WO2021227363A1 - Haloferula sp.β-N-乙酰氨基己糖苷酶在合成人乳寡糖中的应用 - Google Patents

Haloferula sp.β-N-乙酰氨基己糖苷酶在合成人乳寡糖中的应用 Download PDF

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WO2021227363A1
WO2021227363A1 PCT/CN2020/122119 CN2020122119W WO2021227363A1 WO 2021227363 A1 WO2021227363 A1 WO 2021227363A1 CN 2020122119 W CN2020122119 W CN 2020122119W WO 2021227363 A1 WO2021227363 A1 WO 2021227363A1
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protein
hahex74
lactose
seq
nucleic acid
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French (fr)
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江正强
刘翊昊
杨绍青
马俊文
闫巧娟
李婷
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中国农业大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01052—Beta-N-acetylhexosaminidase (3.2.1.52)

Definitions

  • the present invention relates to the technical field of genetic engineering, in particular to the application of Haloferula sp. ⁇ -N-acetylhexosaminidase in the synthesis of human milk oligosaccharides.
  • ⁇ -N-acetylhexosaminidase (EC 3.2.1.52) is a glycoside hydrolase (GH) that can catalyze the cleavage of the ⁇ bond in N-acetylamino- ⁇ -D-hexosamine to produce N-acetylamino - ⁇ -D-Glucosamine (GlcNAc) or N-Acetylamino- ⁇ -D-GalNAc (GalNAc) (Chen et al.
  • GH glycoside hydrolase
  • HMOs Human milk oligosaccharides
  • Lactose-N-triose II lacto-N-triose II, LNT2
  • lactose-N-neotetraose lacto-N-neotetraose
  • the main methods for synthesizing LNT2 and LNnT include whole-cell biotransformation, chemical and enzymatic methods. Due to the integration of the metabolic mechanism of microorganisms and the regional and stereoselectivity of glycosyltransferases, whole-cell biotransformation is currently an effective method for large-scale production of HMOs (Faijes et al. Enzymatic and cell factory approaches to the production of human milk oligosaccharides . Biotechnol. Adv., 2019, 37, 667-697).
  • LNT2 is produced by fermentation of modified Escherichia coli LJ110, the final concentration of which is 1.6g L -1 .
  • Baumgertner et al. F. Baumgertner et al., CN2015800382178 integrated the ⁇ 1,3-N-acetylglucosaminyltransferase gene (LgtA) into E. coli K12 to achieve optimal galactose feeding After 44 hours of reaction in the strategic 30L bioreactor, the concentration of LNT2 reached 15.8g/L.
  • Drouillard et al. Large-scale synthesis of H-antigen oligosaccharides by expressing Helicobacter pylori ⁇ -1,2-fucosyltransferase in metabolically engineered Escherichia coli cells.Angew.Chem., Int.Ed., 2006, 45, 1778-1780 ) LNnT is produced by fermentation of transformed E. coli JM107, the final concentration of which is 0.7g ⁇ L -1 .
  • many scholars have studied the use of Bacillus, etc. as chassis cells to biosynthesize LNnT. For example, Liu Long et al.
  • LNT2 and LNnT mainly relies on artificially synthesized glycoside donors, such as p-nitrophenyl- ⁇ -N-acetylglucosamine (pNP-NAG) and uridine diphosphate-N-acetylglucosamine ( UDP-GlcNAc) and so on.
  • Artificially synthesized donors are expensive and safe, so they are not suitable for the efficient synthesis of food-grade LNT2 and LNnT (Nyffenegger et al.
  • Chitin is a linear polysaccharide formed by ⁇ -N-acetylglucosamine connected by ⁇ -1,4-glycosidic bonds. It is widely found in nature, and its content is second only to cellulose (Lv et al. Highly efficient and selective biocatalytic production of glucosamine from chitin. Green Chem., 2017, 19, 527-535).
  • chitin oligosaccharides has a large amount of ⁇ -N-acetylglucosamine, which can be used as a natural glycoside donor (Nyffenegger et al. Backbone structures in human milk oligosaccharides: trans-glycosylation by metadatanomic ⁇ -N- acetylhexosaminidases.Appl.Microbiol.Biotechnol.,2015,99,7997-8009).
  • ⁇ -N-acetylhexosaminidase is mainly used in biological control (Li Daqi et al., CN201210356675), feed supplementation (Zhou Zhigang et al., CN201210065384) and preparation of chitin oligosaccharides (Jiang Zhengqiang et al., CN201811105536).
  • biological control Li Daqi et al., CN201210356675
  • feed supplementation Zhou Zhigang et al., CN201210065384
  • preparation of chitin oligosaccharides Jiang Zhengqiang et al., CN201811105536
  • ⁇ -N-acetylhexosaminidase to synergize chitinase to convert chitin into LNT2, and then to synthesize LNnT with ⁇ -galactosidase.
  • HEX1 and HEX2 were reacted with chitobiose and lactose at 25°C for 2 hours, and the conversion rates of LNT2 were 2% and 8%, respectively.
  • Zeuner et al. (Zeuner et al. Thermostable ⁇ -galactosidases for the synthesis of human milk oligosaccharides. New Biotechnol., 2016, 33, 355-360) used three thermophilic ⁇ -galactosidases, using lactose and LNT2 as glycoside donors and receptors. LNnT was synthesized separately, but the yields were low, 7.1%, 5.2%, and 1.0%, respectively.
  • Haloferula sp. is a branch of the Verrucomicrobia phylum (Bibi et al. Haloferula luteola sp nov., an endophytic bacterium isolated from the root of a halophyte, Rosa rugosa, and emended description of the System Haloferula. Int. Microbiol., 2011, 61, 1837-1841), there are few reports about this genus in the world, and there is no report on the production of ⁇ -N-acetylhexosaminidase by Haloferula sp.
  • the purpose of the present invention is to provide the application of Haloferula sp. ⁇ -N-acetylhexosaminidase in the synthesis of human milk oligosaccharides.
  • the present invention claims the application of HaHex74 protein or its related biological materials in any of the following:
  • the HaHex74 protein is derived from Haloferula sp., and specifically can be any of the following:
  • amino acid sequence is the protein with positions 94-745 of SEQ ID No. 2 or SEQ ID No. 2;
  • A2 A protein with the same function after substitution and/or deletion and/or addition of one or several amino acid residues from positions 94-745 of SEQ ID No. 2 or the amino acid sequence shown in SEQ ID No. 2 ;
  • (A3) A protein that has 99% or more, 95% or more, 90% or more, 85% or more than 80% homology with the amino acid sequence defined in any one of (A1)-(A2) and has the same function;
  • (A4) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1)-(A3).
  • the relevant biological material may be a nucleic acid molecule capable of expressing the HaHex74 protein or an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant bacteria or a transgenic cell line.
  • the tag refers to a polypeptide or protein expressed by fusion with the target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein.
  • the tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.
  • homology refers to the identity of amino acid sequence.
  • the homology search site on the Internet can be used to determine the identity of the amino acid sequence, such as the BLAST page of the NCBI homepage. For example, in advanced BLAST 2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set Gap existence cost, Per resistance gap cost and Lambda ratio to 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate, and then the identity value (%) can be obtained.
  • the homology of more than 95% may be at least 96%, 97%, or 98% identity.
  • the above 90% homology can be at least 91%, 92%, 93%, 94% identity.
  • the above 85% homology can be at least 86%, 87%, 88%, 89% identity.
  • the above 80% homology can be at least 81%, 82%, 83%, 84% identity.
  • the present invention claims a method for synthesizing lactose-N-trisaccharide II.
  • the method for synthesizing lactose-N-triose II as claimed in the present invention may include the following steps (b1) or (b2) or (b3):
  • Chitinase and HaHex74 protein are used as biological enzymes to catalyze the synthesis of chitin and ⁇ -lactose into lactose-N-triose II (LNT2).
  • the HaHex74 protein is any one of (A1)-(A4) above.
  • the present invention claims a method for synthesizing lactose-N-neotetraose.
  • the method for synthesizing lactose-N-neotetraose as claimed in the present invention may include the following steps (c1) or (c2):
  • (c1) Prepare lactose-N-triose II (LNT2) according to the method described in the second aspect above; then use ⁇ -galactosidase to catalyze lactose-N-triose II (LNT2) and ⁇ -lactose to synthesize lactose- N-Neotetraose (LNnT);
  • c2 Chitinase, HaHex74 protein and ⁇ -galactosidase are used as biological enzymes to catalyze the synthesis of chitin and ⁇ -lactose into lactose-N-triose II (LNT2) and lactose-N-neotetraose ( LNnT).
  • the HaHex74 protein is any one of (A1)-(A4) above.
  • the chitin hydrolysate is a mixture of chitobiose and N-acetylglucosamine.
  • the HaHex74 protein is used as a biological enzyme to catalyze the synthesis of lactose-N-triose II (LNT2) from the chitin hydrolysate and ⁇ -lactose at a pH of 7.5 and/or a temperature of 40°C, and /Or the content of ⁇ -lactose in the reaction system is 0.8M, and/or the content of the HaHex74 protein in the reaction system is 3-5 U/mL (such as 4 U/mL), and/or the reaction time is 10 h.
  • LNT2 lactose-N-triose II
  • the pH is 5.5, and/or the temperature is 55° C., and/or the chitinase
  • the content in the reaction system is 5U/mL, and/or the reaction time is 24h.
  • the pH is natural (pH 7.0)
  • the temperature is 50° C.
  • the content of the ⁇ -galactosidase in the reaction system is 0.1 U/mL
  • the reaction time is 10 h.
  • the optimal pH of the HaHex74 protein is 6.5 and the optimal temperature is 45°C.
  • the HaHex74 protein can be prepared according to a method including the following steps: introducing a nucleic acid molecule encoding the HaHex74 protein into a recipient yeast to obtain a recombinant yeast; and fermenting and cultivating the recombinant yeast according to the following steps, Obtain the HaHex74 protein from the fermentation product:
  • Basic culture stage inoculate the recombinant yeast into BSM medium supplemented with 50g/L glycerol, control pH4.0, temperature 30°C, and start glycerol fed-batch culture when the glycerol concentration is lower than 10g/L stage.
  • Glycerin feeding culture stage feeding 500g/L glycerol, maintaining the glycerol concentration at 10-25g/L, controlling the temperature at 28°C, pH5.0, and dissolved oxygen at 10-20% until the end of fermentation.
  • the step of adding PTM1 to the fermentation system is also included.
  • the added amount of the PTM1 is 4.35 mL/L of the starting fermentation broth.
  • the inoculation amount of the recombinant yeast is 10% by volume.
  • the rotation speed is controlled to be 600 rpm during culture; in (d2), the rotation speed is controlled to be 800 rpm during culture.
  • a seed culture stage may also be included: the recombinant yeast is inoculated into BMGY medium, and cultivated at 30° C. to an OD 600 of about 10.0. During the seed cultivation stage, the rotation speed is controlled to be 200 rpm.
  • obtaining the HaHex74 protein from the fermentation product can be carried out according to a method including the following steps: centrifuging the fermentation product to collect the supernatant; placing the supernatant in a Tris-HCl buffer for dialysis, Centrifugation to obtain the crude liquid; the crude liquid is purified using an agarose weak anion exchange column DE52 to obtain the HaHex74 protein.
