharopolyspora rectivirgula, Pyrococcus furiosus and Thermotoga maritima can be found in Catalytic synthesis of GOS at a high temperature of 70°C-80°C.
  • the existing commercial lactase enzymes generally use lactose as a raw material to form 5%-50% galacto-oligosaccharides.
  • Lactase produced by Bacillus circulans ATCC 31382 (e.g. ), is the lactase with the strongest GOS synthesis ability so far.
  • This enzyme has 4 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 ⁇ -Gal-C and ⁇ -Gal-D are considered to be the most valuable for the production of GOS.
  • lactase identified from Bacillus circulans B2301 has a GOS that catalyzes the formation of 54.5% of lactose at a high temperature above 60°C, and is the lactase with the best GOS synthesis performance among all reported lactases (Zhao Jihua Etc., Food and Fermentation Industry, 2020).
  • the complete reading frame size of the Bacillus circulans B2301 lactase encoding gene is 5 133 bp, encoding 1 710 amino acid residues, without typical bacterial signal peptide sequences, and the highest consistency with the previously reported ⁇ -galactosidase is 93.6%;
  • This enzyme exhibits the highest catalytic activity at 60°C and pH 6.0-6.5.
  • Zn 2+ , Fe 2+ , Cu 2+ , EDTA and SDS show varying degrees of inhibition on the recombinase, and catalyze the synthesis of oligomers.
  • the V max of galactose is 2.47 g/(L ⁇ h) and the K m is 14.37 g/L (Tian Kangming et al., Food and Fermentation Industry, 2020).
  • the coding genes of Bacillus circulans lactase or its mutants were 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. It is difficult for this recombinant bacteria to secrete synthetic lactase into the fermentation broth, which increases the difficulty of preparing lactase enzyme preparations. When expressed in Pichiapastoris GS115, the expression level in shake flask fermentation can reach 70U/mL, but it is also difficult to realize the secretion and expression of lactase, and the separation and purification of the enzyme is extremely difficult.
  • the purpose of the present invention is to obtain a recombinant bacteria with good performance for large-scale fermentation production and ideal lactase synthesis and secretion capabilities on the basis of obtaining excellent galactooligosaccharide special enzyme molecules, which can significantly reduce the fermentation of lactase.
  • the manufacturing cost is simplified, the fermentation manufacturing process of lactase is simplified, and the quality of lactase is significantly improved.
  • one of the technical solutions of the present invention is to provide a variety of lactase enzymes, namely BglD305, BglD305-C, BglD305-D, BglD, BglD-C, BglD-D, BcBG168, BcBG168-C, BcBG168- D;
  • BglD305 comes from Bacillus circulans B2301, which was screened and isolated by the inventor (Zhao Jihua et al. Establishment and preliminary application of a rapid screening method for high transglycosidic activity lactase, Food and Fermentation Industry, 2020).
  • the amino acid sequence is shown in the sequence table SEQ ID NO .2;
  • BglD305-C and BglD305-D are truncated sequences of BglD305 respectively, and the amino acid sequence is shown in SEQ ID NO. 4 and SEQ ID NO. 6 in the sequence table;
  • BglD is from Bacillus circulans ATCC 31382, and the amino acid sequence is shown in SEQ ID NO. 8 in the sequence table; BglD-C and BglD-D are truncated sequences of BglD, respectively, and the amino acid sequence is shown in SEQ ID NO. 10 and As shown in SEQ ID NO.12;
  • BcBG168 is obtained by DNA shuffling modification of BglD305 and BglD
  • BcBG168-C and BcBG168-D are obtained by further deleting the C-terminal partial amino acid sequence on the basis of BcBG168; BcBG168, BcBG168-C and BcBG168-D
  • the amino acid sequence is shown in SEQ ID NO. 14, 16 and 18 in the sequence listing.
  • the second technical solution provided by the present invention is a recombinant vector or a recombinant strain containing the aforementioned lactase encoding gene;
  • the expression vector used in the recombinant vector includes but is not limited to pHY-WZX, pBL-WZX, pHY300plk, pUB110, pE194, pHT1469 (MoBiTec), pWH1520 (Rygus and Hillen, 1991);
  • the expression vector used in the recombinant vector includes, but is 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;
  • 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. Obtained from the signal peptide S aprE (SEQ ID NO.23) of the alkaline protease aprE;
  • the expression host adopted by the recombinant strain includes but is not limited to Bacillus subtilis, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Bacillus licheniformis, etc.;
  • 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, yesZ genes on the genome of Bacillus licheniformis CBB3008 (code CCTCC NO.M208236) ,
  • GenBank accession numbers corresponding to its gene sequence are: MT885340, MT885341, MT885342, MT885336, MT885337, MT885338, MT885339;
  • the present invention provides a recombinant strain with high lactase production-Bacillus 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 recombinant Bacillus licheniformis BCBTBc168D can reach 2208U/mL.
  • the present invention also provides a method for fermentation and production of lactase using the above-mentioned recombinant bacteria:
  • Shake flask culture medium yeast extract 0.5 to 1.5%, peptone 1.2 to 3.6%, glucose 8 to 20%; pH 7.0.
  • Fermentation to produce lactase in fermenter insert the strains into the fermenter culture medium according to the inoculation amount of 5%-10%; during the fermentation process, the fermentation temperature is 33 ⁇ 45°C, and the dissolved oxygen is controlled to 0.1% ⁇ 20%.
  • the pH is 6.0 ⁇ 7.8, 30% ⁇ 60%(w/w) maltose syrup is fed and the reducing sugar content is maintained at 0.1%-5%; the fermentation lasts for 90 ⁇ 120h, the fermentation process is regularly sampled and analyzed, and the fermentation end point is controlled as the fermentation enzyme
  • the live increase value is less than 5-20U/(mL ⁇ h).
  • the medium composition of the fermenter is: 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, pH 6.0 to 8.0.
  • the fermentation broth enzyme activity of the shake flask fermentation lactase can reach 25-54 U/mL; the fermentation broth enzyme activity of the lactase fermented in the fermentor can reach 826-2208 U/mL.
  • the bacteria are removed by plate and frame filtration, and then filtered by an ultrafiltration system to obtain an enzyme solution.
  • the present invention also provides the application of the above-mentioned lactase in the production of galactooligosaccharides.
  • the special enzyme preparation for the production of galacto-oligosaccharides in the present invention is a lactase with extremely high activity of catalyzing lactose to produce galacto-oligosaccharides obtained through gene cloning and artificial evolution; the lactase high-yielding strain of the present invention is selected by microbial strains.
  • the recombinant bacteria obtained by breeding can efficiently synthesize lactase during submerged fermentation and secrete enzyme protein molecules into the culture medium, directly prepare high-activity enzyme preparations from the fermentation supernatant, and apply them to the high-efficiency production of galactooligosaccharides.
  • the expression level of lactase can reach 2208 U/mL under the high-producing bacteria and fermentation process of lactase 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.
  • 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 1.3kb
  • Lane 3 is the mutant strain verified by the correct genome of the protease vpr deletion strain, and the PCR amplification size is 1.3kb;
  • Lane M is the 1kb molecular weight standard
  • Lane 1 is the electrophoresis 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 1.2kb
  • Lane 3 is The electrophoresis pattern confirmed by PCR after the successful knockout of gene lacA2 is 1.2kb
  • Lane 4 is the electrophoretic pattern confirmed by PCR after the successful knockout of gene yesZ, the size is 1.6kb;
  • Lane M is the protein molecular weight standard
  • Lane 1 is the result of direct electrophoresis of the fermentation broth
  • the arrow mark is the lactase in the fermentation broth
  • DP2 galactobiose or lactose
  • DP3 glucosylgalactobiose or galactotriose
  • DP4 glucosylgalactotriose or galactotetraose
  • DP5 glucosylgalactotetraose or half Lactopentaose.
