WO2023041040A1 - 耐高温甘露聚糖酶突变体 - Google Patents

耐高温甘露聚糖酶突变体 Download PDF

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WO2023041040A1
WO2023041040A1 PCT/CN2022/119328 CN2022119328W WO2023041040A1 WO 2023041040 A1 WO2023041040 A1 WO 2023041040A1 CN 2022119328 W CN2022119328 W CN 2022119328W WO 2023041040 A1 WO2023041040 A1 WO 2023041040A1
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mannanase
mutant
enzyme activity
amino acid
high temperature
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French (fr)
Inventor
吴秀秀
李�瑞
李玉强
黄亦钧
李馨培
许丽红
宋清清
辛文
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Qingdao Vland Biotech Group Co Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
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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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
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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
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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
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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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger
    • C—CHEMISTRY; METALLURGY
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma
    • C—CHEMISTRY; METALLURGY
    • 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)
    • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase

Definitions

  • the invention relates to genetic engineering and protein modification technology, in particular to a high temperature resistant mannanase mutant.
  • Mannan is a non-toxic, odorless, high-viscosity high-molecular natural polymer, which is widely used in the fields of medicine, food and cosmetics.
  • mannan present in plant cell walls or seeds is very easy to swell when exposed to water, and its viscosity is greatly increased, which will have a very adverse effect on the development and utilization of these substances.
  • mannan in feed can combine a large amount of water to form a colloidal substance with extremely high viscosity, which affects the hydrolysis of feed by digestive enzymes, thereby reducing the digestion and utilization rate of feed by animals. Therefore, Mannan has become a common anti-nutritional factor in the field of feed.
  • ⁇ -mannanase ( ⁇ -mannanase, EC3.2.1.78) is a glycoside hydrolase that can degrade ⁇ -1,4-mannosidic bonds, and its substrates are linear mannan and glucomannan sugar, galactomannan and galactoglucomannan, etc.
  • Various mannans can be hydrolyzed by ⁇ -mannanase to form mannose and mannan oligosaccharides (Mannan oligosaccharides, MOS, usually composed of 2-10 monosaccharide residues).
  • ⁇ -mannanase is widely found in plants, animals and microorganisms.
  • ⁇ -mannanase is found in the intestinal secretions of some lower animals and the germinated seeds of some plants.
  • most of the ⁇ -mannanases that have been reported are derived from microorganisms, mainly including bacteria, fungi and actinomycetes, among which the ⁇ -mannanases produced by bacteria generally have an optimal reaction pH value of 5.5-7.0, The isoelectric point is 4.0-5.0, and the optimum reaction temperature is 50-70°C.
  • the isoelectric point of ⁇ -mannanase derived from fungi is usually 4.0-5.0, the optimum reaction temperature is 55-75°C, and the optimum reaction pH value is 4.5-5.5, which belongs to acidic ⁇ -mannanase.
  • the purpose of the present invention is to provide a mannanase mutant.
  • the heat resistance of the mutant is significantly improved compared with that of the wild type, which is beneficial to its wide application in the field of feed.
  • the present invention relates to a mannanase mutant comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 1, and compared with SEQ ID NO: 1 at least one position selected from the group Substitutions involving amino acids: 49, 58, 90, 99, 120, 124, 130, 132, 136, 140, 148, 154, 206, 212, 257, 263, 268, 273, 296, 297, 302, 306, 315 , 339, 353, 360, 362.
  • the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or at least 99% identity.
  • the amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, Or at least 99.9% identity.
  • the mutant comprises a substitution or a combination of substitutions selected from the following substitutions and combinations of substitutions:
  • the present invention also relates to a host cell comprising the above-mentioned recombinant expression plasmid.
  • the host cell is Pichia pastoris.
  • the host cell is Trichoderma reesei.
  • the present invention is based on wild-type mannanase M20, and provides D49C/I/N, G58D/E/S, H90L, T99Y, K120Y, N124P, T130D/N/P/Q/R/S, Y132F, S136M, S140L/K, T148K, D154P, F206Y, A212N/S/P, L257F, S263R, D268A/E/G/P, G273P, S302M/E, S306M/Q, T315P, T353L/F, D360E/N/ K, mutants of at least one mutation site in A362R.
  • the enzyme activity residual rate is generally increased by 69.9%-429.6%, and the heat resistance is significantly improved.
  • the N124P single-point mutant and the D360K single-point mutant have the strongest heat resistance, and the enzyme activity residual rates are as high as 51.81% and 60.69%, respectively, achieving unexpected technical effects.
  • the heat resistance of the mannanase mutant provided by the present invention is significantly improved, which is beneficial to its wide application in the field of feed.
  • the invention discloses a mannanase mutant, its preparation method and application, a DNA molecule encoding the mannanase mutant, a carrier, and a host cell.
  • the present invention has used the conventional technology and method that genetic engineering and molecular biology field use, for example MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and the method recorded in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003) .
  • MOLECULAR CLONING A LABORATORY MANUAL, 3nd Ed.
  • CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Ausubel, 2003
  • those skilled in the art may, on the basis of the technical solutions described in the present invention, adopt other conventional methods, experimental schemes and reagents in the field, without being limited to the specific examples of the present invention.
  • the present invention can select following experimental materials and reagents:
  • Enzymes and kits PCR enzyme and ligase were purchased from Takara Company, restriction endonuclease was purchased from Fermentas Company, lyase was purchased from Sigma Company, plasmid extraction kit and gel purification recovery kit were purchased from Omega Company, GeneMorph II was randomly induced The variable kit was purchased from Beijing Bomeisi Biotechnology Co., Ltd.
  • Escherichia coli medium 0.5% yeast extract, 1% peptone, 1% NaCl, pH7.0;
  • Yeast selection medium 2% peptone, 2% agarose;
  • BMGY medium 2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34% YNB, 4 ⁇ 10 -5 % biotin, 1% glycerol;
  • BMMY medium 2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34% YNB, 4 ⁇ 10 -5 % biotin, 0.5% methanol;
  • LB-AMP medium 0.5% yeast extract, 1% peptone, 1% NaCl, 100 ⁇ g/mL ampicillin, pH7.0;
  • LB-AMP plate 0.5% yeast extract, 1% peptone, 1% NaCl, 1.5% agar, 100 ⁇ g/mL ampicillin, pH7.0;
  • Lower medium plate 2% glucose, 0.5% (NH 4 ) 2 SO 4 , 1.5% KH 2 PO 4 , 0.06% MgSO 4 , 0.06% CaCl 2 , 1.5% agar.
  • M20-F1 GCT GAATTC GGCCTCCAATTCACCATTGATGGCG (the underline is the restriction endonuclease EcoRI recognition site);
  • M20-R1 CTG GCGGCCGC TTAGGCGCTATCAATAGCAG (the underline is the recognition site of the restriction enzyme NotI).
  • PCR amplification was carried out with the GeneMorph II random mutation PCR kit (Beijing Bomeisi), and the PCR product was recovered by gel, EcoRI, Not I After enzyme digestion, it was ligated with the pET21a vector after the same enzyme digestion, transformed into Escherichia coli BL21(DE3), spread on LB+Amp plate, and cultured upside down at 37°C. After the transformants appeared, pick them one by one with a toothpick.
  • the present invention also provides D49C/I/N, G58D/E/S, H90L, T99Y, K120Y, N124P, T130D/N/P/Q/R/S, Y132F, S136M, S140L/K, T148K, D154P, At least 2 of F206Y, A212N/S/P, L257F, S263R, D268A/E/G/P, G273P, V296I, T297Y, S302M/E, S306M/Q, T315P, T353L/F, D360E/N/K, A362R at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, Mannanase mutants with at least 15, at least 16, at least 17, at least 18 mutation sites.
  • the amount of enzyme needed to degrade and release 1 ⁇ mol of reducing sugar per minute from the mannan solution with a concentration of 3 mg/mL is one enzyme activity unit U.
  • a standard curve was drawn with the mannose concentration as the Y axis and the absorbance OD value as the X axis.
  • the standard curve needs to be redrawn every time DNS reagent is newly prepared.
  • X D is the activity of mannanase in the diluted enzyme solution, U/ml;
  • a E is the absorbance of the enzyme reaction solution;
  • a B is the absorbance of the enzyme blank solution;
  • K is the slope of the standard curve;
  • C 0 is the standard The intercept of the curve;
  • M is the molar mass of xylose, 180.2g/mol;
  • t is the enzymatic hydrolysis reaction time, min;
  • the enzyme activity detection was carried out according to the above method, and the results showed that the enzyme activity of the fermentation supernatant of the Pichia pastoris recombinant strain obtained by the above construction and expressing the recombinant mannanase M20 and its mutants was 200-500 U/mL.
  • the gene sequences of wild-type mannanase M20 and its mutants were respectively optimized.