  • the DE52 affinity column was equilibrated with buffer A for 5-10 column volumes, the collected crude solution was loaded at a flow rate of 0.5 mL/min, and the buffer A and buffer B were linearly eluted to To OD 280 ⁇ 0.05, collect the fractions with ⁇ -N-acetylhexosaminidase activity, and the purified product obtained by dialysis is the HaHex74 protein.
  • the buffer A is a buffer containing 20 mM Tris-HCl (pH 8.0); the buffer B is a 20 mM Tris-HCl buffer (pH 8.0) containing NaCl (500 mM).
  • nucleic acid molecule encoding the HaHex74 protein may be any of the following DNA molecules:
  • (B3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more than 80% homology with the DNA sequence defined by (B1) or (B2) and encodes the HaHex74 protein.
  • the stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na 3 PO 4 and 1 mM EDTA, at 50°C, 2 ⁇ SSC , Rinsing in 0.1% SDS; also: 50 °C, hybridization in a mixed solution of 7% SDS, 0.5M Na 3 PO 4 and 1 mM EDTA, rinsing in 50 °C, 1 ⁇ SSC, 0.1% SDS; also It is: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na 3 PO 4 and 1 mM EDTA, rinsing in 50°C, 0.5 ⁇ SSC, 0.1% SDS; also: 50°C, in 7% SDS Hybridization in a mixed solution of 0.5M Na 3 PO 4 and 1mM EDTA, rinsing at 50°C, 0.1 ⁇ SSC, 0.1% SDS; also: 50°C, hybridization
  • homology refers to the identity of nucleotide sequences.
  • the homology search site on the Internet can be used to determine the identity of the nucleotide sequence, such as the BLAST page of the NCBI homepage. For example, in advanced BLAST 2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set Gap existence cost, Per resistance gap cost and Lambda ratio to 11, 1 and 0.85 (default value) and perform a search for the identity of a pair of nucleotide sequences to calculate, and then the identity value (%) can be obtained.
  • the homology of more than 95% may be at least 96%, 97%, or 98% identity.
  • the above 90% homology can be at least 91%, 92%, 93%, 94% identity.
  • the above 85% homology can be at least 86%, 87%, 88%, 89% identity.
  • the above 80% homology can be at least 81%, 82%, 83%, 84% identity.
  • nucleic acid molecule can be introduced into the recipient yeast in the form of a recombinant vector.
  • the promoter in the recombinant vector that initiates the transcription of the nucleic acid molecule may be a GAP promoter.
  • the recombinant vector is a recombinant vector obtained by cloning the nucleic acid molecule into a pGAP9K vector (for example, between EcoR I and Not I).
  • the pGAP9K vector is a plasmid obtained by replacing the AOX1 promoter in the pPIC9K vector with the GAP promoter.
  • the sequence of the GAP promoter is shown in SEQ ID No. 1.
  • yeast is Pichia pastoris.
  • Pichia pastoris is Pichia pastoris GS115.
  • the present invention claims a method for preparing the aforementioned HaHex74 protein.
  • the method for preparing the HaHex74 protein may include the following steps: introducing a nucleic acid molecule encoding the HaHex74 protein into a recipient yeast to obtain a recombinant yeast; and fermenting and cultivating the recombinant yeast according to the following steps to obtain the HaHex74 from a fermentation product protein:
  • Basic culture stage inoculate the recombinant yeast into BSM medium supplemented with 50g/L glycerol, control pH4.0, temperature 30°C, and start glycerol fed-batch culture when the glycerol concentration is lower than 10g/L stage.
  • Glycerin feeding culture stage feeding 500g/L glycerol, maintaining the glycerol concentration at 10-25g/L, controlling the temperature at 28°C, pH5.0, and dissolved oxygen at 10-20% until the end of fermentation.
  • the step of adding PTM1 to the fermentation system is also included.
  • the added amount of the PTM1 is 4.35 mL/L of the starting fermentation broth.
  • the inoculation amount of the recombinant yeast is 10% by volume.
  • the rotation speed is controlled to be 600 rpm during culture; in (d2), the rotation speed is controlled to be 800 rpm during culture.
  • a seed culture stage may also be included: the recombinant yeast is inoculated into BMGY medium, and cultivated at 30° C. to an OD 600 of about 10.0. During the seed cultivation stage, the rotation speed is controlled to be 200 rpm.
  • obtaining the HaHex74 protein from the fermentation product can be carried out according to a method including the following steps: centrifuging the fermentation product to collect the supernatant; placing the supernatant in a Tris-HCl buffer for dialysis, Centrifugation to obtain the crude liquid; the crude liquid is purified using an agarose weak anion exchange column DE52 to obtain the HaHex74 protein.
  • the DE52 affinity column was equilibrated with buffer A for 5-10 column volumes, the collected crude solution was loaded at a flow rate of 0.5 mL/min, and the buffer A and buffer B were linearly eluted to To OD 280 ⁇ 0.05, collect the fractions with ⁇ -N-acetylhexosaminidase activity, and the purified product obtained by dialysis is the HaHex74 protein.
  • the buffer A is a buffer containing 20 mM Tris-HCl (pH 8.0); the buffer B is a 20 mM Tris-HCl buffer (pH 8.0) containing NaCl (500 mM).
  • nucleic acid molecule encoding the HaHex74 protein may be any of the following DNA molecules:
  • (B3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more than 80% homology with the DNA sequence defined by (B1) or (B2) and encodes the HaHex74 protein.
  • the stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na 3 PO 4 and 1 mM EDTA, at 50°C, 2 ⁇ SSC , Rinsing in 0.1% SDS; also: 50 °C, hybridization in a mixed solution of 7% SDS, 0.5M Na 3 PO 4 and 1 mM EDTA, rinsing in 50 °C, 1 ⁇ SSC, 0.1% SDS; also It is: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na 3 PO 4 and 1 mM EDTA, rinsing in 50°C, 0.5 ⁇ SSC, 0.1% SDS; also: 50°C, in 7% SDS Hybridization in a mixed solution of 0.5M Na 3 PO 4 and 1mM EDTA, rinsing at 50°C, 0.1 ⁇ SSC, 0.1% SDS; also: 50°C, hybridization
  • homology refers to the identity of nucleotide sequences.
  • the homology search site on the Internet can be used to determine the identity of the nucleotide sequence, such as the BLAST page of the NCBI homepage. For example, in advanced BLAST 2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set Gap existence cost, Per resistance gap cost and Lambda ratio to 11, 1 and 0.85 (default value) and perform a search for the identity of a pair of nucleotide sequences to calculate, and then the identity value (%) can be obtained.
  • the homology of more than 95% may be at least 96%, 97%, or 98% identity.
  • the above 90% homology can be at least 91%, 92%, 93%, 94% identity.
  • the above 85% homology can be at least 86%, 87%, 88%, 89% identity.
  • the above 80% homology can be at least 81%, 82%, 83%, 84% identity.
  • nucleic acid molecule can be introduced into the recipient yeast in the form of a recombinant vector.
  • the promoter in the recombinant vector that initiates the transcription of the nucleic acid molecule may be a GAP promoter.
  • the recombinant vector is a recombinant vector obtained by cloning the nucleic acid molecule into a pGAP9K vector (for example, between EcoR I and Not I).
  • the pGAP9K vector is a plasmid obtained by replacing the AOX1 promoter in the pPIC9K vector with the GAP promoter.
  • the sequence of the GAP promoter is shown in SEQ ID No. 1.
  • yeast is Pichia pastoris.
  • Pichia pastoris is Pichia pastoris GS115.
  • Figure 1 is a high-density fermentation ⁇ -N-acetylhexosaminidase HaHex74 expression and extracellular protein SDS-PAGE analysis.
  • A is the expression level of HaHex74 (( ⁇ ): enzyme activity; ( ⁇ ): protein concentration; ( ⁇ ): wet weight);
  • B is the SDS-PAGE analysis chart
  • Figure 2 is a purification electrophoresis diagram of ⁇ -N-acetylhexosaminidase HaHex74.
  • Figure 3 is a graph showing the determination of optimal reaction pH, pH stability, optimal temperature and temperature stability of ⁇ -N-acetylhexosaminidase HaHex74.
  • A is the optimum reaction pH;
  • B is the pH stability;
  • C is the optimum temperature;
  • D is the temperature stability.
  • citrate buffer pH 5.0-6.0
  • acetic acid-sodium acetate buffer pH 4.0-6.0
  • MES buffer pH 5.5-6.5
  • MOPS buffer Solution pH 6.5-7.5
  • phosphate buffer pH 6.0-8.0
  • Tris-HCl buffer pH 7.0-9.0
  • Glycine-sodium hydroxide buffer pH 8.5-10.5
  • Figure 4 shows the optimal pH (A), optimal temperature (B), optimal reaction time (C), optimal receptor concentration (D) and optimal addition of ⁇ -N-acetylhexosaminidase HaHex74 for the synthesis of LNT2
  • A is the optimum pH; B is the optimum temperature; C is the optimum reaction time; D is the optimum receptor concentration; D is the optimum amount of enzyme.
  • MES buffer pH 5.5-6.5
  • phosphate buffer pH 6.0-8.0
  • Tris-HCl buffer pH 7.0-9.0
  • Figure 5 is a mass spectrum of the product of ⁇ -N-acetylhexosaminidase HaHex74 transglycosidase.
  • Figure 6 is the primary 1 H spectrum of the ⁇ -N-acetylhexosaminidase HaHex74 transglycosidase product.
  • Figure 7 is the primary 13 C spectrum of the ⁇ -N-acetylhexosaminidase HaHex74 transglycosidase product.
  • Figure 8 is a secondary heteronuclear single quantum relationship (HSQC) spectrum of the ⁇ -N-acetylhexosaminidase HaHex74 transglycosidase product.
  • HSQC secondary heteronuclear single quantum relationship
  • FIG. 9 shows the HPLC analysis of synthetic LNT2 (( ⁇ ): conversion rate; ( ⁇ ): LNT2 concentration).
  • FIG 10 shows the HPLC analysis of synthetic LNnT (( ⁇ ): conversion rate; ( ⁇ ): LNnT concentration).
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples, unless otherwise specified, are all conventional methods.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • the activity unit of ⁇ -N-acetylhexosaminidase is defined as: under the above reaction conditions, the amount of enzyme required to generate 1 ⁇ mol of pNP per minute is one enzyme activity unit (1U).
  • Specific enzyme activity is defined as the unit of enzyme activity possessed by 1 mg of protein, expressed as U ⁇ mg -1 .
  • HaHex74 The nucleotide sequence of the HaHex74 gene is "ATG+SEQ ID No.3, 280-2238", encoding the amino acid sequence shown in "Met+SEQ ID No.2, 94-745".
  • the HaHex74 gene has the highest homology with a soil metagenomic ⁇ -N-acetylhexosaminidase (AKC34129), which is 61%. After that, it was sent to Shanghai Shenggong Biological Co., Ltd. to synthesize genes.
  • GAP1 5'-GCAGC GAGCTC ATCCTTTTTTGTAGAAATGTCTTGG-3';
  • GAP2 5'-CGC GGATCC TGTGTTTTGATAGTTGTTCAATTGA- 3 '.
  • the GAP promoter was amplified from the Pichia pastoris GS115 genome, the GAP promoter fragment and pPIC9K vector were digested with Sac I and BamH I, and the modified expression vector was obtained by ligation with T4 DNA ligase, named pGAP9K, i.e. pPIC9K vector Replace the AOX1 promoter in the GAP promoter.