  • the plasmid pHY-WZX used in the present invention is an existing technology, and its construction method has been disclosed in Niu DD, Wang ZX. Development of a pair of bifunctional expression vectors for Escherichia coli and Bacillus licheniformis. J Ind Microbiol Biotechnol (2007) 34: 357-362.DOI 10.1007/s10295-0204-x. The public can also obtain it through the Biocatalysis and Biotransformation Laboratory of the School of Chemical Engineering and Materials, Tianjin University of Science and Technology.
  • the Bacillus licheniformis CBB3008 used in the present invention has been deposited in the China Center for Type Culture Collection (CCTCC), with a deposit date of November 25, 2008, and a deposit number CCTCC NO: M208236.
  • CTCC China Center for Type Culture Collection
  • the present invention is based on two sources of lactase (BglD305 derived from Bacillus circulans B2301 and BglD derived from Bacillus circulans ATCC 31382) molecules as the basis for molecular evolution to obtain high efficiency in synthesizing galacto-oligosaccharides and good expression performance New lactase enzyme molecules (BglD305-C, BglD305-D, BglD-C, BglD-D, BcBG168, BcBG168-C, BcBG168-D);
  • a host cell classified as Bacillus licheniformis (Bacillus licheniformis) CBB3008, has been deposited in the Chinese Type Culture Collection, and the deposit number is CCTCC NO: M208236, and further genetic modification is carried out to knock out it that affects the expression of lactase Multiple genes (alkaline protease coding gene aprE, minor serine protease coding gene vpr, cell wall protease coding gene wpr, regulatory protein coding gene lacR, ⁇ -galactosidase coding gene lacA, ⁇ -galactosidase coding gene Gene lacA2, ⁇ -galactosidase encoding gene yesZ), to obtain a new host strain suitable for high-efficiency expression of lactase;
  • the present invention 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;
  • the coding gene (corresponding to nucleotide sequence 1, 3, 5, 7, 9, 11, 13, 15, 17) cloned into the expression vector constructed in the present invention, genetically transformed into Bacillus licheniformis host strain to obtain lactase-producing recombinant bacteria; establish and optimize fermentation conditions and processes Establish a new method of enzyme separation, purification and refining, and produce lactase products for industrial manufacturing of GOS.
  • the method for constructing a new strain with high lactase production of the present invention is to clone an expression vector through gene cloning technology and obtain an expression plasmid for lactase, and transform it into a new host strain of Bacillus licheniformis to obtain a recombinant strain, thereby realizing lactase High-efficiency secretion and expression; high-efficiency synthesis of lactase in the protein secretion system optimized by recombinant bacteria, and high-efficiency secretion of the synthesized lactase enzyme protein into the medium; through the fermentation production process, lactase is recovered and refined from the fermentation broth to obtain lactose Enzyme products.
  • the coding gene of Bacillus circulans lactase or its mutant is used as the target gene 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 ).
  • 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.
  • the chromosomal DNA extraction method was carried out according to the literature (Zhugejian Wang Zhengxiang. Manual of Industrial Microbiology Experiment Technology, China Light Industry Press, 1994).
  • the plasmid DNA was extracted using a certain concentration of lysozyme to lyse the cell wall using Sigma's plasmid small extraction kit.
  • DNA amplification was carried out in 0.2mL PCR thin-walled tubes.
  • the PCR amplification conditions are: 1 ⁇ (95°C5min); 30 ⁇ (94°C10s, 58°C30s, 72°C30 ⁇ 300s); 1 ⁇ (72°C10min).
  • the extension temperature and time of the PCR reaction are different. Unless otherwise specified, all PCR reactions are performed with Pfu DNA polymerase.
  • lactase The molecular evolution of lactase was carried out using 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.).
  • the lactase gene was partially digested with DNase, and the 100-200bp fragment was 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.
  • 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.
  • the correct transformants were verified by colony PCR verification, plasmid extraction enzyme digestion, and fermentation verification methods.
  • the general procedure is: using Bacillus licheniformis genomic DNA as a template, using apr-up1 (sequence 30) and apr-up2 (sequence 31) and primer apr-dn1 ( Sequence 32) and apr-dn2 (sequence 33) are primers to amplify the upper and lower homology arm fragments respectively to obtain the correct size PCR products and then use gel recovery for purification, and use gel recovery product DNA as a template for overlap By PCR, the deletion mutation box, ⁇ aprE, was obtained.
  • pT2 ts is based on T2(2)-ori (Chen Shouwen et al., Chinese invention patent, ZL201310562150.7) as the starting plasmid, using primer T2-1 (Sequence 28) and T2-2 (sequence 29) after reverse amplification, the PCR product is self-circularized and ligated to obtain the Sma I and Xba I sites of the new plasmid pT2 ts ), transformed into E.
  • deletion plasmid pT2- ⁇ aprE The LB plate containing 20 ⁇ g/mL kanamycin was cultured to obtain the correct deletion plasmid pT2- ⁇ aprE.
  • the deletion plasmid pT2- ⁇ aprE was transformed into Bacillus licheniformis host cells.
  • 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 the deletion, please refer to the above method, design and replace the primer according to the deleted gene sequence).
  • Shake flask fermentation to produce lactase Put 30mL fermentation medium (yeast extract 0.5 ⁇ 1.5%, peptone 1.2 ⁇ 3.6%, glucose 8 ⁇ 20%; pH 7.0) in a 250mL Erlenmeyer flask, inoculate recombinant bacteria, 30 ⁇ 45°C, Incubate at 120-270r/min for 2 to 3 days.
  • fermentation medium yeast extract 0.5 ⁇ 1.5%, peptone 1.2 ⁇ 3.6%, glucose 8 ⁇ 20%; pH 7.0
  • Fermentation tank to produce lactase The composition of the fermentation medium is: 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 is carried out in a 50L ⁇ 10t fermenter, with an inoculum amount of 5%-10%; during the fermentation process, the fermentation temperature is 33 ⁇ 45°C, the dissolved oxygen is controlled at 0.1% ⁇ 20%, and the pH is 6.0 ⁇ 7.8.
  • the bacteria are removed by plate and frame filtration, and then filtered by an ultrafiltration system to obtain the enzyme solution.
  • the enzyme activity determination of lactase is improved in accordance with the 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.
  • 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 an enzyme activity unit (U), expressed in U/mL or U/g.
  • the chromatographic conditions are: the mobile phase is 65% acetonitrile, the flow rate is 1.0mL/min; the TSK-GEL G3000PWXL-CP (7.8mm ⁇ 300mm, 7 ⁇ m) chromatographic column, the column temperature is 25°C; the evaporative light scattering detector, the drift tube temperature is 90 °C, the carrier gas flow rate is 2.2mL/min. 13.
  • the mobile phase is 65% acetonitrile
  • the flow rate is 1.0mL/min
  • the evaporative light scattering detector the drift tube temperature is 90 °C
  • the carrier gas flow rate is 2.2mL/min. 13.
  • Other analysis methods are: the mobile phase is 65% acetonitrile, the flow rate is 1.0mL/min; the TSK-GEL G3000PWXL-CP (7
  • Protease activity determination is carried out in accordance with the national standard method (GB/T 23527-2009);
  • the nucleotide sequence determination is carried out by the Sanger method
  • Glucose content is 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 Wang Zhengxiang. Manual of Industrial Microbiology Experiment Technology, China Light Industry Press, 1994).
  • the above recombinant plasmids were transformed into Bacillus licheniformis CCTCC NO: M208236 by the above-mentioned Bacillus licheniformis genetic transformation method, and corresponding transformants CBB-Bgl-1, CBB-Bgl-2, CBB-Bgl-3, CBB-Bgl- were obtained. 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, The flask fermentation was further carried out and the enzyme production (enzyme activity determination on the supernatant of the fermentation broth) was carried out. The main content and the enzyme production results are shown in Table 1.