  • the optimized gene sequence was synthesized by Shanghai Jierui Bioengineering Co., Ltd., and two restriction sites of KpnI and MluI were added to the 5' and 3' ends of the synthetic sequence, respectively.
  • the synthesized mannanase gene fragment and pSC1G vector were digested with restriction endonucleases KpnI and MluI respectively, the digested products were purified using a gel purification kit, and the above acidic mannan was purified with T4 DNA ligase
  • the enzyme gene was ligated with the digested product of pSC1G vector and transformed into Escherichia coli DH5 ⁇ , selected with ampicillin, and the clone was verified by sequencing. After the sequencing is correct, a recombinant plasmid containing the mannanase gene is obtained.
  • Trichoderma reesei Trichoderma reesei
  • UE Trichoderma reesei
  • inoculate it on a PDA plate and culture it at 30°C for 6 days; 0.5% yeast powder, 1% glucose, 0.1% uridine) liquid medium, 30°C, 220rpm shaking culture for 14-16h;
  • the applicant constructed Trichoderma reesei engineering strains for recombinantly expressing wild-type mannanase M20 and its mutants respectively.
  • the engineering strains of Trichoderma reesei constructed above were inoculated on PDA solid plates, and cultured upside down for 6-7 days in a 30°C constant temperature incubator. Fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH 4 ) 2 SO 4 , 0.09% MgSO 4 , 2% KH 2 PO 4 , 0.04% CaCl 2 , 0.018% Tween-80 , 0.018% trace elements) in a 250mL Erlenmeyer flask, cultivated at 30°C for 48 hours, and then cultivated at 25°C for 48 hours. The fermentation broth is centrifuged to obtain fermentation supernatants respectively containing wild-type mannanase M20 and its mutants.
  • Fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH 4 ) 2 SO 4 , 0.09% MgSO 4 , 2% KH 2 PO 4 , 0.04% Ca
  • the enzyme activity detection was carried out according to the above method, and the results showed that the enzyme activity of the fermentation supernatant of the recombinant Trichoderma reesei recombinant strain expressing the wild-type mannanase M20 and its mutants constructed above was 230-510 U/mL.
  • the fermentation supernatant of the Pichia pastoris recombinant strain obtained from the above construction was subjected to After dilution, the mannan substrates were also prepared with buffer solutions corresponding to the pH value, and the mannanase activity was measured at 37°C, and the enzyme activity was calculated. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated.
  • Enzyme activity residual rate (%) enzyme activity of treated sample/enzyme activity of untreated sample ⁇ 100%.
  • the enzyme activity residual rate of the mannanase single-point mutant provided by the present invention is generally increased by 69.9% after being treated at 80°C for 3 minutes -429.6%, the heat resistance has been significantly improved.
  • the N124P mutant and the D360K mutant have the strongest heat resistance, and after being treated at 80°C for 3 minutes, the enzyme activity residual rates are as high as 51.81% and 60.69%, respectively, achieving unexpected technical effects.
  • the high temperature resistant mannanase mutant provided by the invention can be widely used in the field of feed.

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Abstract

一种耐高温甘露聚糖酶突变体。所述突变体包含D49C/I/N、G58D/E/S、H90L、T99Y、K120Y、N124P、T130D/N/P/Q/R/S、Y132F、S136M、S140L/K、T148K、F206Y、A212N/S/P、L257F、S263R、D268A/E/G/P、G273P、S302M/E、S306M/Q、T315P、T353L/F、D360E/N/K、A362R中至少一个突变位点，耐热性显著高于野生型甘露聚糖酶，可广泛应用于饲料领域。

Description

耐高温甘露聚糖酶突变体
本申请要求于2021年09月18日提交中国专利局、申请号为202111097561.4、发明名称为“耐高温甘露聚糖酶突变体”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
本发明涉及基因工程与蛋白质改造技术，具体涉及一种耐高温甘露聚糖酶突变体。
背景技术
甘露聚糖是一种无毒、无味、粘度大的高分子天然多聚物，在医药、食品和化妆品等领域应用很广。但是，存在于植物细胞壁或种籽中的甘露聚糖遇水后极易膨胀，粘度大幅提高，会对这些物质的开发、利用造成非常不利的影响。例如，在动物养殖过程中，饲料中的甘露聚糖就能结合大量的水，形成粘度极高的胶状物质，影响消化酶对饲料的水解，从而降低动物对饲料的消化利用率，因此，甘露聚糖成为饲料领域中一种常见的抗营养因子。
β-甘露聚糖酶(β-manannase,EC3.2.1.78)是一种能降解β-1,4-甘露糖苷键的糖苷水解酶，其作用底物为线状甘露聚糖、葡甘露聚糖、半乳甘露聚糖及半乳葡甘露聚糖等。各种甘露聚糖经β-甘露聚糖酶水解后，可形成甘露糖和甘露低聚糖(Mannan oligosaccharides,MOS,通常由2-10个单糖残基组成)。
人们对β-甘露聚糖酶的研究、应用开发可以追溯到二十世纪五十年代。Courtios等人于1958年首次从紫花苜蓿(Alfalfa)中发现一种β-甘露聚糖酶，Williams和Doetsch于1960年报道从瘤胃厌氧链球菌(anaerobic rumen Streptococci)中获得一种β-甘露聚糖酶，随后，Reese和Shibata等人于1965年第一次系统性地提出了β-甘露聚糖酶的概念。
β-甘露聚糖酶广泛存在于植物、动物及微生物中，例如，在一些低等动物的肠道分泌液以及一些植物的发芽种子中，都发现有β-甘露聚糖酶的存在。目前，已报道的大多数β-甘露聚糖酶都来源于微生物，主要包括细菌、真菌及放线菌等，其中细菌产生的β-甘露聚糖酶一般最适反应pH值为5.5-7.0，等电点为4.0-5.0，最适反应温度为50-70℃。而真菌来源的β-甘露聚糖酶等电点通常为4.0-5.0，最适反应温度为55-75℃，最适反应pH值为4.5-5.5，属于酸性β-甘露聚糖酶。
由于采用微生物生产β-甘露聚糖酶，具有不受季节限制、表达量高、生产成本相对低廉及易于工业化发酵生产等特点，因而成为β-甘露聚糖酶工业化生产的主要方式。但由于目前在颗粒饲料生产过程中有一个短暂的80-90℃的高温阶段，不耐高温的β-甘露聚糖酶直接添加到动物饲料中进行制粒，其酶活将受到较大损失的，因此，提高热稳定性对目前饲料用β-甘露聚糖酶具有重要的现实意义。
发明内容
本发明的目的是提供一种甘露聚糖酶突变体。所述突变体的耐热性比野生型得到显著提高，有利于其在饲料领域的广泛应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明涉及一种甘露聚糖酶突变体，其包含与SEQ ID NO:1具有至少90％同一性的氨基酸序列，且与SEQ ID NO：1相比在选自下组中的至少一个位置上包含氨基酸的取代：49，58，90，99，120，124，130，132，136，140，148，154，206，212，257，263，268，273，296，297，302，306，315，339，353，360，362。
在本发明的一些实施例中，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少91％，92％，93％，94％，95％，96％，97％，98％，或至少99％的同一性。
在一些更具体的实施例中，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，或至少99.9％的同一性。
在本发明的一些实施例中，所述突变体包含下组中至少一个氨基酸的取代：D49C/I/N，G58D/E/S，H90L，T99Y，K120Y，N124P，T130D/N/P/Q/R/S，Y132F，S136M，S140L/K，T148K，D154P，F206Y，A212N/S/P，L257F，S263R，D268A/E/G/P，G273P，V296I，T297Y，S302M/E，S306M/Q，T315P，N339F，T353L/F，D360E/N/K，A362R。