  • pGAP9K i.e. pPIC9K vector Replace the AOX1 promoter in the GAP promoter.
  • the GAP promoter sequence is shown in SEQ ID No. 1.
  • Upstream primer 5'–CCG GAATTC GAACCAACCATTATTCCATTGCC-3';
  • Downstream primer 5'–AGAAT GCGGCCGC TTACTCAACGGTGATTTCGTGGATA-3'.
  • PCR amplification was carried out with the above primers.
  • the PCR amplification conditions were: 95°C pre-denaturation for 30s; 95°C denaturation for 20s, 58°C annealing for 20s, 72°C extension for 1 min, and 34 cycles; the final 72°C extension for 5 min.
  • the PCR product was recovered by 1% agarose gel electrophoresis, and digested with EcoRI and NotI.
  • the product after the double enzyme digestion is ligated with the vector backbone fragment of the yeast expression vector pGAP9K that has been digested with the same double enzyme using T4 DNA ligase, and transformed into the host E. coli DH5 ⁇ .
  • the recombinant yeast expression vector verified by sequencing was named pGAP9K-HaHex74.
  • the obtained recombinant Pichia pastoris expression vector pGAP9K-HaHex74 was linearized with restriction endonuclease XbaI and then electrotransformed into Pichia pastoris GS115 to form a recombinant bacteria.
  • the GAP promoter sequence in the recombinant expression vector pGAP9K-HaHex74 is the DNA molecule in SEQ ID No. 1 (that is, the AOX1 promoter in the pPIC9K vector is replaced with the GAP promoter by double digestion with Sac I and BamH I to construct a new expression Vector pGAP9K), the complete ORF sequence of the recombinant expression vector pGAP9K-HaHex74 is the DNA molecule in SEQ ID No. 3, in which positions 1-279 are the sequence on the pGAP9K vector, and positions 280-2238 are the HaHex74 gene sequence.
  • Example 1 The recombinant bacteria obtained in Example 1 were coated on MD plates (1.34% YNB, 4 ⁇ 10 -5 % biotin, 2% glucose), and 100 ⁇ L of His + transformants obtained from the MD plates were scraped off with sterilized water Spread on YPD-G418 plates of different concentrations (1% yeast extract, 2% tryptone, 2% glucose, and G418 concentrations of 2, 3, 4, and 6 mg/mL, respectively).
  • the Pichia yeast strain with high enzyme production level obtained in step 1 of Example 2 was subjected to high-density fermentation in a 5L fermentor.
  • the medium used in the fermentation process (seed medium BMGY, fermentation basic medium BSM and glycerin batch feed medium) is prepared according to Pichia Fermentation Process Guidelines (Version B, 053002, Invitrogen).
  • the whole fermentation process adopts two stages of basic culture and glycerol fed-batch culture.
  • Seed culture Select strains with higher enzyme production levels in shake flask fermentation, inoculate 150mL BMGY medium, cultivate at 30°C and 200rpm until OD 600 is about 10.0.
  • (2) Basic culture Inoculate the seed liquid obtained in step (1) into a 5L fermentor (containing 1.35L of basic fermentation medium BSM and 5% (50g/L) glycerin, sterilize the fermentor, and 28% concentration Adjust pH to 4.0 with ammonia water, add PTM1 (known in the art, trace elements, promote yeast expression) 4.35mL/L starting fermentation broth, inoculation volume 10% (v/v), rotating speed 600rpm, temperature 30°C. Wait until the glycerol concentration is lower than At 1% (10g/L), start to add 50% (w/v) glycerol (500g/L) feed medium.
  • PTM1 known in the art, trace elements, promote yeast expression
  • Glycerin fed-batch culture add 50% (w/v) glycerol (500g/L), control the temperature at 28°C, pH 5.0, rotate speed 800rpm, and maintain the glycerol concentration at 1% ⁇ 2.5% (10g/L ⁇ 25g/L), always monitor the dissolved oxygen, and adjust the rotation speed and ventilation to maintain the DO 10%-20% until the end of the fermentation.
  • the fermentation broth was centrifuged at 10,000 ⁇ g for 5 minutes, the supernatant was collected, and 50 mL of the supernatant was placed in Tris-HCl buffer (20mM, pH 8.0) for dialysis overnight. Centrifuge at ⁇ g for 5 min to obtain a crude enzyme solution.
  • the crude enzyme solution uses agarose weak anion exchange column DE52 (DEAE Sepharose Fast Flow) to purify the recombinant protein in one step. Specific steps are as follows:
  • buffer A is a 20mM Tris-HCl buffer (pH 8.0); buffer B is a 20mM Tris-HCl buffer (pH 8.0) containing NaCl (500mM).
  • the purified product obtained from the crude enzyme solution of recombinant bacteria is the recombinant protein HaHex74.
  • the SDS-PAGE purification chart of the crude enzyme solution of the recombinant bacteria and the purified product (recombinant protein HaHex74) obtained is shown in FIG. 2.
  • lane M is the molecular weight standard
  • lane 1 is the recombinant protein HaHex74 crude enzyme
  • lane 2 is the HaHex74 pure enzyme.
  • the results in Figure 2 show that the size of the recombinant protein HaHex74 is approximately 74 kDa, which is consistent with the expected size.
  • the crude enzyme solution and the pure enzyme solution were used as the enzyme solution to be tested, and the corresponding inactivated protein was used as a control to detect the enzyme activity of ⁇ -N-acetylhexosaminidase.
  • the total enzyme activity of the crude enzyme solution shown in Table 1 is 3500 U, and its specific enzyme activity is 360.8 U mg -1 ; the total enzyme activity of the pure enzyme solution is 2890 U, and its specific enzyme activity is 385.3 U mg -1 ; the purification factor is 1.1 .
  • the HaHex74 pure enzyme solution prepared above was used as the enzyme solution to be tested, and the enzyme activity was measured at 40° C. in different buffer systems, and the relative enzyme activity was calculated with the highest enzyme activity being 100%.
  • the various buffers are as follows:
  • HaHex74 has good pH stability, and more than 80% of the enzyme activity remains after 30 minutes of incubation in the pH range of 5.5-9.0.
  • HaHex74 After properly diluting HaHex74 with 50mM phosphate buffer (pH 6.5), keep it at different temperatures (20, 25, 30, 35, 40, 45, 50, 55, 60°C) for 30 minutes, and then quickly place it in ice water to cool 30min, then measure the enzyme activity. Taking the enzyme activity of the untreated ⁇ -N-acetylhexosaminidase as 100%, the relative enzyme activity of HaHex74 after treatment at different temperatures was calculated.
  • colloidal chitin, ethylene glycol chitin, carboxymethyl cellulose (CMC), chitosan solution, p-nitrophenyl- ⁇ -N-acetylglucosamine (pNP-NAG) and p-nitrogen Phenyl- ⁇ -N-acetylgalactoaminoglycoside (pNP-GalNAc), etc. were used as reaction substrates, and the enzyme activity was detected under standard enzyme activity determination conditions, and the substrate specificity of the enzyme was investigated. The results are shown in Table 2.
  • a Method for determining the enzyme activity of chitin oligosaccharides take 0.1 mL of an appropriately diluted enzyme solution and add it to 0.1 mL of 1% (mass to volume ratio, 10 g/L) chitin oligosaccharide substrate solution (use 50 mM, pH 6.5 Phosphate buffer solution), 45°C water bath reaction for 10min.
  • the amount of ⁇ -N-acetylglucosamine released was measured by high performance liquid chromatography (HPLC), and ⁇ -N-acetylglucosamine was used as the standard.
  • HPLC determination conditions are: HPLC-RID detection system (Agilent 1260 infinity II, Agilent Technologies, USA) BP-800Pb++ chromatography column (Benson Polymeric, Reno, NE, 7.8 ⁇ 300mm, USA), distilled water as mobile phase, column temperature 80 °C, the flow rate is 1mL ⁇ min -1 .
  • the activity unit of ⁇ -N-acetylglucosaminidase is defined as: under the above reaction conditions, the amount of enzyme required to generate 1 ⁇ mol of ⁇ -N-acetylglucosamine per minute is one enzyme activity unit (1U).
  • HaHex74 had the highest enzyme activity to pNP-NAG, 385.3 U/mg; followed by chitotriose, chitobiose and chitotetraose. It shows weak activity on colloidal chitin (0.14 U ⁇ mg -1 ), and has no activity on ethylene glycol chitin, chitosan and CMC.
  • the amount of synthesized LNT2 was measured by HPLC.
  • the HPLC measurement conditions are: Waters XBridge BEH Amide 5 ⁇ m chromatographic column (250 ⁇ 4.6mm), 72% acetonitrile, column temperature 45°C, flow rate 0.5mL/min, 45min, differential (RID) detector.
  • Transglycoside product yield (%) concentration of synthesized product (mM)/initial concentration of glycoside donor (mM) ⁇ 100
  • the HaHex74 pure enzyme solution prepared in Example 2 was used as the enzyme solution to be tested, and the LNT2 content was determined in different buffer systems.
  • the various buffers are as follows:
  • the HaHex74 pure enzyme solution prepared in Example 2 was used as the enzyme solution to be tested, and the reaction system was placed in a water bath at different temperatures (20-55° C.) for 1.5 hours to determine the content of LNT2.
  • the HaHex74 pure enzyme solution prepared in Example 2 was used as the enzyme solution to be tested, and the reaction system was placed in a water bath at 40° C. for different times to determine the content of LNT2.
  • the HaHex74 pure enzyme solution prepared in Example 2 was used as the enzyme solution to be tested, ⁇ -lactose of different concentrations was added to the reaction system, and the reaction was carried out in a water bath at 40° C. for 2.5 hours to determine the content of LNT2.
  • the HaHex74 pure enzyme solution prepared in Example 2 was used as the enzyme solution to be tested, and HaHex74 of different concentrations was added to the reaction system, and reacted in a water bath at 40° C. for 2.5 hours to determine the content of LNT2.
  • Fig. 4 E The optimal amount of enzyme for synthesis of LNT2 by HaHex74 is 3-5 U ⁇ mL -1 .
  • the sample was dissolved in pure water, and a Thermo Scientific TM Q Exactive TM mass spectrometer was used to collect a high-resolution primary mass spectrum of the sample in the positive-ion mode of the ESI ion source.
  • transglycoside product was further determined by NMR, and the chemical shifts of 1 H protons (Figure 6) and 13 C ( Figure 7) of the compound were obtained from the one-dimensional 1 H and 13 C spectra. From the two-dimensional heteronuclear single quantum relationship (HSQC) spectrum, the relationship between the carbon and hydrogen atoms of the compound can be obtained ( Figure 8). After data comparison, the product of HaHex74 is consistent with the transglycosidase product of ⁇ -N-acetylhexosaminidase mutant derived from Bifidobacterium bifidum ( et al.
  • 120g ball milled chitin powder (3%, w/v) (Jiang Zhengqiang et al., CN201811105536) was dissolved in 4L 20mM citrate buffer (pH 5.5), and 5U ⁇ mL -1 chitinase (Yang et al. al. Cloning, expression, purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii. Food Chem., 2016,192,1041-1048), as for enzymatic hydrolysis at 55°C for 24h.