  • the expressed enzyme activity of BcBG168 was 103.2% of BglD305 and 109.8% of BglD, respectively.
  • 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%.
  • the mutant cassette 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 ⁇ g/mL kanamycin to obtain Delete the plasmid pT2- ⁇ aprE correctly.
  • the deletion plasmid pT2- ⁇ aprE was transformed into the Bacillus licheniformis host cell CCTCC NO: M208236.
  • 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 and transformed correctly
  • the size of the PCR product is ⁇ 1.5kb ( Figure 1, lane 1).
  • Bacillus licheniformis CCTCC NO: M208236 ⁇ -galactosidase encoding gene lacA deletion Use the method similar to the above aprE gene deletion.
  • Bacillus licheniformis CCTCC NO: M208236 genomic DNA as template, 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 to amplify respectively
  • the upper and lower homology arm fragments are 486bp and 500bp in size, respectively.
  • the mutant cassette 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 ⁇ g/mL kanamycin to obtain Delete the plasmid pT2- ⁇ LacA correctly.
  • the deletion plasmid pT2- ⁇ LacA was transformed into Bacillus licheniformis.
  • the primers lacA-F and lacA-R sequence 40 and 41
  • the PCR product size of the correct transformant is ⁇ 1.2 kb ( Figure 2, lane 2).
  • vpr corresponds to the homology arm primer and the verification primer is the sequence 42-47
  • wpr corresponds to the homology arm primer and the verification primer is sequence 48-53
  • lacR corresponds to the homology arm primer and verification primer is the sequence 54-59
  • lacA2 corresponds to the homology arm primer and verification primer is the sequence 60-65
  • yesZ corresponds to the homology arm
  • the primers and verification primers were sequence 66-71, and different defective mutants were obtained.
  • mutant BCBT03-15 renamed BCBT0529, its genetic background is (CBB3008, ⁇ aprE, ⁇ vpr, ⁇ wpr, ⁇ lacR, ⁇ lacA, ⁇ lacA2, ⁇ yesZ ).
  • Example 3 The effect of knocking out some genes on host cell expression
  • Example 2 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 protease genes vpr and wpr, the total enzyme activity of proteolytic enzymes in the medium was deleted. The vitality drops to 10% of the wild type.
  • 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, and finally It reached 12.12 U/mL, which was 60% higher than the original strain.
  • Table 4 shows the changes in lactase activity of the host bacteria 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 gene aprE and the lactose operon repressor protein gene lacR, the lactase activity in the fermentation broth increased. After further deletion of the related endogenous lactase structural genes, the lactase activity in the fermentation broth was as low as Can not be measured according to existing methods.
  • the plasmid pHY-bgl-12 was transformed into the strains with different endogenous lactase gene deletions obtained in Example 2, and the recombinant bacteria were constructed and subjected to shake flask fermentation.
  • the lactase activity in the fermentation broth was determined. The results are shown in Table 5. .
  • BCBT03-15 that is, BCBT0529, as the host cell, the enzyme activity reached the highest, which is CCTCC NO: M208236 as the host cell 4.47 times the enzyme activity.
  • the expression element is further optimized, and the expression vector is modified by the combination of different promoters and different signal peptides to increase the expression level of lactase.
  • the obtained lactase expression plasmid is pLEBG168 (cloned into the expression plasmid pHSE-008 BcBG168-D coding gene, Figure 3b); the obtained strain BCBTBc168D (cloned into the expression plasmid pHSE-008 BcBG168-D coding gene sequence and then transformed into Bacillus licheniformis host cell BCBT0529 ) Expressing 53.79U/mL lactase activity during shake flask fermentation, which is more than 20 times the enzyme production level of the wild strain, and more than 7 times the host cell genetic modification and signal peptide optimization.
  • Example 5 Lactase fermentation production process under 50L fermentation system
  • the lactase high-producing strain BCBTBc168D was cultured at 37°C for 20-40 hours, and 2-3 single colonies were picked and inoculated into 2 bottles of 5L Erlenmeyer flasks containing 1000mL LB liquid medium at 37°C, 230r/min, and cultured on a shaker for 16 hours. Seed liquid is inoculated into a 50L fully automatic fermentation tank containing 30L 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 is 30L.
  • the dissolved oxygen is maintained at 0.1%-20% by adjusting the speed and aeration, the fermentation temperature is 40-42°C, and the pH is controlled at 6.5 ⁇ 0.5, and 60% (w/w) maltose syrup is added as the carbon source. , And maintain the reducing sugar content 0.5-5%.
  • the typical production process curve of lactase is shown in Figure 4.
  • the enzyme protein in the fermentation broth is the most important protein molecule (Figure 5), and the highest lactase activity in the supernatant of the fermentation broth reaches 1131 U/mL (at 108 h), which is approximately Shake flask fermentation level 21 times.
  • the BCBTBc168D strain prepared lactase under a 10 ton fermentation system after adjusting the operation process accordingly. After the fermentation, the lactase activity in the fermentation broth reached 2208U/mL.
  • liquid dosage form After ultrafiltration, 1% sodium benzoate, 1% potassium sorbate, 2% sodium chloride, 10% sorbitol, and 10% glycerin are added as a stabilizer for the liquid dosage form to obtain a liquid dosage form product.
  • a lactose solution with a concentration of 600g/L is used as a substrate, and the lactase BcBG168-D prepared by the invention is used to catalyze the preparation of galactooligosaccharides, and the total volume of the reaction system is about 30L.
  • the enzyme was added according to the substrate concentration of 20U/g, the pH was adjusted to 6.0, and the reaction temperature was stirred and reacted for 10h in the reactor at a stirring speed of 50r/min at 65°C.
  • Figure 6 is a typical result of the sugar profile analysis of the galactooligosaccharide produced above by the HPLC detection method.