在本发明的一些实施例中，所述突变体包含的取代或取代的组合选自下述取代和取代的组合：
D49C；D49I；D49N；G58D；G58E；G58S；H90L；T99Y；K120Y；N124P；T130D；T130N；T130P；T130Q；T130R；T130S；Y132F；S136M；S140L；S140K；T148K；D154P；F206Y；A212N；A212S；A212P；L257F；S263R；D268A；D268E；D268G；D268P；G273P；V296I；T297Y；S302M；S302E；S306M；S306Q；T315P；N339F；T353L；T353F；D360E；D360N；D360K；A362R；
D49N/G58D；
D49N/H90L；
D49N/T99Y；
D49N/K120Y；
D49N/N124P；
D49N/T130R；
D49N/T130S；
D49N/T130P；
D49N/Y132F；
D49N/S136M；
D49N/S140L；
D49N/S140K；
D49N/T148K；
D49N/D154P；
D49N/F206Y；
D49N/A212N；
D49N/A212S；
D49N/A212P；
D49C/F206Y；
D49I/F206Y；
D49N/L257F；
D49N/D268A；
D49N/D268E；
D49N/D268G；
D49N/D268P；
D49N/G273P；
D49N/S302M；
D49N/S302E；
D49N/S306M；
D49N/S306Q；
D49N/T315P；
D49N/T353L；
D49N/D360E；
D49C/D360E；
D49I/D360E；
D49I/D360E；
D49N/D360N；
D49N/D360K；
D49N/A362R；
G58D/H90L；
G58D/K120Y；
G58D/Y132F；
G58D/T99Y；
G58D/N124P；
G58D/T130P；
G58D/S136M；
G58D/S140K；
G58D/T148K；
G58D/D154P；
G58D/F206Y；
G58D/L257F；
G58D/D268P；
G58D/G273P；
G58D/S302M；
G58D/S302E；
G58D/S306M；
G58D/S306Q；
G58D/T315P；
G58D/T353L；
G58D/D360E；
H90L/A362R；
H90L/G273P；
H90L/S302M；
H90L/S302E；
H90L/S306M；
H90L/S306Q；
T99Y/N124P；
T99Y/T130P；
T99Y/S136M；
T99Y/S140K；
T99Y/T148K；
T99Y/D154P；
T99Y/F206Y；
T99Y/L257F；
T99Y/D268P；
T99Y/G273P；
T99Y/S302M；
T99Y/S302E；
T99Y/S306M；
T99Y/S306Q；
T99Y/T315P；
T99Y/T353L；
T99Y/D360E；
K120Y/N124P；
K120Y/T130P；
K120Y/S136M；
K120Y/S140K；
K120Y/L257F；
K120Y/D268P；
K120Y/T315P；
K120Y/T353L；
K120Y/D360E；
K120Y/A362R；
N124P/T130P；
N124P/S136M；
N124P/S140K；
N124P/L257F；
N124P/D268P；
N124P/T315P；
N124P/T353L；
N124P/D360E；
T130P/S136M；
T130P/S140K；
T130P/L257F；
T130P/D268P；
T130P/T315P；
T130P/T353L；
T130P/D360E；
S136M/S140K；
S136M/L257F；
S136M/D268P；
S136M/T315P；
S136M/T353L；
S136M/D360E；
S140K/L257F；
S140K/D268P；
S140K/T315P；
S140K/T353L；
S140K/D360E；
T148K/D154P；
T148K/F206Y；
T148K/L257F；
T148K/D268P；
T148K/G273P；
T148K/S302M；
T148K/S302E；
T148K/S306M；
T148K/S306Q；
T148K/T315P；
T148K/T353L；
T148K/D360E；
D154P/L257F；
D154P/D268P；
D154P/G273P；
D154P/S302M；
D154P/S302E；
D154P/S306M；
D154P/S306Q；
D154P/T315P；
D154P/T353L；
D154P/D360E；
F206Y/G273P；
F206Y/S302M；
F206Y/S302E；
F206Y/S306M；
F206Y/S306Q；
F206Y/T315P；
F206Y/T353L；
F206Y/D360E；
A212N/D268P；
A212N/T315P；
A212N/T353L；
A212N/D360E；
L257F/D268P；
L257F/T315P；
L257F/T353L；
L257F/D360E；
D268P/T315P；
D268P/T353L；
D268P/D360E；
G273P/S302M；
G273P/S302E；
G273P/S306M；
G273P/S306Q；
G273P/T315P；
G273P/T353L；
G273P/D360E；
G273P/A362R；
S302M/S306M；
S302M/S306Q；
S302M/T315P；
S302M/T353L；
S302M/D360E；
S302M/A362R；
S302E/S306M；
S302E/S306Q；
S302E/T315P；
S302E/T353L；
S302E/D360E；
S302E/A362R；
S306M/T315P；
S306M/T353L；
S306M/D360E；
S306M/A362R；
S306Q/T315P；
S306Q/T353L；
S306Q/D360E；
S306Q/A362R；
T315P/T353L；
T315P/D360E；
T353L/D360E；
T315P/A362R；
T353L/A362R；
D360E/A362R；
D49N/G58D/N124P；
D49N/G58D/T130P；
D49N/T99Y/N124P；
D49N/T99Y/S136M；
D268P/T315P/D360E；
T130P/L257F/D268P；
N124P/T130P/D268P；
N124P/T130P/D360E；
N124P/S302M/S306M；
D49N/G58D/L257F；
D49N/G58D/D268P；
D49N/G58D/A362R；
D49N/T99Y/S140K；
D49N/T99Y/L257F；
D49N/T99Y/D268P；
D49N/G58D/T315P；
D49N/G58D/D360E；
G58D/N124P/T130P；
G58D/N124P/L257F；
G58D/N124P/D268P；
G58D/D268P/T315P；
L257F/D268P/T315P；
N124P/T130P/D360K；
D49N/T99Y/T315P；
D49N/T99Y/T353L；
D49N/T99Y/D360E；
K120Y/N124P/S140K；
K120Y/T130P/L257F；
K120Y/S136M/D268P；
K120Y/S140K/T353L；
N124P/T130P/L257F；
N124P/T130P/D268P；
A212P/L257F/D268P；
D49N/G58D/N124P/T130P；
D49N/G58S/T130P/D268P；
G58S/N124P/T130P/D268P；
G58D/N124P/T130P/D268P；
N124P/T130P/L257F/D268P；
N124P/T130P/S136M/T353L；
N124P/T130P/S136M/D360E；
S136M/S140K/T315P/T353L；
S136M/L257F/T315P/T353L；
S136M/D268P/S306M/T315P；
S136M/T315P/T353L/D360E；
G58D/T99Y/S140K/T315P/T353L；
G58D/N124P/T315P/T353L/D360E；
G58S/T99Y/N124P/T130P/D268P；
G58D/T99Y/N124P/T130P/D268P；
G58D/T130P/T315P/T353L/D360E；
G58D/K120Y/N124P/S140K/S136M；
D49N/T99Y/S136M/G273P/S302M；
T99Y/N124P/T130P/L257F/D268P；
D49N/G58D/T99Y/N124P/T130P；
D49N/G58D/N124P/T130P/D268P；
D49N/G58D/N124P/T130P/T315P；
Y132F/L257F/S263R/D268P/T315P；
Y132F/L257F/S263R/D268P/T315P；
Y132F/L257F/S263R/S306M/T315P；
Y132F/G273P/S263R/S306M/T315P；
Y132F/G273P/S263R/S306M/D360N；
D49N/G58D/N124P/T130P/T353L；
D49N/G58D/N124P/T130P/D360E；
D49N/G58S/N124P/T130P/S136M；
A212P/L257F/S263R/D268P/T315P；
A212P/L257F/S263R/D268P/T315P；
A212P/L257F/S263R/S306M/T315P；
A212P/G273P/S263R/S306M/T315P；
A212P/G273P/S263R/S306M/D360N；
D49N/T99Y/S140K/T353L/D360E；
D49N/T99Y/L257F/S302M/A362R；
D49N/G58D/N124P/T130P/S140K；
D49N/G58D/N124P/T130P/L257F；
G58D/N124P/T130P/L257F/D268P；
D49N/G58D/N124P/T130P/L257F/D268P；
G58D/T99Y/N124P/T130P/L257F/D268P；
G58D/N124P/T130P/S136M/L257F/D268P；
G58D/N124P/T130P/S140K/L257F/D268P；
D154P/L257F/D268P/T315P/T353L/D360E；
D154P/D268P/S306Q/T315P/T353L/D360E；
D154P/F206Y/D268P/S306Q/T353L/D360E；
D49N/N124P/T130P/L257F/T315P/D360E；
D49N/N124P/T130P/L257F/D268P/D360E；
T148K/D154P/D268P/S306Q/T315P/T353L；
T148K/D154P/D268P/S306M/T315P/T353L；
D49N/N124P/T130P/L257F/D268P/T315P；
D49N/N124P/F206Y/L257F/D268P/D360K；
D49N/N124P/F206Y/L257F/D268P/T315P；
N124P/T130P/L257F/D268P/T315P/D360E；
G58D/N124P/T130P/L257F/D268P/T315P；
G58D/N124P/T130P/L257F/D268P/T353L；
G58D/N124P/T130P/L257F/D268P/D360E；
G58D/N124P/T130P/L257F/D268P/T315P；
T148K/D154P/A212P/S306Q/T315P/T353L；
D49N/T130P/L257F/D268P/T315P/D360E；