  • Step 1 The hydrolyzate (Chitobiose mixture and N- acetylglucosamine) centrifuged supernatant, and then concentrated 10-fold, was added NaH 2 PO 4 adjusted to pH 7.5, was added 0.8M ⁇ - lactose and 4U ⁇ mL - 1 HaHex74 (HaHex74 pure enzyme solution obtained in Example 2), reacted at 40°C for a certain time, sampling at intervals, and determined the content of LNT2 by HPLC (see Example 3 for the determination conditions).
  • LNT2 conversion rate (%) the amount of synthesized LNT2 (M)/the amount of chitobiose (M) ⁇ 100%; where M represents the amount of substance.
  • the excess ⁇ -lactose in the reaction product of the above Example 5 was removed by the activated carbon column method (75 ⁇ 1.5cm), using a gradient mixer (TH-2000, Shanghai Qingpu Huxi Instrument Factory), using 0-25% (v/v)
  • the reaction product of Example 5 was subjected to gradient elution with the ethanol solution of the above-mentioned Example 5 at a flow rate of 1 mL min-1 , and the eluent was collected, and the product was detected by TLC (see Example 4 for conditions) and HPLC (see Example 3 for conditions).
  • the solution passed through the activated carbon column was concentrated 10 times to a natural pH (pH 7.0), and 0.1U mL -1 of ⁇ -galactosidases (Zeuner et al. Thermostable ⁇ -galactosidases for the synthesis of human milk oligosaccharides. New Biotechnol., 2016, 33, 355-360) React at 50°C for a certain period of time, sampling at intervals, and inactivate all samples in a boiling water bath for 10 minutes. All samples were analyzed by HPLC (see Example 3 for conditions).
  • LNnT conversion rate (%) synthesized LNnT amount (M)/lactose amount (M) ⁇ 100%; where M represents the amount of substance.
  • the present invention efficiently expresses the ⁇ -N-acetylhexosaminidase gene of a glycoside hydrolase 20 family of Haloferula sp. in Pichia pastoris. After high-density fermentation in a 5L fermentor, the enzyme activity of the fermentation broth can reach 3500 U ⁇ mL -1 , and the protein content is 9.7 g ⁇ L -1 , which is the highest level of ⁇ -N-acetylhexosaminidase expression at present.
  • the newly invented ⁇ -N-acetylhexosaminidase HaHex74 has excellent transglycosidic activity and can efficiently synthesize the important backbone structure of human milk oligosaccharides LNT2 with natural donor chitobiose ((GlcNAc) 2) and ⁇ -lactose
  • the conversion rate is 10-20%, which is the highest level of ⁇ -N-acetylhexosaminidase to synthesize LNT2 from natural donors.
  • chitinase and HaHex74 can efficiently convert chitin powder into LNT2, and further synergize with ⁇ -galactosidase to efficiently synthesize LNnT, the concentrations of which are 8-12g ⁇ L -1 and 1-3g ⁇ L -1, respectively .
  • the ⁇ -N-acetylhexosaminidase of the present invention has high enzyme production level, excellent hydrolysis characteristics and high transglycosidic activity, and has important application value in the enzymatic synthesis of human milk oligosaccharides.

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Abstract

一种Haloferula sp.β-N-乙酰氨基己糖苷酶在合成人乳寡糖中的应用，提供了HaHex74蛋白或其相关生物材料在如下任一中的应用：(a1)合成人乳寡糖；(a2)合成乳糖-N-三糖Ⅱ和/或乳糖-N-新四糖；HaHex74蛋白来源于Haloferula sp.，氨基酸序列如SEQ ID No.2所示。所述β-N-乙酰氨基己糖苷酶产酶水平高、水解特性优异以及转糖苷活性高，在酶法合成人乳寡糖中具有重要的应用价值。

Description

Haloferula sp.β-N-乙酰氨基己糖苷酶在合成人乳寡糖中的应用 技术领域
本发明涉及基因工程技术领域，具体涉及Haloferula sp.β-N-乙酰氨基己糖苷酶在合成人乳寡糖中的应用。
背景技术
β-N-乙酰氨基己糖苷酶(EC 3.2.1.52)是一种糖苷水解酶(GH)，可催化N-乙酰氨基-β-D-己糖胺中β键的裂解，产生N-乙酰氨基-β-D-葡萄糖胺(GlcNAc)或N-乙酰氨基-β-D-半乳糖胺(GalNAc)(Chen et al.Efficient and regioselective synthesis ofβ-GalNAc/GlcNAc-Lactose by a bifunctional transglycosylatingβ-N-Acetylhexosaminidase from Bifidobacterium bifidum.Appl.Environ.Microbiol.,2016,82,5642-5652)。根据氨基酸序列的同源性，现有β-N-乙酰氨基己糖苷酶分属于GH3、20、84和116家族。近年来，由于GH20家族β-N-乙酰氨基己糖苷酶具有较高的转糖苷活性而用于功能性寡糖的合成(Liu,T.,Duan,Y.W.,&Yang,Q.Revisiting glycoside hydrolase family 20β-N-acetyl-D-hexosaminidases:Crystal structures,physiological substrates and specific inhibitors.Biotechnol.Adv.,2018,36,1127-1138)。人乳寡糖(human milk oligosaccharides,HMOs)是天然存在于人乳中的一类主链由2-10个单糖分子组成的低聚糖，其含量仅次于乳糖和脂质(Faijes et al.Enzymatic and cell factory approaches to the production of human milk oligosaccharides.Biotechnol.Adv.,2019,37,667-697)。乳糖-N-三糖Ⅱ(lacto-N-trioseⅡ,LNT2)和乳糖-N-新四糖(lacto-N-neotetraose,LNnT)是HMOs的骨架结构，具有许多功能活性，如抗炎和免疫调节活性(Cheng et al.Human milk oligosaccharides and its acid hydrolysate LNT2 show immunomodulatory effects via TLRs in a dose and structure-dependent way.J.Funct.Foods,2019,59,174-184)。2015年美国食品药品监督管理局(Food and Drug Administration,FDA)批准LNnT为公认安全(Generally Recognized as Safe,GRAS)的新食品原料。2016年，欧盟食品安全局(EFSA)也批准LNnT为新食品原料，相关产品也随之大量上市(Bych et al.Production of HMOs using microbial hosts-from cell engineering to large scale production.Curr.Opin.Biotechnol.,2019,56,130-137)。但是，绿色高效大规模制备LNT2和LNnT仍然存在挑战。
目前合成LNT2和LNnT的主要方法有全细胞生物转化法、化学法和酶法。由于整合了微生物的代谢机制以及糖基转移酶的区域和立体选择性，全细胞生物转化法是目前大规模生产HMOs的有效手段(Faijes et al.Enzymatic and cell factory approaches to the production of human milk oligosaccharides. Biotechnol.Adv.,2019,37,667-697)。
Figure PCTCN2020122119-appb-000001
等(
Figure PCTCN2020122119-appb-000002