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Abstract

提供了生成低聚半乳糖的乳糖酶及其制备和应用，该乳糖酶是以两种来源的乳糖酶分子为基础进行分子进化而获得的低聚半乳糖效率高、表达性能好的乳糖酶新酶分子。还提供了乳糖酶高产菌株，能够在深层发酵时高效合成乳糖酶并将酶蛋白分子分泌到培养基中，从发酵上清中直接制备高活性的酶制剂。

Description

一种低聚半乳糖生产专用酶及其制备与应用
本申请要求于2020年9月29日提交至中国专利局、申请号为CN2020110510561、发明名称为“一种低聚半乳糖生产专用酶及其制备与应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域：
本发明属于酶工程技术领域，具体涉及生成低聚半乳糖的乳糖酶及其制备和应用。
背景技术：
低聚糖又称寡糖类，是指单糖分子通过糖苷键连接的、聚合度为2-10的直链或支链碳水化合物，可简单分为功能性低聚糖和普通低聚糖两大类。其中，功能性低聚糖(Functional oligosaccharides)是指由2-10个相同或不同的单糖以糖苷键聚合而成；具有糖类共有的甜味和感官特性，可直接代替蔗糖作为甜食配料，但不被人体胃酸、胃酶降解，不在小肠吸收，可到达大肠；具有促进人体内的双歧杆菌增殖等生理特性的低聚糖。在功能性低聚糖中，因单糖的异头碳原子(C1或C2)构型使其糖苷键不易被人体肠胃中的水解酶水解消化，又称为非消化性糖。
天然存在的功能性低聚糖，如人乳、牛乳、羊乳等中存在的低聚半乳糖(Galactooligosaccharides，GOS)，是一种重要的益生元，对人类健康发挥了重要作用。上世纪50年代，已有报道利用β-半乳糖苷酶催化乳糖生产低聚半乳糖的工业化技术相关研究，国际上GOS产品于1988年成功上市。
低聚半乳糖的生产，主要以乳糖为原料，利用乳糖酶的转糖基作用在乳糖水解过程中合成含一个葡萄糖或全部为半乳糖分子连接的低聚合度的寡糖，可表示为Gal-(Gal)n-Glc/Gal(n为1-4)。应用于低聚半乳糖生产的乳糖酶(β-半乳糖苷酶的一种，EC 3.2.1.23)能够通过其转半乳糖基作用生成GOS，是一种在乳品工业中非常具有商业价值的酶。商业上一般选择黑曲霉(A.niger)、米曲霉(A.oryzae)、乳酸克鲁维酵母(Kluyveromyces lactis)、脆壁克鲁维酵母(K.fragilis)、罗伦特隐球菌(Cryptococcus laurentii)和环状芽胞杆菌(Bacillus circulans)等菌株，通过深层发酵方法来制备乳糖酶产品。乳糖酶的来源和GOS的生产工艺不同，尽管在酶活组成和转苷活性上均有一定差别，但催化合成的低聚半乳糖主要以β-1,3、β-1,4、β-1,6糖苷键连接，其中以β-1,4糖苷键为主。此外，针对酶 的耐高温性能，研究开发出一些新型的耐高温乳糖酶。如嗜热硫矿硫化叶菌(Sulfolobus solfataricus)、直杆糖多孢菌(Saccharopolyspora rectivirgula)、激烈火球菌(Pyrococcus furiosus)和海栖热袍菌(Thermotoga maritima)等微生物来源的乳糖酶，可以在70℃-80℃高温下催化合成GOS。现有形成商品供应的乳糖酶，一般可以乳糖为原料形成5％-50％的低聚半乳糖。环状芽胞杆菌ATCC 31382生产出的乳糖酶(如
Figure PCTCN2020127693-appb-000001
)，是目前为止合成GOS能力最强的乳糖酶，这个酶在酶制剂产品中有4中不同的存在形式(Song J,Abe K,Imanaka H,Imamura K,Minoda M,Yamaguchi S,and Nakanishi K,Biosci.Biotechnol.Biochem.,2011；75,268-278)，其中，β-Gal-C和β-Gal-D被认为是最有价值用于GOS的生产。此外，从环状芽胞杆菌B2301中鉴定出的乳糖酶，具有在60℃以上的高温条件下催化乳糖形成54.5％的GOS，是目前所有报道的乳糖酶中GOS合成性能最好的乳糖酶(赵继华等，食品与发酵工业，2020)。环状芽胞杆菌B2301乳糖酶编码基因的完整读框大小为5 133bp、编码1 710个氨基酸残基、不含典型细菌信号肽序列、与已有报道的β-半乳糖苷酶的最高一致性为93.6％；此酶在60℃和pH 6.0～6.5下表现出最高催化活性，Zn 2+、Fe 2+、Cu 2+、EDTA和SDS对重组酶表现出不同程度的抑制作用，催化合成低聚半乳糖的V max为2.47g/(L·h)，K m为14.37g/L(田康明等，食品与发酵工业，2020)。
但是，目前用环状芽胞杆菌发酵生产乳糖酶十分不经济，需要很长的发酵时间，通常需要96h-200h；产酶水平也比较低，通常仅能生产出2～5U/mL的乳糖酶(赵继华等，食品与发酵工业，2020)；酶制备技术复杂，乳糖酶大部分酶活存在于细胞内，需要通过复杂的细胞破碎方法将其释放，酶产品质量也因此收到较大的影响。
将环状芽胞杆菌乳糖酶或其突变体的编码基因，在多种宿主细胞进行了表达，以了解乳糖酶的表达水平。例如：在大肠杆菌中进行表达，乳糖酶的表达水平为1～3U/mL，这种重组菌很难将合成的乳糖酶分泌到发酵液中，增加了乳糖酶酶制剂的制备难度。在巴斯德毕赤酵母(Pichiapastoris)GS115中进行表达，摇瓶发酵表达水平可以达到70U/mL，但是同样很难实现乳糖酶的分泌表达，酶的分离纯化极为困难。
发明内容：
本发明的目的是在获得优良低聚半乳糖专用酶分子的基础上，获得具有良好实现大规模发酵生产性能并具有理想乳糖酶合成与分泌能力的重组菌，由此可显著降低乳糖酶的发酵制造成本、简化乳糖酶的发酵制造工艺并显著提高乳糖酶的质量。
为了实现上述目的，本发明的技术方案之一，是提供多种乳糖酶，分别为BglD305、BglD305-C、BglD305-D、BglD、BglD-C、BglD-D、BcBG168、BcBG168-C、BcBG168-D；
其中，BglD305来自发明人自行筛选分离的环状芽胞杆菌B2301(赵继华等.高转苷活性 乳糖酶快速筛选方法的建立与初步应用,食品与发酵工业，2020)，氨基酸序列如序列表SEQ ID NO.2所示；BglD305-C、BglD305-D分别为BglD305的截短序列，氨基酸序列如序列表SEQ ID NO.4和SEQ ID NO.6所示；
其中，BglD来自环状芽胞杆菌ATCC 31382，氨基酸序列如序列表SEQ ID NO.8所示；BglD-C、BglD-D分别为BglD的截短序列，氨基酸序列如序列表SEQ ID NO.10和SEQ ID NO.12所示；
其中，BcBG168为将BglD305和BglD经过DNAshuffling改造重排获得，BcBG168-C和BcBG168-D分别是在BcBG168的基础上进一步删除C-端部分氨基酸序列获得的；BcBG168、BcBG168-C和BcBG168-D的氨基酸序列如序列表SEQ ID NO.14、16和18所示。
本发明提供的技术方案之二，是含有上述乳糖酶编码基因的重组载体或重组菌株；
优选地，所述重组载体采用的表达载体包括但不限于pHY-WZX，pBL-WZX，pHY300plk，pUB110，pE194，pHT1469(MoBiTec)，pWH1520(Rygus and Hillen,1991)；
优选地，所述重组载体采用的表达载体包括但不限于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；