D49N/N124P/L257F/D268P/T315P/D360E；
T148K/D154P/S263R/S306M/T315P/T353L；
T148K/D154P/G273P/S306Q/T315P/T353L；
D49N/N124P/T130P/D268P/T315P/D360E；
G58D/N124P/T130P/L257F/D268P/G273P/D360E；
D49N/G58D/T99Y/N124P/T130P/L257F/D268P；
D49N/G58D/N124P/T130P/S136M/L257F/D268P；
D49N/G58D/N124P/T130P/S140K/L257F/D268P；
D49N/G58D/N124P/T130P/L257F/D268P/T315P；
D49N/G58D/N124P/T130P/L257F/D268P/T353L；
D49N/G58D/N124P/T130P/D268P/T315P/D360E；
D49N/G58D/N124P/T130P/L257F/T315P/D360E；
H90L/N124PT130P/L257F/D268P/G273P/D360E；
H90L/N124P/T130P/L257F/D268P/G273P/A362R；
D49N/G58D/N124P/T130P/L257F/D268P/D360K；
G58D/N124P/T130P/L257F/D268P/T315P/D360E；
D49N/N124P/T130P/L257F/D268P/T315P/D360E；
D49N/G58D/T130P/L257F/D268P/T315P/D360E；
D49N/G58D/N124P/L257F/D268P/T315P/D360E；
D49N/G58D/N124P/T130P/L257F/D268P/D360E；
G58D/T99Y/N124P/T130P/L257F/D268P/S302M/S306M；
G58D/N124P/T130P/S136M/L257F/D268P/S302M/T315P；
G58D/N124P/T130P/S140K/L257F/D268P/S302M/D360E；
D49N/G58D/N124P/T130P/L257F/D268P/T315P/T353L；
D49N/G58D/N124P/T130P/S140K/L257F/D268P/T315P；
D49N/G58D/N124P/T130P/S136M/L257F/D268P/T315P；
D49N/G58D/T99Y/N124P/T130P/L257F/D268P/T315P；
T148K/S140K/D154P/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/N124P/T130P/L257F/D268P/T315P/D360E；
D154P/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
D154P/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
D49N/G58D/N124P/T130P/S140K/L257F/D268P/T353L；
D49N/G58D/N124P/T130P/S136M/L257F/D268P/T353L；
D49N/G58D/H90L/T130P/S136M/L257F/S306M/T353L；
D49N/G58D/H90L/T130P/S136M/G273P/S306M/T353L；
D49N/G58D/N124P/T130P/S136M/S140K/L257F/D268P；
D49N/G58D/N124P/T130P/L257F/245P/G273P/A362R；
H90L/T99Y/N124P/T130P/L257F/D268P/S302M/S306M；
H90L/N124P/T130P/S136M/L257F/D268P/S302M/T315P；
H90L/N124P/T130P/S140K/L257F/D268P/S302M/D360E；
T99Y/N124P/T130P/Y132F/D268P/S302P/S306M/T315P；
D49N/G58D/N124P/T130P/L257F/D268P/T315P/D360E/T353L；
D49N/G58D/N124P/T130P/S136M/L257F/D268P/T315P/D360E；
D49N/G58D/N124P/T130P/S140K/L257F/D268P/T315P/D360E；
D49N/G58D/N124P/T130P/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/T99Y/N124P/T130P/L257F/D268P/T315P/D360E；
T99Y/N124P/T130P/L257F/D268P/S302M/T315P/D360E/A362R；
K120Y/N124P/T130P/S136M/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/N124P/T130P/S136M/S140K/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/N124P/T130P/S136M/L257F/D268P/V296I/T297Y/T315P/D360E；
D154P/L257F/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
D49N/G58D/N124P/T130P/S136M/S140K/L257F/D268P/T315P/D360E；
D154P/L257F/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
D49N/G58D/N124P/T130P/S136M/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/N124P/T130P/S140K/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/N124P/T130P/S136M/L257F/D268P/V296I/T315P/D360E；
D49N/G58D/N124P/T130P/S136M/L257F/D268P/T297Y/T315P/D360E；
D49N/G58D/N124P/T130P/L257F/D268P/G273P/S306M/T315P/T353L/D360E；
D49N/G58D/T99Y/N124P/T130P/L257F/D268P/S302M/T315P/D360E/A362R；
G58D/T99Y/N124P/T130P/S136M/S140K/D154P/L257F/D268P/T315P/D360E/A362R；
T130P/Y132F/S136M/S140K/D268P/G273P/S302M/S306M/T315P/T353L/D360E/A362R；
D49N/G58D/T99Y/N124P/T130P/S136M/S140K/L257F/D268P/T315P/T353L/D360K；
D49N/G58D/H90L/K120Y/N124P/T130P/S136M/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/T99Y/N124P/T130P/S136M/S140K/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/N124P/T130P/T148K/S140K/D154P/L257F/D268P/T315P/T353L/D360E；
D49N/G58D/N124P/T130P/S136M/L257F/D268P/V296I/T297Y/T315P/N339F/D360E；
D49N/G58D/T99Y/N124P/T130P/Y132F/D154P/D268P/G273P/S302P/S306M/T315P；
T99Y/N124P/T130P/Y132F/S136M/S140K/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
H90L/N124P/T130P/Y132F/S136M/S140K/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
H90L/K120Y/T130P/Y132F/S136M/S140K/D268P/S302M/S306M/T315P/T353L/D360K/A362R；
N124P/T130P/Y132F/S136M/S140K/L257F/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
T99Y/S113M/S117K/N124P/T130P/Y132F/D268P/S302P/S306M/T315P/T330L/D337E/A339R；
T99Y/N124P/T130P/Y132F/S136M/S140K/L257F/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
T148K/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
T99Y/T148K/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
T99Y//N124P/T148K/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
D49N/G58D/N124P/T130P/T148K/A212P/L257F/S263R/D268P/G273P/S302M/S30 6M/T315P/T353F/D360E/A362R；
D49N/G58D/T99Y/T130P/T148K/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
D49N/G58D/T99Y/T130P/T148K/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
D49N/G58D/T99Y/T130P/T148K/D154P/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
D49N/G58D/T99Y/T130P/T148K/D154P/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
G58D/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
G58D/H90L/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360K/A362R；
G58D/H90L/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302E/S306M/T315P/T353F/D360E/A362R；
G58D/H90L/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302E/S306M/T315P/T353F/D360N/A362R；
G58D/H90L/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302E/S306M/T315P/T353L/D360K/A362R。
本发明还涉及编码上述甘露聚糖酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达质粒。
本发明还涉及一种宿主细胞，包含上述重组表达质粒。
将上述的质粒转入宿主细胞中，重组表达的甘露聚糖酶突变体的耐热性得到显著提升。
在本发明的一些实施例中，宿主细胞为毕赤酵母(Pichia pastoris)。
