WO2020253426A1 - 比活力提高的木聚糖酶突变体 - Google Patents

比活力提高的木聚糖酶突变体 Download PDF

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WO2020253426A1
WO2020253426A1 PCT/CN2020/090456 CN2020090456W WO2020253426A1 WO 2020253426 A1 WO2020253426 A1 WO 2020253426A1 CN 2020090456 W CN2020090456 W CN 2020090456W WO 2020253426 A1 WO2020253426 A1 WO 2020253426A1
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xylanase
mutant
specific activity
amino acid
present
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PCT/CN2020/090456
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French (fr)
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吴秀秀
宋嵘锡
黄亦钧
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青岛蔚蓝生物集团有限公司
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Priority to US17/619,599 priority Critical patent/US20220380778A1/en
Priority to EP20827264.1A priority patent/EP3988657A4/en
Publication of WO2020253426A1 publication Critical patent/WO2020253426A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • 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
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N9/248—Xylanases
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
    • 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)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • 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
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention relates to the technical fields of genetic engineering and protein modification, in particular to a xylanase mutant with improved specific activity.
  • Xylan is a kind of hybrid poly-penta-carbon sugar, the main chain is connected by multiple xylopyranosyl groups through xylosidic bonds, and a variety of short substituents of different sizes are attached to the side chains.
  • Xylan mainly exists in the secondary wall of plant cells, between lignin and other polysaccharides, and plays a role in connection.
  • Xylan is an important component of plant hemicellulose. It accounts for one third of the total plant carbohydrates. It is the second most abundant renewable material resource after cellulose in nature. Xylan accounts for 7%-12% of dry matter weight in gymnosperms, and 15%-30% of dry matter weight in angiosperms.
  • the main raw materials of livestock and poultry feed in my country are derived from plants, such as corn, wheat, barley, etc., which all contain a certain amount of xylan.
  • arabinoxylan in wheat seed coat accounts for 66% of its total non-starch polysaccharides (NSPs).
  • %, arabinoxylan and ⁇ -glucan in the aleurone layer accounted for 65% and 31% of the total NSPs, respectively.
  • 88% of the NSPs in endosperm cells are arabinoxylans, of which 1/3 are soluble .
  • xylan in feed materials cannot be effectively degraded, which will significantly reduce the digestibility of nutrients, reduce feed intake, and affect the production performance of livestock and poultry.
  • the excretion of viscous feces brings difficulties to sanitation control and increases the incidence of livestock and poultry; at the same time, it can also affect the deposition of egg pigments, make meat and poultry carcasses whiter, and reduce carcass grades.
  • Xylanase refers to the general term for enzymes that can degrade xylan into xylo-oligosaccharides and xylose, which mainly include endo ⁇ -1,4-xylanase, xylosidase, arabinosidase, etc. Among them, endo- ⁇ -1,4-xylanase plays a major role in this type of enzyme. There are many kinds of microorganisms that produce xylanase: filamentous fungi, bacteria, actinomycetes, etc.
  • the bacteria that produce xylanase mainly include Bacillus subtilis, Clostridium thermocellum, Pseudomonas fluorescens, etc.; the actinomycetes that produce xylanase mainly include Streptomyces lividans and C. vulgaris Molds, etc.; the filamentous fungi that produce xylanase mainly include Aspergillus niger, Aspergillus nidulans, Aspergillus punctatus, Trichoderma reesei, Trichoderma konsi and so on.
  • domestic research on xylanase is mainly concentrated on filamentous fungi.
  • Xylanase is widely used in industry. In addition to feed, it can also be used in papermaking, food, textile and biomass energy. Due to the different environments used in different industries, xylanase also needs to have suitable enzymatic properties. For example, the feed industry needs acid-resistant xylanase, but the paper industry prefers alkaline xylanase. Moreover, in addition to enzymatic properties, specific activity is also a key indicator that limits the application of xylanase. Because the higher the specific activity of xylanase itself, the lower the production cost and the lower the price of the enzyme, which will be more conducive to promoting its widespread application. Therefore, screening out xylanase with high specific activity is also the goal that researchers in this field are working on.
  • the present invention provides a xylanase mutant to obtain mutant protein and increase its specific activity, thereby facilitating the wide application of xylanase in the feed field.