et al.Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation.Bioorg.Med.Chem.,2015,23,6799-6806)通过发酵改造后的大肠杆菌LJ110生产LNT2，其终浓度为1.6g L -1。鲍姆格特纳等(F·鲍姆格特纳等,CN201580038218)通过将β1,3-N-乙酰葡糖胺基转移酶基因(LgtA)整合至大肠杆菌K12，在最佳半乳糖补料策略的30L生物反应器中反应44h，LNT2浓度达到15.8g/L。Drouillard等(Drouillard et al.Large-scale synthesis of H-antigen oligosaccharides by expressing Helicobacter pyloriα-1,2-fucosyltransferase in metabolically engineered Escherichia coli cells.Angew.Chem.,Int.Ed.,2006,45,1778-1780)通过发酵改造后的大肠杆菌JM107生产LNnT，其终浓度为0.7g·L -1。此外，许多学者研究利用芽孢杆菌等作为底盘细胞生物合成LNnT，如刘龙等(刘龙等,CN201910146431)通过在枯草芽孢杆菌168△amyE:P43-lacY，P43-lgtB，PxylA-comK的基因组上整合两个β-1,4-半乳糖转移酶基因，并外源表达β-1,3-N-葡糖氨基转移酶基因得到一个重组枯草芽孢杆菌168，该菌在最佳葡萄糖补料策略下反应48h，LNnT浓度达到1.3g/L。Dong等(Dong et al.Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis.Biotechnol.Biofuels,2019,12,212)通过发酵改造后的枯草芽孢杆菌168生产LNnT，其终浓度为4.5g L -1。虽然全细胞生物转化法是目前大规模生产LNT2和LNnT主要手段，但是其得率低，且前期需要对宿主细胞代谢通路进行改造，后期需要对发酵工艺进行优化，生产成本仍居高不下。
化学法合成LNT2和LNnT为了防止副反应的发生，需要较多步骤且合成效率较低，不适合大规模生产(Faijes et al.Enzymatic and cell factory approaches to the production of human milk oligosaccharides.Biotechnol.Adv.,2019,37,667-697)。相比于化学法，酶法合成具有良好的立体和区域选择性，环境友好，且能够有效地保证特定糖苷键构型和将单糖逐个加到糖基受体特定位置的优点，己经成为HMOs合成的有效手段。但目前酶法合成LNT2和LNnT主要依赖于人工合成的糖苷供体，如对硝基苯基-β-N-乙酰氨基葡萄糖苷(pNP-NAG)和尿苷二磷酸-N-乙酰氨基葡萄糖(UDP-GlcNAc)等。人工合成的供体价格昂贵且存在安全性，因此不适合食品级LNT2和LNnT的高效合成(Nyffenegger et al.Backbone structures in human milk oligosaccharides:trans-glycosylation by metagenomicβ-N-acetylhexosaminidases.Appl.Microbiol.Biotechnol.,2015,99,7997-8009)。几丁质是β-N-乙酰氨基葡萄糖通过β-1,4-糖苷键连接而成的线性多聚糖，广泛存在于自然界中，其含量仅次于纤维素(Lv et al.Highly efficient and selective biocatalytic production of glucosamine from chitin.Green Chem.,2017,19,527-535)。 其降解产物，几丁寡糖结构中具有大量β-N-乙酰氨基葡萄糖苷，可作为合成LNT2的天然糖苷供体(Nyffenegger et al.Backbone structures in human milk oligosaccharides:trans-glycosylation by metagenomicβ-N-acetylhexosaminidases.Appl.Microbiol.Biotechnol.,2015,99,7997-8009)。因此，利用β-N-乙酰氨基己糖苷酶协同几丁质酶将几丁质转化为LNT2，再进一步协同β-半乳糖苷酶合成高附加值的LNnT将具有广阔的市场前景。
β-N-乙酰氨基己糖苷酶主要应用于生物防治(李大琪等,CN201210356675)、饲料添加(周志刚等,CN201210065384)和几丁寡糖的制备(江正强等,CN201811105536)。目前未见利用β-N-乙酰氨基己糖苷酶协同几丁质酶将几丁质转化为LNT2，再进一步协同β-半乳糖苷酶合成LNnT的相关专利及文献报道。国际上对β-N-乙酰氨基己糖苷酶和β-半乳糖苷酶合成LNT2和LNnT的报道也相对较少。Nyffenegger等(Nyffenegger et al.Backbone structures in human milk oligosaccharides:trans-glycosylation by metagenomicβ-N-acetylhexosaminidases.Appl.Microbiol.Biotechnol.,2015,99:7997-8009)土壤的宏基因组文库中获得了两个β-N-乙酰氨基葡萄糖苷酶并在大肠杆菌中成功表达(HEX1和HEX2)，HEX1和HEX2利用几丁二糖和乳糖在25℃下反应2h，LNT2转化率分别为2％和8％。Zeuner等(Zeuner et al.Thermostableβ-galactosidases for the synthesis of human milk oligosaccharides.New Biotechnol.,2016,33,355-360)利用三个嗜热β-半乳糖苷酶，以乳糖和LNT2为糖苷供体和受体，分别合成了LNnT，但其得率均较低，分别为7.1％、5.2％和1.0％。
Haloferula sp.是Verrucomicrobia菌门的一个分支(Bibi et al.Haloferula luteola sp nov.,an endophytic bacterium isolated from the root of a halophyte,Rosa rugosa,and emended description of the genus Haloferula.Int.J.Syst.Evol.Microbiol.,2011,61,1837-1841)，目前国际上关于此属的报道较少，且尚无Haloferula sp.产β-N-乙酰氨基己糖苷酶的报道。
发明公开
本发明的目的是提供Haloferula sp.β-N-乙酰氨基己糖苷酶在合成人乳寡糖中的应用。
第一方面，本发明要求保护HaHex74蛋白或其相关生物材料在如下任一中的应用：
(a1)合成人乳寡糖；
(a2)合成乳糖-N-三糖Ⅱ和/或乳糖-N-新四糖。
所述HaHex74蛋白来源于Haloferula sp.，具体可为如下任一：
(A1)氨基酸序列为SEQ ID No.2的第94-745位或SEQ ID No.2的蛋白质；
(A2)将SEQ ID No.2的第94-745位或SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质；
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
所述相关生物材料可为能够表达所述HaHex74蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
上述蛋白质中，所述标签是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述蛋白质中，同源性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述蛋白质中，所述95％以上的同源性可为至少96％、97％、98％的同一性。所述90％以上的同源性可为至少91％、92％、93％、94％的同一性。所述85％以上的同源性可为至少86％、87％、88％、89％的同一性。所述80％以上的同源性可为至少81％、82％、83％、84％的同一性。
第二方面，本发明要求保护一种合成乳糖-N-三糖Ⅱ的方法。
本发明要求保护的合成乳糖-N-三糖Ⅱ的方法，可包括如下步骤(b1)或(b2)或(b3)：
(b1)以HaHex74蛋白为生物酶，催化几丁质水解物和β-乳糖合成乳糖-N-三糖Ⅱ(LNT2)；
(b2)以几丁质酶催化几丁质水解，得到几丁质水解物；然后以HaHex74蛋白催化所述几丁质水解物和β-乳糖合成乳糖-N-三糖Ⅱ(LNT2)；
(b3)以几丁质酶和HaHex74蛋白为生物酶，催化几丁质和β-乳糖合成乳糖-N-三糖Ⅱ(LNT2)。
所述HaHex74蛋白为如上(A1)-(A4)中任一所述蛋白质。
第三方面，本发明要求保护一种合成乳糖-N-新四糖的方法。
本发明所要求保护的合成乳糖-N-新四糖的方法，可包括如下步骤(c1)或(c2)：
(c1)按照前文第二方面所述方法制备得到乳糖-N-三糖Ⅱ(LNT2)；然后以β-半乳糖苷酶催化乳糖-N-三糖Ⅱ(LNT2)和β-乳糖合成乳糖-N-新四糖 (LNnT)；
(c2)以几丁质酶、HaHex74蛋白和β-半乳糖苷酶为生物酶，催化几丁质和β-乳糖合成乳糖-N-三糖Ⅱ(LNT2)和乳糖-N-新四糖(LNnT)。
所述HaHex74蛋白为如上(A1)-(A4)中任一所述蛋白质。
进一步地，在所述(b1)和所述(b2)中，所述几丁质水解物为几丁二糖和N-乙酰氨基葡萄糖混合物。
进一步地，以所述HaHex74蛋白为生物酶，催化所述几丁质水解物和β-乳糖合成乳糖-N-三糖Ⅱ(LNT2)时的pH为7.5，和/或温度为40℃，和/或β-乳糖在反应体系中的含量为0.8M，和/或所述HaHex74蛋白在反应体系中的含量为3-5U/mL(如4U/mL)，和/或反应时间为10h。
进一步地，在以所述几丁质酶催化几丁质水解，得到所述几丁质水解物的过程中，pH为5.5，和/或温度为55℃，和/或所述几丁质酶在反应体系中的含量为5U/mL，和/或反应时间为24h。
进一步地，在以所述β-半乳糖苷酶催化乳糖-N-三糖Ⅱ(LNT2)和β-乳糖合成乳糖-N-新四糖(LNnT)的过程中，pH自然(pH 7.0)，和/或温度为50℃，和/或所述β-半乳糖苷酶在反应体系中的含量为0.1U/mL，和/或反应时间为10h。
在上述各方面中，所述HaHex74蛋白的最适pH为6.5、最适温度为45℃。
在上述各方面中，所述HaHex74蛋白可按照包括如下步骤的方法制备得到：将编码所述HaHex74蛋白的核酸分子导入受体酵母，得到重组酵母；按照如下步骤对所述重组酵母进行发酵培养，从发酵产物中获得所述HaHex74蛋白：
(d1)基础培养阶段：将所述重组酵母接种到添加有50g/L甘油的BSM培养基中培养，控制pH4.0，温度30℃，待甘油浓度低于10g/L时开始甘油流加培养阶段。
(d2)甘油流加培养阶段：流加500g/L甘油，维持甘油浓度为10-25g/L，控制温度28℃，pH5.0，溶氧量为10-20％，直至发酵终点。
在(d1)中，还包括向发酵体系中加入PTM1的步骤。所述PTM1的加入量为4.35mL/L起始发酵液。
在(d1)中，所述重组酵母的接种量为10％体积百分含量。
在(d1)中，培养时控制转速600rpm；在(d2)中，培养时控制转速800rpm。
在(d1)之前，还可包括种子培养阶段：将所述重组酵母接种到BMGY培养基中，30℃培养至OD 600为10.0左右。在所述种子培养阶段，控制转速200rpm。
进一步地，从所述发酵产物中获得所述HaHex74蛋白可按照包括如下步骤的方法进行：将所述发酵产物离心收集上清液；将所述上清液置于Tris-HCl缓冲液中透析，离心得到粗液；将所述粗液使用琼脂糖弱阴离子交换柱DE52纯化得到所述HaHex74蛋白。
更进一步地，用缓冲液A平衡DE52亲和柱5-10个柱体积，将收集的所述粗液 以0.5mL/min流速上样，用所述缓冲液A与缓冲液B线性洗脱至至OD 280<0.05，收集具有β-N-乙酰氨基己糖苷酶活力的组分，透析得到纯化产物即为所述HaHex74蛋白。
其中，所述缓冲液A为含有20mM Tris-HCl缓冲液(pH 8.0)；所述缓冲液B为含有NaCl(500mM)的20mM Tris-HCl缓冲液(pH 8.0)。
进一步地，编码所述HaHex74蛋白的核酸分子可为如下任一所述的DNA分子：
(B1)SEQ ID No.3的第280-2238位或SEQ ID No.3所示的DNA分子；
(B2)在严格条件下与b1)或b2)限定的DNA分子杂交且编码所述HaHex74蛋白的DNA分子；
(B3)与(B1)或(B2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述HaHex74蛋白的DNA分子。
上述基因中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
上述核酸分子中，同源性是指核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定核苷酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对核苷酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述核酸分子中，所述95％以上的同源性可为至少96％、97％、98％的同一性。所述90％以上的同源性可为至少91％、92％、93％、94％的同一性。所述85％以上的同源性可为至少86％、87％、88％、89％的同一性。所述80％以上的同源性可为至少81％、82％、83％、84％的同一性。