在本发明的一些实施例中，宿主细胞为里氏木霉(Trichoderma reesei)。
本发明还提供了上述甘露聚糖酶突变体在饲料领域中的应用。
本发明以野生型甘露聚糖酶M20为基础，提供了包含D49C/I/N，G58D/E/S，H90L，T99Y，K120Y，N124P，T130D/N/P/Q/R/S，Y132F，S136M，S140L/K，T148K，D154P，F206Y，A212N/S/P，L257F，S263R，D268A/E/G/P，G273P，S302M/E，S306M/Q，T315P，T353L/F，D360E/N/K，A362R中至少一个突变位点的突变体。与野生型甘露聚糖酶相比，本发明提供的突变体在80℃条件下处理3min后，酶活残留率普遍提高了69.9％-429.6％，耐热性得到显著提高。其中，N124P单点突变体和D360K单点突变体的耐热性最强，酶活残留率分别高达51.81％和60.69％，取得了意料不到的技术效果。
综上，本发明提供的甘露聚糖酶突变体的耐热性得到显著提高，有利于其在饲料领域中的广泛应用。
具体实施方式
本发明公开了一种甘露聚糖酶突变体、其制备方法及应用、编码该甘露聚糖酶突变体的DNA分子、载体、宿主细胞，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING：A LABORATORY MANUAL，3nd Ed.(Sambrook,2001)和CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Ausubel,2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本发明所记载的技术方案的基础上，采用本领域其它常规的方法、实验方案和试剂，而不限于本发明具体实施例的限定。例如，本发明可选用如下实验材料和试剂：
菌株与载体：大肠杆菌DH5α、毕赤酵母GS115、载体pPIC9k、Amp、G418购自Invitrogen公司。
酶与试剂盒：PCR酶及连接酶购买自Takara公司，限制性内切酶购自Fermentas公司，裂解酶购自Sigma，质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司，GeneMorph II随机诱变试剂盒购自北京博迈斯生物科技有限公司。
培养基配方：
大肠杆菌培养基(LB培养基)：0.5％酵母提取物，1％蛋白胨，1％NaCl，pH7.0；
酵母培养基(YPD培养基)：1％酵母提取物，2％蛋白胨，2％葡萄糖；
酵母筛选培养基(MD培养基)：2％蛋白胨，2％琼脂糖；
BMGY培养基：2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10 -5％生物素，1％甘油；
BMMY培养基：2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10 -5％生物素，0.5％甲醇；
LB-AMP培养基：0.5％酵母提取物，1％蛋白胨，1％NaCl，100μg/mL氨苄青霉素，pH7.0；
LB-AMP平板：0.5％酵母提取物，1％蛋白胨，1％NaCl，1.5％琼脂，100μg/mL氨苄青霉素，pH7.0；
上层培养基：0.1％MgSO 4，1％KH 2PO 4，0.6％(NH 4) 2SO 4，1％葡萄糖，18.3％山梨醇，0.35％琼脂糖；
下层培养基平板：2％葡萄糖，0.5％(NH 4) 2SO 4，1.5％KH 2PO 4，0.06％MgSO 4，0.06％CaCl 2，1.5％琼脂。
下面结合实施例，进一步阐述本发明：
实施例1野生型甘露聚糖酶基因的克隆与表达质粒的构建
以黑曲霉(Aspergillus niger)基因组为模板进行PCR扩增，PCR引物M20-F1、M20-R1如下：
M20-F1：GCT GAATTCGGCCTCCAATTCACCATTGATGGCG(下划线为限制性内切酶EcoRI识别位点)；
M20-R1：CTG GCGGCCGCTTAGGCGCTATCAATAGCAG(下划线为限制性内切酶NotI识别位点)。
胶回收PCR产物，连接pEASY-T载体，转化至大肠杆菌DH5α中，挑取正确的转化子进行测序。测序结果显示，扩增得到的基因片段的核苷酸序列为SEQ ID NO：2，其编码的氨基酸序列为SEQ ID NO：1，从而说明表达质粒构建成功，命名为pPIC9K-M20。通过NCBI BLAST比对发现，SEQ ID NO：1与来源于黑曲霉的酸性甘露聚糖酶序列相似性高达100％，从而确定通过PCR获得的基因为酸性甘露聚糖酶基因，命名为M20。
实施例2耐高温甘露聚糖酶突变体的筛选
为了提高酸性甘露聚糖酶M20的耐热性，申请人通过定向进化技术对该酶进行了大量突变的筛选。
以M20基因为模板，利用实施例1中所述引物M20-F1、M20-R1，用GeneMorph II随机突变PCR试剂盒(北京博迈斯)进行PCR扩增，胶回收PCR产物，EcoRI、Not I进行酶切处理后与经同样酶切后的pET21a载体连接，转化至大肠杆菌BL21(DE3)中，涂布于LB+Amp平板，37℃倒置培养，待转化子出现后，用牙签逐个挑至96孔板，每个孔中加入150ul含有0.1mM IPTG的LB+Amp培养基，37℃220rpm培养6h左右，离心弃上清，菌体用缓冲液重悬，反复冻融破壁，获得含有酸性甘露聚糖酶的大肠杆菌细胞裂解液。
分别取出30ul裂解液至两块新的96孔板，将其中一块于75℃处理5min；然后将两块96孔板都加入30ul底物，于37℃反应30min后，DNS法测定生成的还原糖，分别计算不同突变子的酶活水平。
实验结果表明，不同的突变子高温处理后保持的活性不同。有些突变对甘露聚糖酶M20的耐热性没有影响，有些突变甚至使其耐热性变得更差了；另外还有些突变，虽然能提高甘露聚糖酶的耐热性，但突变后其酶学性质发生了显著的变化，这些均不符合要求。最终，得到既能显著提高甘露聚糖酶的耐热性，又不会影响其原有酶学性质的突变位点：D49C、D49I、D49N、G58D、G58E、G58S、H90L、T99Y、K120Y、N124P、T130D、T130N、T130P、T130Q、T130R、T130S、Y132F、S136M、S140L、S140K、T148K、D154P、F206Y、A212N、A212S、A212P、L257F、S263R、D268A、D268E、D268G、D268P、G273P、S302M、S302E、S306M、S306Q、T315P、T353L、T353F、D360E、D360N、D360K、A362R。
在野生型甘露聚糖酶M20的基础上，本发明提供了分别含D49C、D49I、D49N、G58D、G58E、G58S、H90L、T99Y、K120Y、N124P、T130D、T130N、T130P、T130Q、T130R、T130S、Y132F、S136M、S140L、S140K、T148K、D154P、F206Y、A212N、 A212S、A212P、L257F、S263R、D268A、D268E、D268G、D268P、G273P、S302M、S302E、S306M、S306Q、T315P、T353L、T353F、D360E、D360N、D360K、A362R单个突变位点的甘露聚糖酶突变体。
本发明还提供了包含D49C/I/N，G58D/E/S，H90L，T99Y，K120Y，N124P，T130D/N/P/Q/R/S，Y132F，S136M，S140L/K，T148K，D154P，F206Y，A212N/S/P，L257F，S263R，D268A/E/G/P，G273P，V296I，T297Y，S302M/E，S306M/Q，T315P，T353L/F，D360E/N/K，A362R中至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个，至少11个，至少12个，至少13个，至少14个，至少15个，至少16个，至少17个，至少18个突变位点的甘露聚糖酶突变体。
参照突变体的氨基酸序列，分别得到所述甘露聚糖酶突变体的编码核苷酸序列。
实施例3甘露聚糖酶在毕赤酵母中的表达
3.1表达质粒的构建
依照毕赤酵母的密码偏爱性对野生型甘露聚糖酶M20及其突变体的基因序列进行优化，基因由上海捷瑞生物工程有限公司合成，并且在合成序列5’和3’两端分别加上EcoRI和NotI两个酶切位点。
按照实施例1中的方法，将合成的突变体的基因序列分别进行EcoRI和NotI双酶切，然后与经同样酶切后的pPIC-9K载体16℃过夜连接，并转化大肠杆菌DH5a，涂布于LB+Amp平板，37℃倒置培养，待转化子出现后，菌落PCR验证阳性克隆子，经测序验证后获得了正确的重组突变体表达质粒。
3.2毕赤酵母工程菌株的构建
3.2.1酵母感受态制备
将毕赤酵母GS115菌株进行YPD平板活化，30℃培养48h后接种活化的GS115单克隆于6mLYPD液体培养基中，30℃、220rpm，培养约12h后转接菌液于装有30mLYPD液体培养基的三角瓶中，30℃、220rpm培养约5h，经紫外分光光度计检测其菌体密度，待其OD600值在1.1–1.3范围后，4℃、9000rpm离心2min，分别收集4mL菌体至灭菌EP管中，轻轻弃上清，用灭菌的滤纸吸干残留的上清后用预冷的1mL灭菌水重悬菌体，4℃、9000rpm离心2min，轻轻弃上清，重复用1mL灭菌水洗一遍后，4℃、9000rpm离心2min，轻轻弃上清，预冷的1mL山梨醇(1mol/L)重悬菌体；4℃、9000rpm离心2min，轻轻弃上清，预冷的100-150μl山梨醇(1mol/L)轻柔重悬菌体。
3.2.2转化和筛选
分别将实施例1和实施例3.1中构建获得的表达质粒用Sac I进行线性化，线性化片段纯化回收后通过电穿孔法分别转化毕赤酵母GS115，在MD平板上筛选得到毕赤酵母重组菌株，然后在含不同浓度遗传霉素的YPD平板(0.5mg/mL-8mg/mL)上筛选多拷贝的转化子。
将获得的转化子分别转接于BMGY培养基中，30℃、250rpm振荡培养1d；再转 入BMMY培养基中，30℃、250rpm振荡培养；每天添加0.5％的甲醇，诱导表达4d；9000rpm离心10min去除菌体，即得到分别含野生型甘露聚糖酶M20及其突变体的发酵上清液。
3.3甘露聚糖酶活力检测方法
(1)甘露聚糖酶酶活单位的定义
在37℃、pH值为5.5的条件下，每分钟从浓度为3mg/mL的甘露聚糖溶液中降解释放1umol还原糖所需要的酶量为一个酶活力单位U。
(2)酶活测定方法
(2.1)标准曲线的绘制：
吸取乙酸—乙酸钠缓冲溶液4.0mL，加入DNS试剂5.0mL，沸水浴加热5min。用自来水冷却至室温，用水定容至25.0mL，制成标准空白样。
分别吸取甘露糖溶液(5.5)1.00、2.00、3.00、4.00、5.00、6.00和7.00mL，分别用乙酸—乙酸钠缓冲溶液定容至100mL，配制成浓度为0.10—0.70mg/mLD-甘露糖标准溶液。
分别吸取上述浓度系列的甘露糖标准溶液各2.00mL(做二个平行)，分别加入到刻度试管中，再分别加入2mL乙酸—乙酸钠缓冲溶液和5mLDNS试剂。电磁振荡3s，沸水浴加热5min。然后用自来水冷却到室温，再用水定容至25mL。以标准空白样为对照调零，在540nm处测定吸光度OD值。
以甘露糖浓度为Y轴、吸光度OD值为X轴，绘制标准曲线。每次新配制DNS试剂均需要重新绘制标准曲线。
(2.2)酶活力测定：
吸取10.0mL甘露聚糖溶液，37℃平衡10min。
吸取10.0mL经过适当稀释的酶液，37℃平衡10min。
吸取2.00mL经过适当稀释的酶液(已经过37℃平衡)，加入到刻度试管中，再加入5mLDNS试剂，电磁振荡3s。然后加入2.0mL甘露聚糖溶液，37℃保温30min，沸水浴加热5min。用自来水冷却至室温，加水定容至25mL，电磁振荡3s。以标准空白样为空白对照，在540nm处测定吸光度A B。
吸取2.0mL经过适当稀释的酶液(已经过37℃平衡)，加入到刻度试管中，再加入2.0mL甘露聚糖溶液(已经过37℃平衡)，电磁振荡3s，37℃精确保温30min。加入5.0mLDNS试剂，电磁振荡3s，酶解反应。沸水浴加热5min，用自来水冷却至室温，加水定容至25mL，电磁振荡3s。以标准空白样为空白对照，在540nm处测定吸光度A E。
酶活计算公式：