  • the present invention relates to a xylanase mutant comprising an amino acid sequence having at least 90% identity with SEQ ID NO:1, and compared with SEQ ID NO:1 in at least one position selected from the following group Contains amino acid substitutions: 78,143,148,163,177,206.
  • the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared with SEQ ID NO:1. 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% compared with SEQ ID NO:1, Or at least 99.9% identity.
  • the mutant comprises a substitution of at least one amino acid in the following group: M78F, V143I, R148K, F163W, I177V, V206L.
  • substitution or combination of substitutions contained in the mutant is selected from the following substitutions and combinations of substitutions:
  • the present invention also relates to DNA molecules encoding the aforementioned xylanase mutants.
  • the present invention also relates to a recombinant expression vector containing the above-mentioned DNA molecule.
  • the present invention also relates to a host cell comprising the above-mentioned recombinant expression vector.
  • the host cell is Pichia pastoris.
  • the present invention also provides a preparation method of the above-mentioned xylanase mutant, including:
  • Step 1 Obtain a DNA molecule encoding a xylanase mutant, the xylanase mutant comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:1, and compared with SEQ ID NO:1 At least one position selected from the group consisting of at least one amino acid substitution: 78,143,148,163,177,206;
  • Step 2 Fusion the DNA molecule obtained in step 1 with an expression vector, construct a recombinant expression vector, and transform a host cell;
  • Step 3 Induce the host cell containing the recombinant expression vector to express the fusion protein, and separate and purify the expressed fusion protein.
  • the xylanase mutant described in step 1 contains at least one amino acid substitution in the following group: M78F, V143I, R148K, F163W, I177V, V206L.
  • the host cell described in step 2 is Pichia pastoris.
  • the invention also provides the application of the xylanase mutant in feed.
  • the present invention is based on wild-type xylanase PT, and provides a mutant containing at least one mutation site among M78F, V143I, R148K, F163W, I177V, and V206L.
  • the specific activity of the single-point mutant provided by the present invention is generally increased by 12.3%-71.1%, and the specific activity of the combined mutants is generally increased by 84.0%-106.4%, and the effect is significant, which is beneficial Reduce the production cost of xylanase and promote the wide application of xylanase in feed.
  • the invention discloses a xylanase mutant, its preparation method and application, DNA molecules, vectors, and host cells encoding the xylanase mutant.
  • Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve .
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the present invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003) .
  • These general references provide definitions and methods known to those skilled in the art.
  • those skilled in the art can use other conventional methods, experimental schemes, and reagents in the field on the basis of the technical solutions described in the present invention, and are not limited to the specific embodiments of the present invention.
  • the present invention can choose the following experimental materials and reagents:
  • Escherichia coli DH5 ⁇ , Pichia pastoris GS115, vectors pPIC9k, Amp, and G418 were purchased from Invitrogen.
  • Enzymes and kits PCR enzymes and ligases were purchased from Takara, restriction endonucleases were purchased from Fermentas, plasmid extraction kits and gel purification recovery kits were purchased from Omega, GeneMorph II random mutagenesis kits were purchased from Beijing Bomax Biotechnology Co., Ltd.
  • E. coli culture medium 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;
  • Yeast medium 1% yeast extract, 2% peptone, 2% glucose;
  • Yeast selection medium 2% peptone, 2% agarose;
  • BMGY medium 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 ⁇ 10 -5 biotin, 1% glycerol;
  • BMMY medium 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 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, pH 7.0;
  • LB/Amp plate 0.5% yeast extract, 1% peptone, 1% NaCl, 1.5% agar, 100 ⁇ g/mL ampicillin, pH 7.0;
  • Example 1 Screening of mutants of high specific activity xylanase
  • the amino acid sequence of wild-type xylanase PT derived from the eukaryotic chytrid phylum Neomethea genus is SEQ ID NO: 1, and its coding nucleotide sequence is SEQ ID NO: 2.
  • the protein is a right-hand half-grip formed by a folded sheet, and two catalytic residues are located in a crack formed by a highly twisted ⁇ -sheet that can accommodate xylan sugar chains.
  • the applicant further mutated the gene.