更优选地，所述重组载体采用的表达载体为pHSE-008质粒，是以pHY-WZX为基础表达载体的骨架，整合来源于地衣芽胞杆菌淀粉酶启动子P amyL(SEQ ID NO.20)和碱性蛋白酶aprE的信号肽S aprE(SEQ ID NO.23)所得；
优选地，所述重组菌株采用的表达宿主包括但不限于枯草芽胞杆菌、环状芽胞杆菌、巨大芽胞杆菌、短小芽胞杆菌、解淀粉芽胞杆菌、谷氨酸棒杆菌、地衣芽胞杆菌等；
更优选地，所述重组菌株采用的表达宿主为突变株BCBT0529，是在地衣芽胞杆菌CBB3008(编号CCTCC NO.M208236)的基因组上敲除aprE，vpr，wpr，lacR，lacA，lacA2，yesZ基因所得，其基因序列对应的GenBank登录号分别为：MT885340、MT885341、MT885342、MT885336、MT885337、MT885338、MT885339；
优选地，本发明提供一株高产乳糖酶的重组菌株-地衣芽胞杆菌BCBTBc168D，所述菌株是将BcBG168-D编码基因整合在pHSE-008质粒上，并在宿主细胞突变株BCBT0529中进行表达所得；采用地衣芽胞杆菌重组菌BCBTBc168D发酵制备乳糖酶BcBG168-D的表达水平可以达到2208U/mL。
本发明还提供采用上述重组菌发酵生产乳糖酶的方法：
(1)摇瓶发酵生产乳糖酶：接种重组菌至摇瓶培养基，30～45℃、120-270r/min下培养2～3天；
摇瓶培养基：酵母膏0.5～1.5％，蛋白胨1.2～3.6％，葡萄糖8～20％；pH 7.0。
(2)发酵罐发酵生产乳糖酶：将菌种按接种量为5％-10％接入发酵罐培养基；发酵过程中采用发酵温度33～45℃，控制溶氧为0.1％～20％，pH为6.0～7.8，流加30％～60％(w/w)麦芽糖浆并维持还原糖含量在0.1％-5％；发酵持续90～120h，发酵过程定时取样分析，发酵终点控制为发酵酶活增加值小于5-20U/(mL·h)。
发酵罐培养基组成为：麦芽糖浆1％～5％，棉籽粉0％～5％，玉米浆0％-4％，豆饼粉0.5～5％，硫酸铵0.1～5％，pH 6.0～8.0。
经发酵后，摇瓶发酵的乳糖酶的发酵液酶活可达25-54U/mL；发酵罐发酵的乳糖酶的发酵液酶活可达826-2208U/mL。
进一步地，发酵结束后，经板框过滤除去菌体，再经超滤系统过滤后获得酶液。
本发明还提供上述乳糖酶在生产低聚半乳糖中的应用。
有益效果：
本发明中的低聚半乳糖生产专用酶制剂为通过基因克隆与人工进化获得的、具有极高催化乳糖生成低聚半乳糖的活性的乳糖酶；本发明乳糖酶高产菌株为通过微生物菌种选育获得的重组菌，能够在深层发酵时高效合成乳糖酶并将酶蛋白分子分泌到培养基中，从发酵上清中直接制备高活性的酶制剂，应用于低聚半乳糖的高效生产。
本发明的乳糖酶高效制备方法，在本发明提供的乳糖酶高产菌和发酵工艺下，乳糖酶的表达水平可以达到2208U/mL。本发明有助于降低乳糖酶的发酵制造成本、简化发酵制造工艺并提高乳糖酶酶制剂的质量。
附图说明：
图1.蛋白酶编码基因敲除的验证图谱
泳道M为1kb分子量标准；泳道1为aprE基因删除后菌株基因组验证正确的突变菌株，PCR扩增大小1.5kb；泳道2为蛋白酶wpr基因删除后菌株基因组验证正确的突变菌株，PCR扩增大小为1.3kb；泳道3为蛋白酶vpr删除菌株基因组验证正确的突变菌株，PCR扩增大小为1.3kb；
图2.内源性乳糖酶等编码基因敲除的验证图谱
泳道M为1kb分子量标准；泳道1为基因lacR成功敲除后PCR确认的电泳图谱，大小为1.3kb；泳道2为基因lacA成功敲除后PCR确认的电泳图谱，大小为1.2kb；泳道3为基因lacA2成功敲除后PCR确认的电泳图谱，大小为1.2kb；泳道4为基因yesZ成功敲除后PCR确认的电泳图谱，大小为1.6kb；
图3.表达载体的物理图谱
a：优化后的表达载体pHES-008；b：乳糖酶表达质粒pLEBG168；
图4.乳糖酶发酵产酶曲线图；
图5.发酵液的蛋白电泳图谱
泳道M为蛋白质分子量标准；泳道1为发酵液直接电泳的结果图，箭头标记部位为发酵液中的乳糖酶；
图6.乳糖酶作用乳糖后生成的低聚半乳糖HPLC糖谱图
DP2：半乳二糖或乳糖，DP3：葡糖基半乳二糖或半乳三糖；DP4：葡糖基半乳三糖或半乳四糖；DP5：葡糖基半乳四糖或半乳五糖。
具体实施方式：
为了使本专利的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本专利进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本专利，并不用于限定本发明。
本发明所使用的质粒pHY-WZX为现有技术，其构建方法已公开在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.公众也可通过天津科技大学化工与材料学院生物催化与生物转化实验室获得。
本发明所采用的地衣芽胞杆菌(Bacillus licheniformis)CBB3008，已保藏于中国典型培养物保藏中心(简称CCTCC)，保藏日期2008年11月25日，保藏编号CCTCC NO：M208236。
本发明以两种来源的乳糖酶(来源于环状芽胞杆菌B2301的BglD305和来源于环状芽胞杆菌ATCC 31382的BglD)分子为基础进行分子进化，获得合成低聚半乳糖效率高、表达性能好的乳糖酶新酶分子(BglD305-C、BglD305-D、BglD-C、BglD-D、BcBG168、BcBG168-C、BcBG168-D)；
本发明对一种宿主细胞，其分类命名为地衣芽胞杆菌(Bacillus licheniformis)CBB3008，已保藏于中国典型培养物保藏中心，保藏编号CCTCC NO：M208236，进一步进行遗传改造，敲除其中影响乳糖酶表达的多个基因(碱性蛋白酶编码基因aprE，次要丝氨酸蛋白酶编码基因vpr，胞壁蛋白酶编码基因wpr，调控蛋白编码基因lacR，β-半乳糖苷酶编码基因lacA，β-半乳糖苷酶编码基因lacA2，β-半乳糖苷酶编码基因yesZ)，获得适合乳糖酶高效表达的新宿主菌株；
本发明构建并优化适合乳糖酶分泌表达的表达载体，为在pHY-WZX的基础上优化构建，表达载体中有引导乳糖酶高表达的优选启动子和高效介导乳糖酶分泌表达的信号肽；
本发明将环状芽胞杆菌(B.circulans)乳糖酶或其突变体(氨基酸序列2、4、6、8、 10、12、14、16、18)的编码基因(对应核苷酸序列1、3、5、7、9、11、13、15、17)克隆入本发明所构建的表达载体中，遗传转化入地衣芽胞杆菌宿主菌株中获得产乳糖酶重组菌；建立并优化发酵条件与工艺，建立新型酶分离纯化与精制方法，生产出乳糖酶产品，用于GOS的工业化制造。
本发明用于乳糖酶高产新菌种的构建方法，是通过基因克隆技术在表达载体中进行克隆和获得乳糖酶的表达质粒，并转化入地衣芽胞杆菌新宿主菌中获得重组菌，实现乳糖酶的高效分泌表达；在通过重组菌优化的蛋白分泌系统高效合成乳糖酶，将合成的乳糖酶酶蛋白高效分泌到培养基中；通过发酵生产工艺，从发酵液中回收、精制乳糖酶，获得乳糖酶产品。
本发明采用的主要实验方法如下：
1、基因克隆、分子进化与表达质粒的构建
常规分子克隆操作参考文献方法(Sambrook等。Molecular Cloning：A Laboratory Manual,1989)进行。环状芽胞杆菌乳糖酶或其突变体的编码基因作为本发明中的目标基因；基础表达载体为pHY-WZX(王正祥，牛丹丹。中国发明专利，ZL200510051648；Niu&Wang.J Ind Microbiol Biotechnol,2007)。乳糖酶或其突变体的编码基因经PCR扩增后克隆入表达载体中，获得表达乳糖酶的系列重组质粒。