Figure PCTCN2022119328-appb-000001
式中：X D为稀释酶液中甘露聚糖酶的活力，U/ml；A E为酶反应液的吸光度；A B为酶空白液的吸光度；K为标准曲线的斜率；C 0为标准曲线的截距；M为木糖的摩尔质量，180.2g/mol；t为酶解反应时间，min；N为酶液稀释倍数；1000为转化因子， 1mmol＝1000μmol。
(3)酶活测定结果
按照上述方法进行酶活检测，结果显示：上述构建得到的重组表达甘露聚糖酶M20及其突变体的毕赤酵母重组菌株发酵上清液的酶活为200-500U/mL。
实施例4甘露聚糖酶在里氏木霉中的表达
首先依照木霉的密码子偏爱性，分别对野生型甘露聚糖酶M20以及其突变体的基因序列进行优化。优化后的基因序列由上海捷瑞生物工程有限公司合成，并且在合成序列5’和3’两端分别加上KpnI和MluI两个酶切位点。
4.1表达载体的构建
将合成后的甘露聚糖酶基因片段与pSC1G载体分别用限制性内切酶KpnI和MluI进行酶切，使用凝胶纯化试剂盒将酶切产物纯化，并用T4DNA连接酶分别将上述酸性甘露聚糖酶基因与pSC1G载体的酶切产物连接并转化大肠杆菌DH5α，用氨苄青霉素进行选择，并对克隆进行测序验证。测序正确后，即得到含有甘露聚糖酶基因的重组质粒。
4.2里氏木霉重组菌株的构建
(1)原生质体制备
取宿主菌里氏木霉(Trichoderma reesei)UE孢子悬液，接种于PDA平板上，30℃培养6天；待其产孢丰富后，切取约1cm×1cm的菌落置于含120mL YEG+U(0.5％酵母粉、1％葡萄糖、0.1％尿苷)的液体培养基中，30℃，220rpm振荡培养14～16h；
用无菌纱布过滤收集菌丝体，并用无菌水清洗一次；将菌丝体置于含有20mL 10mg/mL裂解酶液(Sigma L1412)的三角瓶中，30℃，90rpm作用1-2h；用显微镜观察检测原生质体转化进展；
将预冷的20mL 1.2M山梨醇(1.2M山梨醇，50mM Tris-Cl，50mM CaCl 2)加入上述三角瓶中，轻轻摇匀，用无菌Miracloth滤布过滤收集滤液，3000rpm，4℃离心10min；弃上清，加入预冷的5mL 1.2M山梨醇溶液悬浮菌体，3000rpm，4℃离心10min；弃上清，加入适量预冷的1.2M山梨醇悬浮分装(200μL/管，原生质体浓度为10 8个/mL)。
(2)表达载体转化
以下操作均在冰上进行，分别取10μg上述构建得到的重组质粒加入到含有200μL原生质体溶液的7mL无菌离心管中，然后加入50μL 25％PEG(25％PEG，50mM Tris-Cl，50mM CaCl 2)，轻弹管底混匀，冰上放置20min；加入2mL 25％PEG，混匀后室温放置5min；加入4mL 1.2M山梨醇，轻轻混匀后倒入熔化并保持在55℃的上层培养基中；轻轻混匀后铺在制备好的下层培养基平板上，30℃培养5～7d至有转化子长出，将生长出的转化子挑至下层培养基平板进行复筛，菌落边缘形态较光滑的菌株为阳性转化子。
按照上述方法，申请人分别构建得到重组表达野生型甘露聚糖酶M20及其突变体的里氏木霉工程菌株。
(3)发酵验证和酶活测定
将上述构建得到的里氏木霉工程菌株分别接种至PDA固体平板，在30℃恒温培养箱倒置培养6-7天，待孢子丰富后，分别取两块直径1cm的菌丝块接种于含有50mL发酵培养基(1.5％葡萄糖，1.7％乳糖，2.5％玉米浆，0.44％(NH 4) 2SO 4，0.09％MgSO 4，2％KH 2PO 4，0.04％CaCl 2，0.018％吐温-80，0.018％微量元素)的250mL三角瓶中，30℃培养48小时，然后25℃培养48小时。将发酵液离心，即得到分别含野生型甘露聚糖酶M20及其突变体的发酵上清液。
按照上述方法进行酶活检测，结果显示：上述构建得到的重组表达野生型甘露聚糖酶M20及其突变体的里氏木霉重组菌株发酵上清液的酶活为230-510U/mL。
实施例5甘露聚糖酶的酶学性质鉴定及分析
1、最适作用pH分析
采用pH值分别为2.0、2.5、3.0、4.0、4.5、5.0、5.5、6.0、7.0、8.0的磷酸氢二钠-柠檬酸缓冲液，对上述构建得到的毕赤酵母重组菌株发酵上清液进行稀释，甘露聚糖底物也分别用对应pH值的缓冲液配制，在37℃下进行甘露聚糖酶活力测定，计算酶活，以最高酶活为100％，计算相对酶活。
结果显示，野生型甘露聚糖酶M20及其突变体的最适作用pH值均为4.0，突变并未导致甘露聚糖酶M20的最适作用pH值发生变化。
2、最适反应温度分析
分别在30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃、90℃，pH5.5条件下，对上述构建得到的毕赤酵母重组菌株发酵上清液进行甘露聚糖酶酶活测定，以最高酶活为100％，计算相对酶活。
结果显示，野生型甘露聚糖酶M20的最适作用温度为70℃；而甘露聚糖酶突变体的最适作用温度为72-77℃。
3、耐热性分析
分别将上述毕赤酵母重组菌株发酵上清液用pH5.5的乙酸-乙酸钠缓冲液稀释至约20U/mL，在80℃条件下处理3min后，测定其甘露聚糖酶酶活，以未处理样品的酶活为100％，计算酶活残留率。结果如表1所示。
酶活残留率(％)＝处理后样品的酶活/未处理样品的酶活×100％。
表1甘露聚糖酶突变体耐热性比较
甘露聚糖酶单点突变体 酶活残留率
野生型M20 11.46％
D49I 20.34％
D49C 21.92％
D49N 31.74％
G58D 27.79％
G58E 23.22％
G58S 23.82％
H90L 19.47％
T99Y 21.93％
K120Y 32.83％
N124P 51.81％
T130D 26.14％
T130N 23.51％
T130Q 21.87％
T130R 19.97％
T130P 37.31％
T130S 22.13％
Y132F 23.49％
S136M 26.74％
S140L 24.79％
S140K 19.48％
T148K 29.37％
D154P 25.93％
F206Y 28.67％
A212N 23.06％
A212S 24.01％
A212P 21.46％
L257F 37.52％
S263R 43.75％
D268A 23.88％
D268G 19.79％
D268E 20.07％
D268P 28.73％
G273P 25.35％
S302M 20.20％
S302E 40.78％
S306M 39.51％
S306Q 25.98％
T315P 20.03％
T353L 22.47％
T353F 33.48％
D360E 29.47％
D360N 39.01％
D360K 60.69％
A362R 35.01％
从表1的结果可以看出，与野生型甘露聚糖酶M20相比，本发明提供的甘露聚糖酶单点突变体在80℃条件下处理3min后的酶活残留率普遍提高了69.9％-429.6％，耐热性得到显著提高。其中，N124P突变体和D360K突变体的耐热性最强，在80℃条件下处理3min后，酶活残留率分别高达51.81％和60.69％，取得了意料不到的技术效果。
本发明提供的耐高温甘露聚糖酶突变体可广泛应用于饲料领域。
以上对本发明所提供的耐高温甘露聚糖酶突变体进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2022119328-appb-000002
Figure PCTCN2022119328-appb-000003

Claims (10)

  1. 一种甘露聚糖酶突变体，其特征在于，所述突变体包含与SEQ ID NO:1具有至少90％同一性的氨基酸序列，且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代：49，58，90，99，120，124，130，132，136，140，148，154，206，212，257，263，268，273，296，297，302，306，315，339，353，360，362。
  2. 如权利要求1所述的突变体，其特征在于，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少91％，92％，93％，94％，95％，96％，97％，98％，或至少99％的同一性。
  3. 如权利要求1所述的突变体，其特征在于，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，或至少99.9％的同一性。
  4. 如权利要求1-3任一所述的突变体，其特征在于，所述突变体包含下组中至少一个氨基酸的取代：D49C/I/N，G58D/E/S，H90L，T99Y，K120Y，N124P，T130D/N/P/Q/R/S，Y132F，S136M，S140L/K，T148K，D154P，F206Y，A212N/S/P，L257F，S263R，D268A/E/G/P，G273P，V296I，T297Y，S302M/E，S306M/Q，T315P，N339F，T353L/F，D360E/N/K，A362R。
  5. 如权利要求4所述的突变体，其特征在于，所述突变体包含的取代或取代的组合选自下述取代和取代的组合：
    D49C；D49I；D49N；G58D；G58E；G58S；H90L；T99Y；K120Y；N124P；T130D；T130N；T130P；T130Q；T130R；T130S；Y132F；S136M；S140L；S140K；T148K；D154P；F206Y；A212N；A212S；A212P；L257F；S263R；D268A；D268E；D268G；D268P；G273P；V296I；T297Y；S302M；S302E；S306M；S306Q；T315P；N339F；T353L；T353F；D360E；D360N；D360K；A362R；
    D49N/G58D；
    D49N/H90L；
    D49N/T99Y；
    D49N/K120Y；
    D49N/N124P；
    D49N/T130R；
    D49N/T130S；
    D49N/T130P；
    D49N/Y132F；
    D49N/S136M；
    D49N/S140L；
    D49N/S140K；
    D49N/T148K；
    D49N/D154P；
    D49N/F206Y；
    D49N/A212N；
    D49N/A212S；
    D49N/A212P；
    D49C/F206Y；
    D49I/F206Y；
    D49N/L257F；
    D49N/D268A；
    D49N/D268E；
    D49N/D268G；
    D49N/D268P；
    D49N/G273P；
    D49N/S302M；
    D49N/S302E；
    D49N/S306M；
    D49N/S306Q；
    D49N/T315P；
    D49N/T353L；
    D49N/D360E；
    D49C/D360E；
    D49I/D360E；
    D49I/D360E；
    D49N/D360N；
    D49N/D360K；
    D49N/A362R；
    G58D/H90L；
    G58D/K120Y；
    G58D/Y132F；
    G58D/T99Y；
    G58D/N124P；
    G58D/T130P；
    G58D/S136M；
    G58D/S140K；
    G58D/T148K；
    G58D/D154P；
    G58D/F206Y；
    G58D/L257F；
    G58D/D268P；
    G58D/G273P；
    G58D/S302M；
    G58D/S302E；
    G58D/S306M；
    G58D/S306Q；
    G58D/T315P；
    G58D/T353L；
    G58D/D360E；
    H90L/A362R；
    H90L/G273P；
    H90L/S302M；
    H90L/S302E；
    H90L/S306M；
    H90L/S306Q；
    T99Y/N124P；
    T99Y/T130P；
    T99Y/S136M；
    T99Y/S140K；