进一步地，所述核酸分子可通过重组载体的形式导入所述受体酵母中。
其中，所述重组载体中启动所述核酸分子转录的启动子可为GAP启动子。
更进一步地，所述重组载体为将所述核酸分子克隆到pGAP9K载体(如EcoR I和Not I之间)中后得到的重组载体。其中，所述pGAP9K载体为将pPIC9K载体中的AOX1启动子替换为GAP启动子后所得质粒。
所述GAP启动子的序列如SEQ ID No.1所示。
进一步地，所述酵母为毕赤酵母。
更进一步地，所述毕赤酵母为毕赤酵母GS115。
第四方面，本发明要求保护前文所述HaHex74蛋白的制备方法。
所述HaHex74蛋白的制备方法可包括如下步骤：将编码所述HaHex74蛋白的核酸分子导入受体酵母，得到重组酵母；按照如下步骤对所述重组酵母进行发酵培养，从发酵产物中获得所述HaHex74蛋白：
(d1)基础培养阶段：将所述重组酵母接种到添加有50g/L甘油的BSM培养基中培养，控制pH4.0，温度30℃，待甘油浓度低于10g/L时开始甘油流加培养阶段。
(d2)甘油流加培养阶段：流加500g/L甘油，维持甘油浓度为10-25g/L，控制温度28℃，pH5.0，溶氧量为10-20％，直至发酵终点。
在(d1)中，还包括向发酵体系中加入PTM1的步骤。所述PTM1的加入量为4.35mL/L起始发酵液。
在(d1)中，所述重组酵母的接种量为10％体积百分含量。
在(d1)中，培养时控制转速600rpm；在(d2)中，培养时控制转速800rpm。
在(d1)之前，还可包括种子培养阶段：将所述重组酵母接种到BMGY培养基中，30℃培养至OD 600为10.0左右。在所述种子培养阶段，控制转速200rpm。
进一步地，从所述发酵产物中获得所述HaHex74蛋白可按照包括如下步骤的方法进行：将所述发酵产物离心收集上清液；将所述上清液置于Tris-HCl缓冲液中透析，离心得到粗液；将所述粗液使用琼脂糖弱阴离子交换柱DE52纯化得到所述HaHex74蛋白。
更进一步地，用缓冲液A平衡DE52亲和柱5-10个柱体积，将收集的所述粗液以0.5mL/min流速上样，用所述缓冲液A与缓冲液B线性洗脱至至OD 280<0.05，收集具有β-N-乙酰氨基己糖苷酶活力的组分，透析得到纯化产物即为所述HaHex74蛋白。
其中，所述缓冲液A为含有20mM Tris-HCl缓冲液(pH 8.0)；所述缓冲液B为含有NaCl(500mM)的20mM Tris-HCl缓冲液(pH 8.0)。
进一步地，编码所述HaHex74蛋白的核酸分子可为如下任一所述的DNA分子：
(B1)SEQ ID No.3的第280-2238位或SEQ ID No.3所示的DNA分子；
(B2)在严格条件下与b1)或b2)限定的DNA分子杂交且编码所述HaHex74蛋白的DNA分子；
(B3)与(B1)或(B2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述HaHex74蛋白的DNA分子。
上述基因中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在 7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
上述核酸分子中，同源性是指核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定核苷酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对核苷酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述核酸分子中，所述95％以上的同源性可为至少96％、97％、98％的同一性。所述90％以上的同源性可为至少91％、92％、93％、94％的同一性。所述85％以上的同源性可为至少86％、87％、88％、89％的同一性。所述80％以上的同源性可为至少81％、82％、83％、84％的同一性。
进一步地，所述核酸分子可通过重组载体的形式导入所述受体酵母中。
其中，所述重组载体中启动所述核酸分子转录的启动子可为GAP启动子。
更进一步地，所述重组载体为将所述核酸分子克隆到pGAP9K载体(如EcoR I和Not I之间)中后得到的重组载体。其中，所述pGAP9K载体为将pPIC9K载体中的AOX1启动子替换为GAP启动子后所得质粒。
所述GAP启动子的序列如SEQ ID No.1所示。
进一步地，所述酵母为毕赤酵母。
更进一步地，所述毕赤酵母为毕赤酵母GS115。
附图说明
图1为高密度发酵β-N-乙酰氨基己糖苷酶HaHex74表达量和胞外蛋白SDS-PAGE分析图。A为HaHex74表达量((■)：酶活；(▲)：蛋白浓度；(●)：湿重)；B为SDS-PAGE分析图
图2为β-N-乙酰氨基己糖苷酶HaHex74的纯化电泳图。
图3为β-N-乙酰氨基己糖苷酶HaHex74的最适反应pH、pH稳定性、最适温度和温度稳定性的测定曲线图。A为最适反应pH；B为pH稳定性；C为最适温度；D为温度稳定性。其中，(■)柠檬酸缓冲液(pH 5.0-6.0)、(●)乙酸-乙酸钠缓冲液(pH 4.0-6.0)、(▲)MES缓冲液(pH 5.5-6.5)、(▼)MOPS缓冲液(pH 6.5-7.5)、(◆)磷酸缓冲液(pH 6.0-8.0)、
Figure PCTCN2020122119-appb-000003
Tris-HCl缓冲液(pH 7.0-9.0)、
Figure PCTCN2020122119-appb-000004
甘氨酸-氢氧化钠缓冲液(pH 8.5-10.5)。
图4为β-N-乙酰氨基己糖苷酶HaHex74的合成LNT2最适pH(A)、最适温度(B)、最适反应时间(C)、最适受体浓度(D)和最适加酶量(E)的测定曲线图。A为最适pH；B为最适温度；C为最适反应时间；D为最适受体浓度；D为最适加酶量。其中(■)MES缓冲液(pH 5.5-6.5)、(▲)磷酸缓冲液(pH 6.0-8.0)、 (▼)Tris-HCl缓冲液(pH 7.0-9.0)。
图5为β-N-乙酰氨基己糖苷酶HaHex74转糖苷产物的一级质谱图。
图6为β-N-乙酰氨基己糖苷酶HaHex74转糖苷产物的一级 1H谱图。
图7为β-N-乙酰氨基己糖苷酶HaHex74转糖苷产物的一级 13C谱图。
图8为β-N-乙酰氨基己糖苷酶HaHex74转糖苷产物的二级异核单量子关系(HSQC)谱图。
图9为合成LNT2的HPLC分析((■)：转化率；(●)：LNT2浓度)。
图10为合成LNnT的HPLC分析((■)：转化率；(●)：LNnT浓度)。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中β-N-乙酰氨基己糖苷酶酶活测定方法如下：
取0.1mL适当稀释的酶液，加入到0.1mL浓度为2mM的pNP-NAG底物溶液(用50mM，pH 6.5的磷酸缓冲液配制)中，45℃水浴反应10min，测定其在410nm下的吸光度(OD 410)(Yang et al.Biochemical characterization of the first fungal glycoside hydrolyase family 3β-N-acetylglucosaminidase from Rhizomucor miehei.J.Agric.Food Chem.,2014,62,5181-5190)。
β-N-乙酰氨基己糖苷酶的活力单位定义为：在上述反应条件下，每分钟反应生成1μmol的pNP所需要的酶量为一个酶活力单位(1U)。
比酶活定义为1mg蛋白所具有的酶活力单位，表示为U·mg -1。
1个β-N-乙酰氨基己糖苷酶酶活单位的定义：在pH 6.5、45℃条件下，每分钟分解浓度为2mM的pNP-NAG底物溶液(用50mM，pH 6.5的磷酸缓冲液配制)释放1μmol的pNP所需要的酶量，酶活计算公式为：H＝Cx×n/(T×V)，其中，H代表酶活力(U/mL)，Cx代表生成pNP物质的量(μmol)，n代表酶液的稀释倍数，T代表反应时间(min)，V代表加入稀释后的酶液体积(mL)。
实施例1、β-N-乙酰氨基己糖苷酶基因的发掘及工程菌的构建
1、β-N-乙酰氨基己糖苷酶基因的确定
通过CAZy数据库查找已报道的β-N-乙酰氨基己糖苷酶的蛋白序列，将序列进行同源序列比对分析，得到来源于Haloferula sp.的一个糖苷水解酶20家族的β-N-乙酰氨基己糖苷酶基因(命名为HaHex74)。HaHex74基因的核苷酸序列为“ATG+SEQ ID No.3的第280-2238”，编码“Met+SEQ ID No.2的第94-745位”所示氨基酸序列。HaHex74基因与一个土壤宏基因组β-N-乙酰氨基己糖苷酶(AKC34129)的同源性最高，为61％。之后送去上海生工生物有限公司合成基 因。
2、表达β-N-乙酰氨基己糖苷酶工程菌的构建
2.1、根据毕赤酵母GS115基因组中三磷酸甘油醛脱氢酶(GAP)启动子基因序列设计引物：
GAP1：5’-GCAGC GAGCTCATCCTTTTTTGTAGAAATGTCTTGG-3’；
GAP2：5’-CGC GGATCCTGTGTTTTGATAGTTGTTCAATTGA-3’。
从毕赤酵母GS115基因组中扩增GAP启动子，将GAP启动子片段和pPIC9K载体用Sac I和BamH Ⅰ双酶切，经T4DNA连接酶连接得到改造后的表达载体，命名为pGAP9K，即将pPIC9K载体中的AOX1启动子替换为GAP启动子。其中，GAP启动子序列如SEQ ID No.1所示。
2.2、根据β-N-乙酰氨基己糖苷酶的序列设计表达引物，上下游引物分别添加上EcoRI和NotI酶切位点上下游引物分别如下：
上游引物：5’–CCG GAATTCGAACCAACCATTATTCCATTGCC-3’；
下游引物：5’–AGAAT GCGGCCGCTTACTCAACGGTGATTTCGTGGATA-3’。
用上述引物进行PCR扩增，PCR扩增条件为：95℃预变性30s；95℃变性20s，58℃退火20s，72℃延伸1min，循环34次；最后72℃后延伸5min。PCR产物经过1％琼脂糖凝胶电泳回收，以EcoRI和NotI双酶切。将该双酶切后的产物与经相同双酶切过的酵母表达载体pGAP9K的载体骨架片段以T4DNA连接酶进行连接，转化至宿主大肠杆菌DH5α。挑选菌落PCR(PCR所用的引物和扩增条件与本段前述PCR的相同)验证为阳性的转化子测序。将测序验证正确的重组酵母表达载体命名为pGAP9K-HaHex74。将得到的重组巴斯德毕赤酵母表达载体pGAP9K-HaHex74以限制性内切酶XbaI线性化后得电转化巴斯德毕赤酵母GS115，构成重组菌。
重组表达载体pGAP9K-HaHex74中GAP启动子序列为SEQ ID No.1中的DNA分子(即通过Sac I和BamH Ⅰ双酶切将pPIC9K载体中的AOX1启动子替换为GAP启动子，构建新的表达载体pGAP9K)，重组表达载体pGAP9K-HaHex74完整的ORF序列为SEQ ID No.3中的DNA分子，其中，第1-279位为pGAP9K载体上的序列，第280-2238位为HaHex74基因序列。
实施例2、β-N-乙酰氨基己糖苷酶的制备及其酶学性质
1、β-N-乙酰氨基己糖苷酶的高拷贝筛选
将实施例1中得到的重组菌涂布MD板(1.34％YNB，4×10 -5％生物素，2％葡萄糖)，从MD平板得到的His +转化子经灭菌水刮取后取100μL涂布于不同浓度的YPD-G418平板(1％酵母提取物，2％胰蛋白胨，2％葡萄糖，G418浓度分别为2，3，4，6mg/mL)。30℃下培养3～5d后，挑取转化子于BMGY培养基摇瓶培养16～18h，3000rpm离心5min，收集菌体，用BMGY培养基重悬菌体至OD 600为1.0左右，分批流加甘油使目标蛋白表达。以上培养条件为100mL三角 瓶装量20mL培养基，温度30℃，转速200rpm。每24h加甘油至终浓度为1％以持续3天，测定发酵液酶活，并通过SDS-PAGE分析HaHex74重组蛋白表达情况。
2、毕赤酵母重组菌高密度发酵
将实施例2步骤1获得的产酶水平高的毕赤酵母菌株在5L发酵罐中进行高密度发酵。发酵过程中所用到的培养基(种子培养基BMGY、发酵基本培养基BSM和甘油分批补料培养基)参照Pichia Fermentation Process Guidelines(Version B，053002，Invitrogen)配制。整个发酵过程采用基础培养和甘油流加培养两个阶段。
(1)种子培养：选择摇瓶发酵中产酶水平较高的菌株，接种150mL BMGY培养基中，30℃，200rpm培养至OD 600为10.0左右。