2、染色体DNA的提取
染色体DNA提取方法按文献(诸葛键王正祥。工业微生物实验技术手册，中国轻工业出版社，1994)进行。
3、质粒DNA的提取
质粒DNA的提取在用一定浓度的溶菌酶裂解细胞壁后使用Sigma公司的质粒小提试剂盒进行。
4、基因扩增
DNA扩增在0.2mL PCR薄壁管中进行。PCR扩增条件为：1×(95℃5min)；30×(94℃10s，58℃30s，72℃30～300s)；1×(72℃10min)。依据不同扩增长度，PCR反应的延伸温度和时间有所不同。除特别注明外，所有PCR反应皆使用Pfu DNA聚合酶进行。
5、酶分子人工进化
乳糖酶的分子进化按照文献方法(Stemmer W P C.Rapid evolution of a protein in vitro by DNA shuffling[J].Nature,1994,370(6488):389-391.)，采用DNAshuffling进行。首先将乳糖酶基因用DNA酶进行部分酶切，通过密度梯度法回收100～200bp的片段，混合后不加引物进行基因扩增15～25个循环，然后加入两端特异性引物，对全长基因进行扩增；用PCR产物纯化试剂盒(Sigma)进行纯化，纯化后的DNA克隆入表达载体pHY-WZX中，采用 CaCl 2法(诸葛健王正祥编著工业微生物实验技术手册，中国轻工业出版社，1994)转化入大肠杆菌JM109中；测定乳糖酶酶活并进行比较。
6、重叠PCR
参照文献(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)进行。通用步骤为：用片段F1和片段F2的引物(P1+P2；P3+P4，引物P2和P3为反向互补的序列)介导PCR的扩增，获得基因片段；胶回收纯化扩增片段F1和F2；将纯化得到的两个片段F1和F2稀释适当倍数，以1:1摩尔比混合作为模板，以引物P1+P4介导新的PCR反应，获得全长序列。
7、地衣芽胞杆菌遗传转化
参照文献(徐敏,马骏双,王正祥。高渗透压对细菌电转化率的影响。无锡轻工大学学报，2004(04):98-100)介绍的方法进行。主要步骤如下：接种新鲜单菌落到液体LB培养基中，37℃ 200r/min过夜培养，转接5％菌液到新的LB培养基中继续培养至OD600为0.75-0.90。菌体冰浴10min后4℃、6000r/min离心10min收集菌体。用预冷的电转洗液(0.5mol/L山梨醇，0.5mol/L的甘露醇和10％甘油)反复洗涤细胞4次。将细胞沉淀悬浮于1mL预冷的电转洗液中，完成感受态细胞制备。取1μL质粒DNA和约100μL感受态细胞混匀，立即电击(1800v,5ms)，随即加入电转复苏液(含0.65mol/L山梨醇和0.45mol/L甘露醇的LB培养基)，37℃、160r/min复苏后涂布对应抗性的LB平板，于合适温度培养至单菌落长出。正确转化子经菌落PCR验证、质粒提取酶切、发酵验证功能等方法验证。
8、地衣芽胞杆菌特定基因的删除
参考文献方法(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)进行。以地衣芽胞杆菌CCTCC NO：M208236中aprE基因删除为例的通用步骤为：以地衣芽胞杆菌基因组DNA为模板，利用apr-up1(序列30)及apr-up2(序列31)和引物apr-dn1(序列32)及apr-dn2(序列33)为引物，分别扩增上、下两个同源臂片段，得到正确大小的PCR产物后分别采用胶回收进行纯化，以胶回收产物DNA为模板进行重叠PCR，获得删除突变盒，△aprE。将该突变盒纯化后Xba I酶切后，克隆入质粒pT2 ts(pT2 ts是以T2(2)-ori(陈守文等，中国发明专利，ZL201310562150.7)为出发质粒，采用引物T2-1(序列28)、T2-2(序列29)进行反向扩增后，PCR产物自身环化、连接获得新质粒pT2 ts)的Sma I和Xba I位点，转化大肠杆菌JM109感受态细胞，在含20μg/mL卡那霉素的LB平板进行培养，获得正确的删除质粒pT2-△aprE。按照地衣芽胞杆菌的遗传转化方法中描述的步骤，将 删除质粒pT2-△aprE转化入地衣芽胞杆菌宿主细胞。经两次同源重组以后，以同源臂两侧设计引物apr-F：(序列34)和apr-R(序列35)，并以这对引物进行菌落PCR，进行转化子的验证(其他基因的删除可参考上述方法，根据删除的基因序列设计并替换引物即可)。
9、发酵试验
摇瓶发酵生产乳糖酶：在250mL三角瓶中装30mL发酵培养基(酵母膏0.5～1.5％，蛋白胨1.2～3.6％，葡萄糖8～20％；pH 7.0)，接种重组菌，30～45℃、120-270r/min下培养2～3天。
发酵罐发酵生产乳糖酶：发酵培养基组成为：麦芽糖浆1％～5％，棉籽粉0％～5％，玉米浆0％-4％，豆饼粉0.5～5％，硫酸铵0.1～5％，pH 6.0～8.0；发酵在50L～10t发酵罐中进行，接种量为5％-10％；发酵过程中采用发酵温度33～45℃，控制溶氧为0.1％～20％，pH为6.0～7.8，流加30％～60％(w/w)麦芽糖浆并维持还原糖含量在0.1％-5％；发酵持续到90～120h，发酵过程定时取样分析，发酵终点控制为发酵酶活增加值小于5-20U/(mL·h)。
10、乳糖酶酶制剂的制备方法
发酵结束后，经板框过滤除去菌体，再经超滤系统过滤后获得酶液。
11、乳糖酶酶活测定
乳糖酶的酶活测定，按照国标GB/T 33409-2016进行改进。一般过程是，反应在pH5.0和40℃下进行，以乳糖为底物。采用生物传感仪测定葡萄糖的释放量。
乳糖酶的酶活定义为：在pH 5.0和40℃下每分钟分解乳糖生成1微摩尔葡萄糖所需的酶量，定义为一个酶活力单位(U)，以U/mL或U/g表示。
12、低聚半乳糖的合成与产物分析
采用300g/L～800g/L乳糖为底物，添加5U/g～20U/g的乳糖酶，反应在50℃-70℃下进行，定时取样。反应原料和低聚半乳糖形成与含量的分析，采用HPLC法分析酶促产物特征与生成。色谱条件为：流动相为65％的乙腈，1.0mL/min流速；TSK-GEL G3000PWXL-CP(7.8mm×300mm，7μm)色谱柱，柱温25℃；蒸发光散射检测器，漂移管温度90℃，载气流速2.2mL/min。13、其它分析方法
蛋白酶酶活测定，按照国标法(GB/T 23527-2009)进行；
基因和氨基酸序列比对，采用DNAMAN软件进行；
核苷酸序列测定采用Sanger方法进行；
序列蛋白含量按文献方法(Bradford.Anal Chem,1976)进行；
葡萄糖含量用酶电极法测定(SBA-90,山东)；
细胞密度用分光光度计(UV-2000，美国)在600nm下测定；
蛋白电泳按文献方法进行(诸葛键王正祥。工业微生物实验技术手册，中国轻工业出版社，1994)。
以下将通过具体实施例对本发明做进一步的解释说明。
实施例1：乳糖酶的分子进化
以序列表SEQ ID NO.1和SEQ ID NO.7所示的BglD305和BglD编码基因为模板，采用DNAshuffling方法对其进行分子进化。经过酶活筛选后，获得了酶活水平显著提高的乳糖酶酶分子BcBG168(核苷酸序列SEQ ID NO.13)，其氨基酸序列(氨基酸序列SEQ ID NO.14)。