    T99Y/T148K；
    T99Y/D154P；
    T99Y/F206Y；
    T99Y/L257F；
    T99Y/D268P；
    T99Y/G273P；
    T99Y/S302M；
    T99Y/S302E；
    T99Y/S306M；
    T99Y/S306Q；
    T99Y/T315P；
    T99Y/T353L；
    T99Y/D360E；
    K120Y/N124P；
    K120Y/T130P；
    K120Y/S136M；
    K120Y/S140K；
    K120Y/L257F；
    K120Y/D268P；
    K120Y/T315P；
    K120Y/T353L；
    K120Y/D360E；
    K120Y/A362R；
    N124P/T130P；
    N124P/S136M；
    N124P/S140K；
    N124P/L257F；
    N124P/D268P；
    N124P/T315P；
    N124P/T353L；
    N124P/D360E；
    T130P/S136M；
    T130P/S140K；
    T130P/L257F；
    T130P/D268P；
    T130P/T315P；
    T130P/T353L；
    T130P/D360E；
    S136M/S140K；
    S136M/L257F；
    S136M/D268P；
    S136M/T315P；
    S136M/T353L；
    S136M/D360E；
    S140K/L257F；
    S140K/D268P；
    S140K/T315P；
    S140K/T353L；
    S140K/D360E；
    T148K/D154P；
    T148K/F206Y；
    T148K/L257F；
    T148K/D268P；
    T148K/G273P；
    T148K/S302M；
    T148K/S302E；
    T148K/S306M；
    T148K/S306Q；
    T148K/T315P；
    T148K/T353L；
    T148K/D360E；
    D154P/L257F；
    D154P/D268P；
    D154P/G273P；
    D154P/S302M；
    D154P/S302E；
    D154P/S306M；
    D154P/S306Q；
    D154P/T315P；
    D154P/T353L；
    D154P/D360E；
    F206Y/G273P；
    F206Y/S302M；
    F206Y/S302E；
    F206Y/S306M；
    F206Y/S306Q；
    F206Y/T315P；
    F206Y/T353L；
    F206Y/D360E；
    A212N/D268P；
    A212N/T315P；
    A212N/T353L；
    A212N/D360E；
    L257F/D268P；
    L257F/T315P；
    L257F/T353L；
    L257F/D360E；
    D268P/T315P；
    D268P/T353L；
    D268P/D360E；
    G273P/S302M；
    G273P/S302E；
    G273P/S306M；
    G273P/S306Q；
    G273P/T315P；
    G273P/T353L；
    G273P/D360E；
    G273P/A362R；
    S302M/S306M；
    S302M/S306Q；
    S302M/T315P；
    S302M/T353L；
    S302M/D360E；
    S302M/A362R；
    S302E/S306M；
    S302E/S306Q；
    S302E/T315P；
    S302E/T353L；
    S302E/D360E；
    S302E/A362R；
    S306M/T315P；
    S306M/T353L；
    S306M/D360E；
    S306M/A362R；
    S306Q/T315P；
    S306Q/T353L；
    S306Q/D360E；
    S306Q/A362R；
    T315P/T353L；
    T315P/D360E；
    T353L/D360E；
    T315P/A362R；
    T353L/A362R；
    D360E/A362R；
    D49N/G58D/N124P；
    D49N/G58D/T130P；
    D49N/T99Y/N124P；
    D49N/T99Y/S136M；
    D268P/T315P/D360E；
    T130P/L257F/D268P；
    N124P/T130P/D268P；
    N124P/T130P/D360E；
    N124P/S302M/S306M；
    D49N/G58D/L257F；
    D49N/G58D/D268P；
    D49N/G58D/A362R；
    D49N/T99Y/S140K；
    D49N/T99Y/L257F；
    D49N/T99Y/D268P；
    D49N/G58D/T315P；
    D49N/G58D/D360E；
    G58D/N124P/T130P；
    G58D/N124P/L257F；
    G58D/N124P/D268P；
    G58D/D268P/T315P；
    L257F/D268P/T315P；
    N124P/T130P/D360K；
    D49N/T99Y/T315P；
    D49N/T99Y/T353L；
    D49N/T99Y/D360E；
    K120Y/N124P/S140K；
    K120Y/T130P/L257F；
    K120Y/S136M/D268P；
    K120Y/S140K/T353L；
    N124P/T130P/L257F；
    N124P/T130P/D268P；
    A212P/L257F/D268P；
    D49N/G58D/N124P/T130P；
    D49N/G58S/T130P/D268P；
    G58S/N124P/T130P/D268P；
    G58D/N124P/T130P/D268P；
    N124P/T130P/L257F/D268P；
    N124P/T130P/S136M/T353L；
    N124P/T130P/S136M/D360E；
    S136M/S140K/T315P/T353L；
    S136M/L257F/T315P/T353L；
    S136M/D268P/S306M/T315P；
    S136M/T315P/T353L/D360E；
    G58D/T99Y/S140K/T315P/T353L；
    G58D/N124P/T315P/T353L/D360E；
    G58S/T99Y/N124P/T130P/D268P；
    G58D/T99Y/N124P/T130P/D268P；
    G58D/T130P/T315P/T353L/D360E；
    G58D/K120Y/N124P/S140K/S136M；
    D49N/T99Y/S136M/G273P/S302M；
    T99Y/N124P/T130P/L257F/D268P；
    D49N/G58D/T99Y/N124P/T130P；
    D49N/G58D/N124P/T130P/D268P；
    D49N/G58D/N124P/T130P/T315P；
    Y132F/L257F/S263R/D268P/T315P；
    Y132F/L257F/S263R/D268P/T315P；
    Y132F/L257F/S263R/S306M/T315P；
    Y132F/G273P/S263R/S306M/T315P；
    Y132F/G273P/S263R/S306M/D360N；
    D49N/G58D/N124P/T130P/T353L；
    D49N/G58D/N124P/T130P/D360E；
    D49N/G58S/N124P/T130P/S136M；
    A212P/L257F/S263R/D268P/T315P；
    A212P/L257F/S263R/D268P/T315P；
    A212P/L257F/S263R/S306M/T315P；
    A212P/G273P/S263R/S306M/T315P；
    A212P/G273P/S263R/S306M/D360N；
    D49N/T99Y/S140K/T353L/D360E；
    D49N/T99Y/L257F/S302M/A362R；
    D49N/G58D/N124P/T130P/S140K；
    D49N/G58D/N124P/T130P/L257F；
    G58D/N124P/T130P/L257F/D268P；
    D49N/G58D/N124P/T130P/L257F/D268P；
    G58D/T99Y/N124P/T130P/L257F/D268P；
    G58D/N124P/T130P/S136M/L257F/D268P；
    G58D/N124P/T130P/S140K/L257F/D268P；
    D154P/L257F/D268P/T315P/T353L/D360E；
    D154P/D268P/S306Q/T315P/T353L/D360E；
    D154P/F206Y/D268P/S306Q/T353L/D360E；
    D49N/N124P/T130P/L257F/T315P/D360E；
    D49N/N124P/T130P/L257F/D268P/D360E；
    T148K/D154P/D268P/S306Q/T315P/T353L；
    T148K/D154P/D268P/S306M/T315P/T353L；
    D49N/N124P/T130P/L257F/D268P/T315P；
    D49N/N124P/F206Y/L257F/D268P/D360K；
    D49N/N124P/F206Y/L257F/D268P/T315P；
    N124P/T130P/L257F/D268P/T315P/D360E；
    G58D/N124P/T130P/L257F/D268P/T315P；
    G58D/N124P/T130P/L257F/D268P/T353L；
    G58D/N124P/T130P/L257F/D268P/D360E；
    G58D/N124P/T130P/L257F/D268P/T315P；
    T148K/D154P/A212P/S306Q/T315P/T353L；
    D49N/T130P/L257F/D268P/T315P/D360E；
    D49N/N124P/L257F/D268P/T315P/D360E；
    T148K/D154P/S263R/S306M/T315P/T353L；
    T148K/D154P/G273P/S306Q/T315P/T353L；
    D49N/N124P/T130P/D268P/T315P/D360E；
    G58D/N124P/T130P/L257F/D268P/G273P/D360E；
    D49N/G58D/T99Y/N124P/T130P/L257F/D268P；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P；
    D49N/G58D/N124P/T130P/S140K/L257F/D268P；
    D49N/G58D/N124P/T130P/L257F/D268P/T315P；