(2)基础培养：将步骤(1)所得的种子液接种到5L发酵罐中(装有1.35L发酵基本培养基BSM和5％(50g/L)的甘油，发酵罐灭菌，28％浓氨水调pH 4.0，加入PTM1(本领域公知，微量元素，促进酵母表达)4.35mL/L起始发酵液，接种量10％(v/v)，转速600rpm，温度30℃。待甘油浓度低于1％(10g/L)时，开始流加50％(w/v)的甘油(500g/L)补料培养基。
(3)甘油流加培养：流加50％(w/v)的甘油(500g/L)，控制温度28℃，pH 5.0，转速800rpm，维持甘油浓度为1％～2.5％(10g/L～25g/L)，始终监视溶氧，适时调节转速和通气量维持DO10％～20％，直至发酵终点。
发酵过程中取样测定菌体湿重、蛋白含量和酶活。结果显示，发酵168h酶活达到最高，发酵上清液酶活3500U/mL，蛋白浓度9.7g/L(图1)。
3、β-N-乙酰氨基己糖苷酶的纯化
高密度发酵结束后，取发酵液在10,000×g条件下离心5min，收集上清液，取50mL上清液置于Tris-HCl缓冲液(20mM，pH 8.0)中透析过夜，透析结束后于10,000×g离心5min，获得粗酶液。粗酶液使用琼脂糖弱阴离子交换柱DE52(DEAE Sepharose Fast Flow)一步纯化重组蛋白。具体步骤如下：
用缓冲液A平衡DE52亲和柱5-10个柱体积，将收集的酶液以0.5mL/min流速上样，使用蛋白纯化系统(
Figure PCTCN2020122119-appb-000005
GE Healthcare,USA)，将缓冲液A与缓冲液B线性洗脱至OD 280<0.05(洗脱程序：0-30min，A 100％；30-120min，A 100％-0，B 0-100％；120-135min，B 100％；％表示体积百分含量)，收集具有β-N-乙酰氨基己糖苷酶活力的组分，SDS-PAGE检验纯度，透析得到纯化产物。
其中，缓冲液A为含有20mM Tris-HCl缓冲液(pH 8.0)；缓冲液B为含有NaCl(500mM)的20mM Tris-HCl缓冲液(pH 8.0)。
重组菌的粗酶液得到的纯化产物为重组蛋白HaHex74。
重组菌的粗酶液及其得到的纯化产物(重组蛋白HaHex74)的SDS-PAGE纯化图如图2所示。图2中，泳道M为分子量标准，泳道1为重组蛋白HaHex74 粗酶，泳道2为HaHex74纯酶。图2的结果表明，重组蛋白HaHex74的大小约为74kDa，与预期大小一致。分别将粗酶液和纯酶液作为待测酶液，并以对应灭活的蛋白作为对照，检测β-N-乙酰氨基己糖苷酶的酶活力。表1所示粗酶液的总酶活力为3500U，其比酶活为360.8U mg -1；纯酶液的总酶活力为2890U，其比酶活为385.3U mg -1；纯化倍数为1.1。
表1 β-N-乙酰氨基己糖苷酶HaHex74的纯化表
Figure PCTCN2020122119-appb-000006
4、β-N-乙酰氨基己糖苷酶的酶学性质
(1)最适pH的测定
将上述制备得到的HaHex74纯酶液作为待测酶液，将其分别在不同缓冲液体系中，于40℃下进行酶活测定，以最高酶活力为100％计算相对酶活。各种缓冲如下：
1)柠檬酸缓冲液(pH 5.0-6.0)
2)乙酸-乙酸钠缓冲液(pH 4.0-6.0)
3)MES缓冲液(pH 5.5-6.5)
4)MOPS缓冲液(pH 6.5-7.5)
5)磷酸缓冲液(pH 6.0-8.0)
6)Tris-HCl缓冲液(pH 7.0-9.0)
7)甘氨酸-氢氧化钠缓冲液(pH 8.5-10.5)。
结果见图3中A：HaHex74的最适pH为6.5。
(2)pH稳定性测定
将HaHex74用上述缓冲液进行稀释，置于35℃水浴锅中处理30min，将其迅速置于冰水中冷却30min，然后测定酶活力。以未经处理的β-N-乙酰氨基己糖苷酶的酶活力作为100％，计算经过不同pH处理后HaHex74的相对酶活力。
结果如图3中B所示：HaHex74具有良好的pH稳定性，在pH 5.5-9.0范围内保温30min后仍残留80％以上的酶活力。
(3)最适温度的测定
将HaHex74用50mM磷酸缓冲液(pH 6.5)适当稀释后，分别在不同温度(20、25、30、35、40、45、50、55、60、65、70℃)测定酶活力。以最高酶活力为100％，计算相对酶活力。
结果见图3中C：HaHex74的最适温度为45℃。
(4)温度稳定性的测定
将HaHex74用50mM磷酸缓冲液(pH 6.5)适当稀释后，分别在不同温度(20、25、30、35、40、45、50、55、60℃)保温30min，将其迅速置于冰水 中冷却30min，然后测定酶活力。以未经处理的β-N-乙酰氨基己糖苷酶的酶活力作为100％，计算经过不同温度处理后HaHex74的相对酶活力。
结果见图3中D：HaHex74在45℃以下具有良好的稳定性。
(4)底物特异性
分别以胶体几丁质、乙二醇几丁质、羧甲基纤维素(CMC)、壳聚糖溶液、对硝基苯基-β-N-乙酰氨基葡萄糖苷(pNP-NAG)和对硝基苯基-β-N-乙酰氨基半乳糖苷(pNP-GalNAc)等作为反应底物，在标准酶活力测定条件下检测酶活，考察酶的底物特异性，结果见表2。
表2 β-N-乙酰氨基己糖苷酶HaHex74的底物特异性
底物 比酶活(U mg -1) 相对活性(％)
pNP-NAG 385.3±3.0 100
pNP-GalNAc 51.8±1.8 13.4
(GlcNAc) 2 73.4±1.0 a 19.1
(GlcNAc) 3 112.8±2.2 29.3
(GlcNAc) 4 54.3±1.2 14.1
(GlcNAc) 5 44.2±2.3 11.5
胶体几丁质 0.14±0.06 0.04
乙二醇几丁质 - -
壳聚糖 - -
CMC - -
注： a对几丁寡糖酶活测定方法：取0.1mL适当稀释的酶液，加入到0.1mL1％(质量体积比，10g/L)的几丁寡糖底物溶液中(用50mM，pH 6.5的磷酸缓冲液配制)，45℃水浴反应10min。采用高效液相色谱(HPLC)法测定所释放的β-N-乙酰氨基葡萄糖量，以β-N-乙酰氨基葡萄糖作为标准。HPLC测定条件为：HPLC-RID检测系统(Agilent 1260 infinity II,Agilent Technologies,USA)BP-800Pb++层析柱(Benson Polymeric,Reno,NE,7.8×300mm,USA)，蒸馏水作为流动相，柱温80℃，流速为1mL·min -1。β-N-乙酰氨基葡萄糖苷酶的活力单位定义为：在上述反应条件下，每分钟反应生成1μmol的β-N-乙酰氨基葡萄糖所需要的酶量为一个酶活力单位(1U)。
结果显示：HaHex74对pNP-NAG的酶活最高，为385.3U/mg；其次是几丁三糖、几丁二糖和几丁四糖。对胶体几丁质显示出微弱的活性(0.14U·mg -1)，对乙二醇几丁质、壳聚糖和CMC没有显示出活性。
实施例3、合成LNT2条件的优化
合成LNT2的量利用HPLC测定。HPLC测定条件为：Waters XBridge BEH Amide 5μm色谱柱(250×4.6mm)，72％乙腈，柱温45℃，流速0.5mL/min，45min，示差(RID)检测器。
转糖苷产物得率(％)＝合成产物的浓度(mM)/糖苷供体的初始浓度(mM)×100
1、最适pH的测定
将实施例2制备得到的HaHex74纯酶液作为待测酶液，将其分别在不同缓冲液体系中，测定LNT2含量。各种缓冲如下：
1)MES缓冲液(pH 5.5-6.5)
2)磷酸缓冲液(pH 6.0-8.0)
3)Tris-HCl缓冲液(pH 7.0-9.0)
结果见图4中A：HaHex74合成LNT2最适pH为7.5。
2、最适温度的测定
将实施例2制备得到的HaHex74纯酶液作为待测酶液，将反应体系置于不同温度(20-55℃)下水浴反应1.5h，测定LNT2含量。
结果见图4中B：HaHex74合成LNT2最适温度为40℃。
3、最适反应时间的测定
将实施例2制备得到的HaHex74纯酶液作为待测酶液，将反应体系置于40℃下水浴反应不同时间，测定LNT2含量。
结果见图4中C：HaHex74合成LNT2最适反应时间为2.5h。
4、最适乳糖浓度的测定
将实施例2制备得到的HaHex74纯酶液作为待测酶液，将不同浓度的β-乳糖加入反应体系中，40℃下水浴反应2.5h，测定LNT2含量。
结果见图4中D：HaHex74合成LNT2最适乳糖浓度为0.8M。
5、最适加酶量的测定
将实施例2制备得到的HaHex74纯酶液作为待测酶液，在反应体系中加入不同浓度的HaHex74，40℃下水浴反应2.5h，测定LNT2含量。
结果见图4中E：HaHex74合成LNT2最适加酶量为3-5U·mL -1。
实施例4、合成LNT2的纯化和结构鉴定
实施例3优化条件反应后的产物通过HPLC纯化。收集的产物经TLC分析验证纯度(展层剂为正丁醇：乙醇：水＝2:1:1(v/v/v)，显色剂为甲醇：硫酸＝95:5(v/v))，冷冻干燥后的样品为白黄色粉末。
将样品溶解于纯水中，使用Thermo Scientific TM Q Exactive TM质谱仪，在ESI离子源positive-ion模式下，采集样品的高分辨率一级质谱图。
将样品溶解于D 2O中并转移到核磁专用试管中，加入DSS(3-(三甲基硅基)-1-丙磺酸钠)作为内标，使用500MHz Varian V NMR SYSTEM TM设备，在298K温度条件下采集信号。使用Agilent DD2 500MHz核磁共振波谱仪分析所有样品的一维 1H和 13C光谱，二维 1H- 13C异核单量子相干波谱(heteronuclear single quantum coherencespectroscopy，HSQC)使用Agilent提供的标准脉 冲序列和相关参数获得。
结果表明，纯化后的产物在一级高分辨率质谱图中在质荷比(m/z)[M+Na] +为568.3处呈现单一离子峰(图5)，表明该产物的分子量为545.3，这与LNT2的分子量(545)一致。之前HaHex74合成产物HPLC分析显示在目标产物处有一个主峰和一个次要峰，经质谱分析显示其为同分异构体。
进一步通过NMR测定该转糖苷产物的结构，由一维 1H及 13C图谱可获得该化合物 1H质子的化学位移(图6)和 13C的化学位移(图7)。由二维异核单量子关系(HSQC)谱图可获得化合物碳氢原子之间的关系(图8)。经数据比对，HaHex74产物与双歧杆菌(Bifidobacterium bifidum)来源的β-N-乙酰氨基己糖苷酶突变株转糖苷产物一致(
Figure PCTCN2020122119-appb-000007
et al.Glycosynthase principle transformed into biocatalytic process technology:Lacto-N-triose II production with engineered exo-hexosaminidase.ACS Catal.2019,9,5503-5514)，表明该化合物为LNT2。
实施例5、LNT2的制备
1、以几丁质为起始原料
将120g球磨几丁质粉末(3％，w/v)(江正强等，CN201811105536)溶于4L 20mM的柠檬酸缓冲液中(pH 5.5)，加入5U·mL -1的几丁质酶(Yang et al.Cloning,expression,purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii.Food Chem.,2016,192,1041-1048)，至于55℃下酶解24h。
2、LNT2的制备
将步骤1水解产物(几丁二糖和N-乙酰氨基葡萄糖混合物)离心取上清，再浓缩10倍，加入NaH 2PO 4调节pH至7.5，加入0.8M的β-乳糖和4U·mL -1HaHex74(实施例2获得的HaHex74纯酶液)，40℃下反应一定时间，间隔时间取样，利用HPLC测定LNT2含量(测定条件见实施例3)。
LNT2转化率(％)＝合成LNT2量(M)/几丁二糖的量(M)×100％；其中M表示物质的量。
结果显示，反应10h，LNT2的量达到最高，转化率为13-20％，其浓度达到8-12g·L -1(图9)。
实施例6、LNnT的制备
上述实施例5反应产物中多余β-乳糖利用活性炭柱法(75×1.5cm)去除，使用梯度混合仪(TH-2000，上海青浦沪西仪器厂)，利用0-25％(v/v)的乙醇溶液以1mL min -1的流速将上述实施例5反应产物进行梯度洗脱，收集洗脱液，利用TLC(条件见实施例4)和HPLC检测产物(条件见实施例3)。将经过活性炭柱的溶液浓缩10倍，pH自然(pH 7.0)，加入0.1U mL -1的β-半乳糖苷酶 (Zeuner et al.Thermostableβ-galactosidases for the synthesis of human milk oligosaccharides.New Biotechnol.,2016,33,355-360)于50℃下反应一定时间，间隔取样，所有样品与沸水浴中灭活10min。将所有样品进行HPLC分析(条件见实施例3)。