通过对BglD305、BglD和BcBG168的编码基因进行不同程度的截短，并对改造序列和原序列进行高效表达，通过PCR扩增技术扩增相应的基因序列，并克隆入表达载体pHY-WZX中，获得乳糖酶表达质粒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。以上述地衣芽胞杆菌遗传转化方法将上述重组质粒分别转化到地衣芽胞杆菌CCTCC NO：M208236，获得相应的转化子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，进一步进行摇瓶发酵并进行产酶(对发酵液上清进行酶活测定)情况分析，主要内容与产酶结果见表1。
在相同条件下，BcBG168的表达酶活分别是BglD305的103.2％和BglD的109.8％。
乳糖酶的C端截短的乳糖酶在同样的表达条件下均呈现上升趋势，酶活力较原始基因序列提高40％、78％；35％、69％和31％，70％。
表1不同来源及C端截短的乳糖酶的表达效率
Figure PCTCN2020127693-appb-000002
实施例2：表达宿主细胞的遗传修饰
地衣芽胞杆菌CCTCC NO：M208236中aprE基因的删除。以地衣芽胞杆菌CCTCC NO：M208236基因组DNA为模板，利用apr-up1(序列30)及apr-up2(序列31)和引物apr-dn1(序列32)及apr-dn2(序列33)为引物，分别扩增上下两个同源臂片段，大小分别为667bp和495bp。得到正确大小的PCR产物后分别采用胶回收进行纯化，以胶回收产物DNA为模板进行重叠PCR，获得大小为～1.2kb的删除突变盒△aprE。将该突变盒纯化后Xba I酶切后，克隆入质粒pT2 ts的Sma I和Xba I位点，转化大肠杆菌JM109感受态细胞，在含20μg/mL卡那霉素的LB平板进行培养，获得正确的删除质粒pT2-△aprE。按照“地衣芽胞杆菌的遗传转化”方法中描述的步骤，将删除质粒pT2-△aprE转化入地衣芽胞杆菌宿主细胞CCTCC NO：M208236。经两次同源重组以后，以同源臂两侧设计引物apr-F(序列34)和apr-R(序列35)，并以这对引物进行菌落PCR验证获得正确的转化子BCBT01，正确转化子的PCR产物大小为～1.5kb(图1，泳道1)。
地衣芽胞杆菌CCTCC NO：M208236β-半乳糖苷酶编码基因lacA的删除。采用上述aprE基因删除相似的方法进行。以地衣芽胞杆菌CCTCC NO：M208236基因组DNA为模板，利用lacA-up1及lacA-up2(序列36和序列37)和引物lacA-dn1及lacA-dn2(序列38和序列39)为引物，分别扩增上下两个同源臂片段，大小分别为486bp和500bp。得到正确大小的PCR产物后分别采用胶回收进行纯化，以胶回收产物DNA为模板进行重叠PCR，获得大小为936bp的删除突变盒△lacA。将该突变盒纯化后Xba I酶切后，克隆入质粒pT2 ts的Sma I和Xba I位点，转化大肠杆菌JM109感受态细胞，在含20μg/mL卡那霉素的LB平板进行培养，获得正确的删除质粒pT2-△LacA。按照“地衣芽胞杆菌的遗传转化”方法中描述的步骤，将删除质粒pT2-△LacA转化入地衣芽胞杆菌。经两次同源重组以后，以同源臂两侧引物lacA-F和lacA-R(序列40和序列41)，进行菌落PCR验证获得正确的转化子，正确转化子的PCR产物大小为～1.2kb(图2，泳道2)。
采用上述相似地方法，对地衣芽胞杆菌CCTCC NO：M208236基因组中的vpr、wpr、lacR、lacA2和yesZ基因进行不同组合的删除，其中，vpr对应同源臂引物及验证引物为序列42-47；wpr对应同源臂引物及验证引物为序列48-53；lacR对应同源臂引物及验证引物为序列54-59；lacA2对应同源臂引物及验证引物为序列60-65；yesZ对应同源臂引物及验证引物为序列66-71，获得不同缺陷型的突变株，其中突变株BCBT03-15,重新命名为BCBT0529，其遗传背景为(CBB3008，ΔaprE，Δvpr，Δwpr，ΔlacR，ΔlacA，ΔlacA2，ΔyesZ)。
实施例3敲除部分基因对宿主细胞表达的影响
(1)部分蛋白酶敲除对胞外蛋白酶酶活的影响
对实施例2获得的不同突变株进行摇瓶发酵试验，并分析其胞外蛋白酶酶活。如表2所示，在删除碱性蛋白酶编码基因aprE后，培养基中蛋白水解酶的总酶活降低了80％，进一步删除两种蛋白酶基因vpr、wpr后培养基中蛋白水解酶的总酶活力降至野生型的10％。
表2突变菌株碱性蛋白酶活力测定
Figure PCTCN2020127693-appb-000003
(2)部分蛋白酶敲除对乳糖酶表达的影响
将实施例1中获得的产酶活力最高的重组菌CBB-Bgl-12中携带的表达质粒pHY-bgl-12转化入实施例2中获得的蛋白质水解酶基因删除的宿主菌中，获得相应的重组菌，以CCTCC NO：M208236为对照，重组菌摇瓶发酵结果见表3。删除碱性蛋白酶基因aprE后发酵液中乳糖酶酶活力明显增加，达到10.68U/mL，提高了41.08％；删除另外两个蛋白水解酶基因vpr、wpr后发酵液中乳糖酶活力进一步提高，最终达到了12.12U/mL，较出发菌株提高了60％。
表3删除蛋白酶后乳糖酶的表达水平
Figure PCTCN2020127693-appb-000004
(3)部分基因敲除对乳糖酶表达的影响
宿主菌在其内源性乳糖酶相关基因突变后，在摇瓶发酵条件下的乳糖酶酶活变化如表4所示。可以看到，在删除碱性蛋白酶基因aprE和乳糖操纵子阻遏蛋白基因lacR后发酵液中乳糖酶活力有所提高，进一步删除相关内源性乳糖酶结构基因后，发酵液中乳糖酶活力低至按照现有方法测定不到。
表4内源性乳糖酶基因突变菌株乳糖酶酶活力
Figure PCTCN2020127693-appb-000005
Figure PCTCN2020127693-appb-000006
*n.d.:酶活未检测到
实施例4：乳糖酶的高效表达
在实施例2中获得的不同内源性乳糖酶基因删除的菌株中转入质粒pHY-bgl-12，构建重组菌并进行摇瓶发酵，测定发酵液中乳糖酶活力，结果如表5所示。宿主细胞内源性乳糖酶相关基因删除后，发现本发明的乳糖酶的表达水平大幅度提升，其中BCBT03-15，即BCBT0529作为宿主细胞时，酶活达到最高，是CCTCC NO：M208236作为宿主细胞酶活的4.47倍。
表5内源性乳糖酶删除后乳糖酶的表达水平
Figure PCTCN2020127693-appb-000007
Figure PCTCN2020127693-appb-000008
*n.d.：酶活未检测到。
在确定最优宿主细胞的基础上，进一步进行表达元件的优化，采用不同启动子与不同信号肽的组合对表达载体进行改造，提高乳糖酶的表达水平。