    D49N/G58D/N124P/T130P/L257F/D268P/T353L；
    D49N/G58D/N124P/T130P/D268P/T315P/D360E；
    D49N/G58D/N124P/T130P/L257F/T315P/D360E；
    H90L/N124PT130P/L257F/D268P/G273P/D360E；
    H90L/N124P/T130P/L257F/D268P/G273P/A362R；
    D49N/G58D/N124P/T130P/L257F/D268P/D360K；
    G58D/N124P/T130P/L257F/D268P/T315P/D360E；
    D49N/N124P/T130P/L257F/D268P/T315P/D360E；
    D49N/G58D/T130P/L257F/D268P/T315P/D360E；
    D49N/G58D/N124P/L257F/D268P/T315P/D360E；
    D49N/G58D/N124P/T130P/L257F/D268P/D360E；
    G58D/T99Y/N124P/T130P/L257F/D268P/S302M/S306M；
    G58D/N124P/T130P/S136M/L257F/D268P/S302M/T315P；
    G58D/N124P/T130P/S140K/L257F/D268P/S302M/D360E；
    D49N/G58D/N124P/T130P/L257F/D268P/T315P/T353L；
    D49N/G58D/N124P/T130P/S140K/L257F/D268P/T315P；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P/T315P；
    D49N/G58D/T99Y/N124P/T130P/L257F/D268P/T315P；
    T148K/S140K/D154P/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/N124P/T130P/L257F/D268P/T315P/D360E；
    D154P/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
    D154P/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
    D49N/G58D/N124P/T130P/S140K/L257F/D268P/T353L；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P/T353L；
    D49N/G58D/H90L/T130P/S136M/L257F/S306M/T353L；
    D49N/G58D/H90L/T130P/S136M/G273P/S306M/T353L；
    D49N/G58D/N124P/T130P/S136M/S140K/L257F/D268P；
    D49N/G58D/N124P/T130P/L257F/245P/G273P/A362R；
    H90L/T99Y/N124P/T130P/L257F/D268P/S302M/S306M；
    H90L/N124P/T130P/S136M/L257F/D268P/S302M/T315P；
    H90L/N124P/T130P/S140K/L257F/D268P/S302M/D360E；
    T99Y/N124P/T130P/Y132F/D268P/S302P/S306M/T315P；
    D49N/G58D/N124P/T130P/L257F/D268P/T315P/D360E/T353L；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P/T315P/D360E；
    D49N/G58D/N124P/T130P/S140K/L257F/D268P/T315P/D360E；
    D49N/G58D/N124P/T130P/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/T99Y/N124P/T130P/L257F/D268P/T315P/D360E；
    T99Y/N124P/T130P/L257F/D268P/S302M/T315P/D360E/A362R；
    K120Y/N124P/T130P/S136M/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/N124P/T130P/S136M/S140K/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P/V296I/T297Y/T315P/D360E；
    D154P/L257F/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
    D49N/G58D/N124P/T130P/S136M/S140K/L257F/D268P/T315P/D360E；
    D154P/L257F/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/N124P/T130P/S140K/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P/V296I/T315P/D360E；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P/T297Y/T315P/D360E；
    D49N/G58D/N124P/T130P/L257F/D268P/G273P/S306M/T315P/T353L/D360E；
    D49N/G58D/T99Y/N124P/T130P/L257F/D268P/S302M/T315P/D360E/A362R；
    G58D/T99Y/N124P/T130P/S136M/S140K/D154P/L257F/D268P/T315P/D360E/A362R；
    T130P/Y132F/S136M/S140K/D268P/G273P/S302M/S306M/T315P/T353L/D360E/A362R；
    D49N/G58D/T99Y/N124P/T130P/S136M/S140K/L257F/D268P/T315P/T353L/D360K；
    D49N/G58D/H90L/K120Y/N124P/T130P/S136M/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/T99Y/N124P/T130P/S136M/S140K/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/N124P/T130P/T148K/S140K/D154P/L257F/D268P/T315P/T353L/D360E；
    D49N/G58D/N124P/T130P/S136M/L257F/D268P/V296I/T297Y/T315P/N339F/D360E；
    D49N/G58D/T99Y/N124P/T130P/Y132F/D154P/D268P/G273P/S302P/S306M/T315P；
    T99Y/N124P/T130P/Y132F/S136M/S140K/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
    H90L/N124P/T130P/Y132F/S136M/S140K/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
    H90L/K120Y/T130P/Y132F/S136M/S140K/D268P/S302M/S306M/T315P/T353L/D360K/A362R；
    N124P/T130P/Y132F/S136M/S140K/L257F/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
    T99Y/S113M/S117K/N124P/T130P/Y132F/D268P/S302P/S306M/T315P/T330L/D337E/A339R；
    T99Y/N124P/T130P/Y132F/S136M/S140K/L257F/D268P/S302M/S306M/T315P/T353L/D360E/A362R；
    T148K/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    T99Y/T148K/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    T99Y//N124P/T148K/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    D49N/G58D/N124P/T130P/T148K/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    D49N/G58D/T99Y/T130P/T148K/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    D49N/G58D/T99Y/T130P/T148K/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    D49N/G58D/T99Y/T130P/T148K/D154P/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    D49N/G58D/T99Y/T130P/T148K/D154P/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    G58D/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360E/A362R；
    G58D/H90L/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302M/S306M/T315P/T353F/D360K/A362R；
    G58D/H90L/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302E/S306M/T315P/T353F/D360E/A362R；
    G58D/H90L/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302E/S306M/T315P/T353F/D360N/A362R；
    G58D/H90L/T99Y/N124P/T130P/D154P/F206Y/A212P/L257F/S263R/D268P/G273P/S302E/S306M/T315P/T353L/D360K/A362R。
  6. 编码权利要求1-5任一所述的突变体的DNA分子。
  7. 包含权利要求6所述DNA分子的重组表达质粒。
  8. 一种宿主细胞，其特征在于，所述的宿主细胞包含权利要求7所述的重组表达质粒。
  9. 如权利要求7所述的宿主细胞，其特征在于，所述的宿主细胞为毕赤酵母(Pichiapastoris)或里氏木霉(Trichoderma reesei)。
  10. 权利要求1-5任一所述甘露聚糖酶突变体在饲料领域中的应用。
PCT/CN2022/119328 2021-09-18 2022-09-16 耐高温甘露聚糖酶突变体 Ceased WO2023041040A1 (zh)

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