LNnT转化率(％)＝合成LNnT量(M)/乳糖的量(M)×100％；其中M表示物质的量。
结果显示，经过碳柱并浓缩的LNT2溶液中含有20mMβ-乳糖和90mM LNT2，反应10h，LNnT的量达到最高，转化率为7-21％，其浓度达到1-3g·L -1(图10)。
以上，仅为本发明较佳的具体实施方式，但发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求书的保护范围为准。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。
工业应用
本发明将Haloferula sp.的一个糖苷水解酶20家族的β-N-乙酰氨基己糖苷酶基因在毕赤酵母中高效表达。经5L发酵罐高密度发酵后，发酵液的酶活力可达3500U·mL -1，蛋白含量为9.7g·L -1，为目前β-N-乙酰氨基己糖苷酶表达的最高水平。新发明的β-N-乙酰氨基己糖苷酶HaHex74具有优异的转糖苷活性，能够以天然供体几丁二糖((GlcNAc) 2)和β-乳糖高效合成人乳寡糖重要的骨架结构LNT2，转化率为10-20％，为目前β-N-乙酰氨基己糖苷酶利用天然供体合成LNT2的最高水平。利用几丁质酶和HaHex74可高效将几丁质粉末转化为LNT2，进一步协同β-半乳糖苷酶可高效合成LNnT，其浓度分别为8-12g·L -1和1-3g·L -1。本发明的β-N-乙酰氨基己糖苷酶产酶水平高、水解特性优异以及转糖苷活性高，在酶法合成人乳寡糖中具有重要的应用价值。

Claims (31)

  1. HaHex74蛋白或其相关生物材料在如下任一中的应用：
    (a1)合成人乳寡糖；
    (a2)合成乳糖-N-三糖Ⅱ和/或乳糖-N-新四糖；
    所述HaHex74蛋白为如下任一：
    (A1)氨基酸序列为SEQ ID No.2的第94-745位或SEQ ID No.2的蛋白质；
    (A2)将SEQ ID No.2的第94-745位或SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
    (A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且来源于Haloferula sp.的具有相同功能的蛋白质；
    (A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白；
    所述相关生物材料为能够表达所述HaHex74蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
  2. 根据权利要求1所述的应用，其特征在于：所述HaHex74蛋白是按照包括如下步骤的方法制备得到的：将编码所述HaHex74蛋白的核酸分子导入受体酵母，得到重组酵母；按照如下步骤对所述重组酵母进行发酵培养，从发酵产物中获得所述HaHex74蛋白：
    (d1)基础培养阶段：将所述重组酵母接种到添加有50g/L甘油的BSM培养基中培养，控制pH4.0，温度30℃，待甘油浓度低于10g/L时开始甘油流加培养阶段。
    (d2)甘油流加培养阶段：流加500g/L甘油，维持甘油浓度为10-25g/L，控制温度28℃，pH5.0，溶氧量为10-20％，直至发酵终点。
  3. 根据权利要求2所述的应用，其特征在于：从所述发酵产物中获得所述HaHex74蛋白是按照包括如下步骤的方法进行的：将所述发酵产物离心收集上清液；将所述上清液置于Tris-HCl缓冲液中透析，离心得到粗液；将所述粗液使用琼脂糖弱阴离子交换柱DE52纯化得到所述HaHex74蛋白。
  4. 根据权利要求2或3所述的应用，其特征在于：编码所述HaHex74蛋白的核酸分子为如下任一所述的DNA分子：
    (B1)SEQ ID No.3的第280-2238位或SEQ ID No.3所示的DNA分子；
    (B2)在严格条件下与(B1)限定的DNA分子杂交且编码所述HaHex74蛋白的DNA分子；
    (B3)与(B1)或(B2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述HaHex74蛋白的DNA分子。
  5. 根据权利要求4所述的应用，其特征在于：所述核酸分子是通过重组载体的形式导入所述受体酵母中的。
  6. 根据权利要求5所述的应用，其特征在于：所述重组载体中启动所述核酸分子转录的启动子为GAP启动子。
  7. 根据权利要求6所述的应用，其特征在于：所述GAP启动子的序列如SEQ ID No.1所示。
  8. 根据权利要求2-7中任一所述的应用，其特征在于：所述酵母为毕赤酵母。
  9. 根据权利要求8所述的应用，其特征在于：所述毕赤酵母为毕赤酵母GS115。
  10. 一种合成乳糖-N-三糖Ⅱ的方法，包括如下步骤(b1)或(b2)或(b3)：
    (b1)以HaHex74蛋白为生物酶，催化几丁质水解物和β-乳糖合成乳糖-N-三糖Ⅱ；
    (b2)以几丁质酶催化几丁质水解，得到几丁质水解物；然后以HaHex74蛋白催化所述几丁质水解物和β-乳糖合成乳糖-N-三糖Ⅱ；
    (b3)以几丁质酶和HaHex74蛋白为生物酶，催化几丁质和β-乳糖合成乳糖-N-三糖Ⅱ；
    所述HaHex74蛋白为如下任一：
    (A1)氨基酸序列为SEQ ID No.2的第94-745位或SEQ ID No.2的蛋白质；
    (A2)将SEQ ID No.2的第94-745位或SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
    (A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且来源于Haloferula sp.的具有相同功能的蛋白质；
    (A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
  11. 一种合成乳糖-N-新四糖的方法，包括如下步骤(c1)或(c2)：
    (c1)按照权利要求10所述方法制备得到乳糖-N-三糖Ⅱ；然后以β-半乳糖苷酶催化乳糖-N-三糖Ⅱ和β-乳糖合成乳糖-N-新四糖；
    (c2)以几丁质酶、HaHex74蛋白和β-半乳糖苷酶为生物酶，催化几丁质和β-乳糖合成乳糖-N-三糖Ⅱ和乳糖-N-新四糖；
    所述HaHex74蛋白为如下任一：
    (A1)氨基酸序列为SEQ ID No.2的第94-745位或SEQ ID No.2的蛋白质；
    (A2)将SEQ ID No.2的第94-745位或SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
    (A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质；
    (A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
  12. 根据权利要求10或11所述的方法，其特征在于：所述(b1)和所述(b2)中，所述几丁质水解物为几丁二糖和N-乙酰氨基葡萄糖混合物。
  13. 根据权利要求10-12中任一所述的方法，其特征在于：以所述HaHex74蛋白为生物酶，催化所述几丁质水解物和β-乳糖合成乳糖-N-三糖Ⅱ时的pH为7.5，和/或温度为40℃，和/或β-乳糖在反应体系中的含量为0.8M，和/或所述HaHex74蛋白在反应体系中的含量为3-5U/mL，和/或反应时间为10h。
  14. 根据权利要求10-13中任一所述的方法，其特征在于：在以所述几丁质酶催化几丁质水解，得到所述几丁质水解物的过程中，pH为5.5，和/或温度为55℃，和/或所述几丁质酶在反应体系中的含量为5U/mL，和/或反应时间为24h。
  15. 根据权利要求10-14任一所述的方法，其特征在于：在以所述β-半乳糖苷酶催化乳糖-N-三糖Ⅱ和β-乳糖合成乳糖-N-新四糖的过程中，温度为50℃，和/或所述β-半乳糖苷酶在反应体系中的含量为0.1U/mL，和/或反应时间为10h。
  16. 根据权利要求10-15中任一所述的方法，其特征在于：所述HaHex74蛋白是按照包括如下步骤的方法制备得到的：将编码所述HaHex74蛋白的核酸分子导入受体酵母，得到重组酵母；按照如下步骤对所述重组酵母进行发酵培养，从发酵产物中获得所述HaHex74蛋白：
    (d1)基础培养阶段：将所述重组酵母接种到添加有50g/L甘油的BSM培养基中培养，控制pH4.0，温度30℃，待甘油浓度低于10g/L时开始甘油流加培养阶段。
    (d2)甘油流加培养阶段：流加500g/L甘油，维持甘油浓度为10-25g/L，控制温度28℃，pH5.0，溶氧量为10-20％，直至发酵终点。
  17. 根据权利要求16所述的方法，其特征在于：从所述发酵产物中获得所述HaHex74蛋白是按照包括如下步骤的方法进行的：将所述发酵产物离心收集上清液；将所述上清液置于Tris-HCl缓冲液中透析，离心得到粗液；将所述粗液使用琼脂糖弱阴离子交换柱DE52纯化得到所述HaHex74蛋白。
  18. 根据权利要求16或17所述的方法，其特征在于：编码所述HaHex74蛋白的核酸分子为如下任一所述的DNA分子：
    (B1)SEQ ID No.3的第280-2238位或SEQ ID No.3所示的DNA分子；
    (B2)在严格条件下与(B1)限定的DNA分子杂交且编码所述HaHex74蛋白的DNA分子；
    (B3)与(B1)或(B2)限定的DNA序列具有99％以上、95％以上、90％以上、 85％以上或者80％以上同源性且编码所述HaHex74蛋白的DNA分子。
  19. 根据权利要求18所述的方法，其特征在于：所述核酸分子是通过重组载体的形式导入所述受体酵母中的。
  20. 根据权利要求19所述的方法，其特征在于：所述重组载体中启动所述核酸分子转录的启动子为GAP启动子。
  21. 根据权利要求20所述的方法，其特征在于：所述GAP启动子的序列如SEQ ID No.1所示。
  22. 根据权利要求16-21中任一所述的方法，其特征在于：所述酵母为毕赤酵母。
  23. 根据权利要求22所述的方法，其特征在于：所述毕赤酵母为毕赤酵母GS115。
  24. 一种制备权利要求1中所述HaHex74蛋白的方法，包括如下步骤：将编码所述HaHex74蛋白的核酸分子导入受体酵母，得到重组酵母；按照如下步骤对所述重组酵母进行发酵培养，从发酵产物中获得所述HaHex74蛋白：
    (d1)基础培养阶段：将所述重组酵母接种到添加有50g/L甘油的BSM培养基中培养，控制pH4.0，温度30℃，待甘油浓度低于10g/L时开始甘油流加培养阶段。
    (d2)甘油流加培养阶段：流加500g/L甘油，维持甘油浓度为10-25g/L，控制温度28℃，pH5.0，溶氧量为10-20％，直至发酵终点。
  25. 根据权利要求24所述的方法，其特征在于：从所述发酵产物中获得所述HaHex74蛋白是按照包括如下步骤的方法进行的：将所述发酵产物离心收集上清液；将所述上清液置于Tris-HCl缓冲液中透析，离心得到粗液；将所述粗液使用琼脂糖弱阴离子交换柱DE52纯化得到所述HaHex74蛋白。
  26. 根据权利要求24或25所述的方法，其特征在于：编码所述HaHex74蛋白的核酸分子为如下任一所述的DNA分子：
    (B1)SEQ ID No.3的第280-2238位或SEQ ID No.3所示的DNA分子；
    (B2)在严格条件下与(B1)限定的DNA分子杂交且编码所述HaHex74蛋白的DNA分子；
    (B3)与(B1)或(B2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述HaHex74蛋白的DNA分子。
  27. 根据权利要求26所述的方法，其特征在于：所述核酸分子是通过重组载体的形式导入所述受体酵母中的。
  28. 根据权利要求27所述的方法，其特征在于：所述重组载体中启动所述核酸分子转录的启动子为GAP启动子。
  29. 根据权利要求28所述的方法，其特征在于：所述GAP启动子的序列如SEQ ID No.1所示。
  30. 根据权利要求24-29中任一所述的方法，其特征在于：所述酵母为毕 赤酵母。
  31. 根据权利要求30所述的方法，其特征在于：所述毕赤酵母为毕赤酵母GS115。
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