以质粒pHY-WZX为基础表达载体的骨架，选用3种不同的组成型启动子，其分别为P cry(序列19，苏云金芽胞杆菌杀虫蛋白基因启动子)、P amyL(序列20，地衣芽胞杆菌淀粉酶基因启动子)、P 43(序列21，枯草芽胞杆菌胞苷脱氨酶基因启动子)以及6种不同的信号肽，其分别为S amyL(序列22，地衣芽胞杆菌淀粉酶基因信号肽)、S aprE(序列23，地衣芽胞杆菌碱性蛋白酶信号肽)、S amyQ(序列24，解淀粉芽胞杆菌淀粉酶基因信号肽)、S amyE(序列25，枯草芽胞杆菌淀粉酶基因信号肽)、S nprE(序列26，地衣芽胞杆菌中性蛋白酶基因信号肽)、S chi(序列27，地衣芽胞杆菌几丁质酶基因信号肽)，以不同的组合方式替换pHY-WZX上原有的启动子和信号肽，构建成18种新的表达载体pHSE-001～018(具体见表6)，克隆入BcBG168-D编码基因序列后转化地衣芽胞杆菌宿主细胞BCBT0529，获得系列重组菌，进行摇瓶发酵并测定酶活力，结果汇总于表6。所测试的18种启动子和信号肽组合形式，皆能介导乳糖酶BcBG168-D在地衣芽胞杆菌中分泌表达。其中，来源于地衣芽胞杆菌淀粉酶启动子P amyL和碱性蛋白酶aprE的信号肽组合构成的表达质粒pHSE-008(图3a)，能够介导最高的酶表达，所获得的乳糖酶表达质粒为pLEBG168(在表达质粒pHSE-008中克隆入BcBG168-D编码基因，图3b)；所获得的菌株BCBTBc168D(在表达质粒pHSE-008中克隆入BcBG168-D编码基因序列后转化地衣芽胞杆菌宿主细胞BCBT0529)在摇瓶发酵时表达53.79U/mL的乳糖酶酶活，是野生菌株产酶水平的20倍以上，是宿主细胞遗传改造和信号肽优化前的7倍以上。
表6不同的启动子信号肽组合对乳糖酶BcBG168-D表达的影响
Figure PCTCN2020127693-appb-000009
Figure PCTCN2020127693-appb-000010
实施例5：50L发酵体系下的乳糖酶发酵生产工艺
将乳糖酶高产菌株BCBTBc168D在37℃培养20～40h，挑取2-3个单菌落接种于2瓶装有1000mL LB液体培养基的5L三角瓶中，37℃、230r/min，摇床培养16h作为种子液，按照5％接种量接种于装有30L发酵培养基(麦芽糖浆4％，棉籽粉2.5％，豆饼粉3.5％，硫酸铵0.5％，pH6.5)的50L全自动发酵罐中，工作发酵体积30L，过程中通过调节转速、通气量维持溶氧为0.1％～20％，发酵温度40-42℃，并控制pH 6.5±0.5，流加60％(w/w)麦芽糖浆作为碳源，并维持还原糖含量0.5-5％。定时取样分析残糖量和酶活力，发酵至120小时，酶活力增加速度低于5U/(mL·h)停止发酵，下罐并制备酶制剂。
乳糖酶的典型产酶进程曲线见图4，发酵液中酶蛋白为最主要的蛋白分子(图5)，发酵液上清中的乳糖酶酶活最高达到1131U/mL(108h时)，约为摇瓶发酵水平的21倍。
相似地，将BglD305-D和BglD-D在地衣芽胞杆菌淀粉酶启动子P amyL和碱性蛋白酶AprE的信号肽S aprE组合介导下于以pHSE-008为表达载体在地衣芽胞杆菌BCBT0529中进行表达，分别获得的乳糖酶高产菌株BCBT305D和BCBTatccD，在上述发酵条件下的乳糖酶产酶水平，分别达到820U/mL和870U/mL。
实施例6：乳糖酶的发酵生产制备
按照实施例5中50L发酵罐的工艺，相应调整操作流程后BCBTBc168D菌株在10吨发酵体系下制备乳糖酶。发酵结束后，发酵液中乳糖酶酶活达到2208U/mL。
发酵结束后，发酵液中按照1.0％加入生物絮凝剂(聚丙烯酰胺:碱式氯化铝＝8:1)，絮凝完成后添加2％的硅藻土TS-20#，然后进行板框过滤除菌。选择截留分子量为30kDa的膜材料进行超滤浓缩，40℃条件下，操作压力0.05MPa，工作2h。
超滤后添加1％苯甲酸钠，1％山梨酸钾，2％氯化钠，10％山梨醇，10％甘油作为液体剂型的稳定剂，获得液体剂型产品。
将上述液体剂型稳定剂中的10％的甘油替换为3％的乳糖和2％的硫酸钠，其他组分不变，经喷雾干燥制备固体剂型产品。
上述百分比(％)均为w/v。
实施例7：乳糖酶在低聚半乳糖制备中的应用
以浓度为600g/L的乳糖溶液为底物，用本发明制备的乳糖酶BcBG168-D催化制备低聚半乳糖，反应体系为总体积约为30L。按照20U/g底物浓度添加酶，调整pH为6.0，反应温度在65℃下于反应釜中以50r/min的搅拌速度搅拌并反应10h。
反应产物中低聚半乳糖的含量达到50％以上。图6为通过HPLC检测方法对上述所生产出的低聚半乳糖产品糖谱分析的典型结果。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。

Claims (13)

  1. 一种乳糖酶，其特征在于，所述乳糖酶氨基酸序列如序列表SEQ ID NO.2、4、6、8、10、12、14、16或18所示。
  2. 权利要求1所述的一种乳糖酶，其特征在于，所述乳糖酶编码基因如序列表SEQ ID NO.1、3、5、7、9、11、13、15或17所示。
  3. 含有权利要求2所述乳糖酶编码基因的重组载体或重组菌株。
  4. 如权利要求3所述的重组载体，其特征在于，采用的表达载体包括但不限于pHY-WZX，pBL-WZX，pHY300plk，pUB110，pE194，pHT1469，pWH1520；
    或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。
  5. 如权利要求3所述的重组载体，其特征在于，采用的表达载体pHSE-018是以pHY-WZX为基础表达载体的骨架，整合来源于地衣芽胞杆菌淀粉酶启动子P amyL和碱性蛋白酶aprE的信号肽所得；
    所述P amyL如SEQ ID NO.20所示，所述碱性蛋白酶aprE的信号肽如SEQ ID NO.23所示。
  6. 如权利要求3所述的重组菌株，其特征在于，所述重组菌株采用的表达宿主包括但不限于枯草芽胞杆菌、环状芽胞杆菌、巨大芽胞杆菌、短小芽胞杆菌、解淀粉芽胞杆菌、谷氨酸棒杆菌、地衣芽胞杆菌。
  7. 如权利要求6所述的重组菌株，其特征在于，所述重组菌株采用的表达宿主为突变株BCBT0529，是在地衣芽胞杆菌CBB3008，编号CCTCC NO.M208236的基因组上敲除aprE，vpr，wpr，lacR，lacA，lacA2，yesZ基因所得；
    aprE，vpr，wpr，lacR，lacA，lacA2，yesZ基因的GenBank登录号分别为：MT885340、MT885341、MT885342、MT885336、MT885337、MT885338、MT885339。
  8. 如权利要求7所述的重组菌株，其特征在于，具体为地衣芽胞杆菌BCBTBc168D，是将SEQ ID NO.17所示的BcBG168-D编码基因整合在pHSE-008质粒上，并在宿主细胞突变株BCBT0529中进行表达所得。
  9. 权利要求3所述重组菌发酵生产乳糖酶的方法，其特征在于，摇瓶发酵生产乳糖酶：接种重组菌至摇瓶培养基，30～45℃、120-270r/min下培养2～3天；
    摇瓶培养基组成：酵母膏0.5～1.5％，蛋白胨1.2～3.6％，葡萄糖8～20％；pH 7.0。
  10. 权利要求3所述重组菌发酵生产乳糖酶的方法，其特征在于，发酵罐发酵生产乳糖 酶：将菌种按接种量为5％-10％接入发酵罐培养基；发酵过程中采用发酵温度33～45℃，控制溶氧为0.1％～20％，pH为6.0～7.8，流加30％～60％麦芽糖浆并维持还原糖含量在0.1％-5％；发酵持续90～120h，发酵终点控制为发酵酶活增加值小于5-20U/(mL·h)。
    发酵罐培养基组成为：麦芽糖浆1％～5％，棉籽粉0％～5％，玉米浆0％-4％，豆饼粉0.5～5％，硫酸铵0.1～5％，pH 6.0～8.0。
  11. 权利要求9或10所述重组菌发酵生产乳糖酶的方法，其特征在于，发酵结束后，经板框过滤除去菌体，再经超滤系统过滤后获得酶液。
  12. 权利要求3所述重组载体或重组菌株在生产乳糖酶中的应用。
  13. 权利要求1或2所述乳糖酶在生产低聚半乳糖中的应用。
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