  • PT-F1 GGC GAATTC CAAAGTTTCTGTAGTTCAGCTTCTC (underline is the recognition site of restriction enzyme EcoRI);
  • PT-R1 ATA GCGGCCGC TTATCATTAATCACCAATGTAAACCT (underlined is the restriction enzyme NotI recognition site).
  • PT gene SEQ ID NO: 2
  • the above primers to perform PCR amplification with the GeneMorph II Random Mutation PCR Kit (Stratagene), gel to recover the PCR product, and digest EcoRI and NotI with the same enzyme digestion After connecting the pET21a vector, it was transformed into E.
  • the present invention provides mutants containing single mutation sites of M78F, V143I, R148K, F163W, I177V, and V206L.
  • the present invention also provides xylanase mutants comprising at least 2, at least 3, at least 4, at least 5, and at least 6 mutation sites of M78F, V143I, R148K, F163W, I177V, and V206L, for example: M78F/F163W, V143I/I177V, F163W/I177V, M78F/R148K, R148K/V206L, R148K/F163W, I177V/V206L two-point mutants, M78F/V143I/F163W, M78F/F163W/I177V, M78F/R148K/F163W, V143I/R148K/V206L, R148K/F163W/V206L three-point mutants, M78F/V143I/R148K/F163W, M78F/F163W/I177V/V206L, V143I/R148K/F163W/V206L, V143I/R148
  • the gene sequence SEQ ID NO: 2 of PT and the gene sequence of the above mutants were optimized and synthesized, and two EcoRI and NotI were added to the 5'and 3'ends of the synthetic sequence. Restriction sites.
  • Pichia pastoris GS115 strain was activated on YPD plate, incubated at 30°C for 48 hours, and then inoculated with activated GS115 monoclonal in 6mL YPD liquid medium, 30°C, 220rpm, about 12 hours, then transferred to the bacterial solution and filled with 30mL YPD liquid culture Incubate at 30°C and 220rpm for about 5 hours in a basic Erlenmeyer flask, and detect the cell density by UV spectrophotometer.
  • the expression plasmids constructed in 2.1 were linearized with Sac I. After the linearized fragments were purified and recovered, they were transformed into Pichia pastoris GS115 by electroporation. The recombinant strains of Pichia pastoris were screened on MD plates, and then genetically modified at different concentrations. Multiple copies of transformants were screened on YPD plates (0.5 mg/mL-8 mg/mL) with mycin.
  • the obtained transformants were transferred to BMGY medium and cultured with shaking at 30°C and 250rpm for 1d; then transferred to BMMY medium and cultured with shaking at 30°C and 250rpm; 0.5% methanol was added every day to induce expression for 4d; centrifuged at 9000rpm After removing the bacteria in 10 minutes, the fermentation supernatant containing wild-type xylanase PT and xylanase mutants was obtained.
  • the applicant separately constructed and obtained Pichia engineering strains that recombinantly express wild-type xylanase PT and the above-mentioned xylanase mutant.
  • the amount of enzyme required to release 1 ⁇ mol reducing sugar from a xylan solution with a concentration of 5 mg/ml per minute is an enzyme activity unit U.
  • X D is the activity of xylanase in the diluted enzyme solution, U/ml
  • a E is the absorbance of the enzyme reaction solution
  • AB is the absorbance of the enzyme blank solution
  • K is the slope of the standard curve
  • C 0 is the standard Intercept of the curve
  • M is the molar mass of xylose, 150.2g/mol
  • t is the enzymatic hydrolysis reaction time, min
  • N is the enzyme dilution multiple
  • the xylanase enzyme activity in the fermentation supernatant of the Pichia pastoris engineering bacteria was detected according to the above method. The results showed that the enzyme activity of the fermentation supernatant of the Pichia pastoris engineered strain recombinantly expressing wild-type xylanase PT and its mutants was 180-400 U/mL.
  • Coomassie Brilliant Blue (Bradford) binding method is a compound method combining colorimetric method and pigment method to determine protein content.
  • Coomassie Brilliant Blue G-250 is brownish red in acidic solution, and turns blue when combined with protein, and conforms to Beer's law within a certain concentration range of protein, and can be colorimetrically measured at 595nm. It can be absorbed in a large amount within 3 to 5 minutes and is stable for at least 1 hour. In the range of 10 ⁇ 1000 ⁇ g/mL, the absorbance value is directly proportional to the protein concentration.
  • the xylanase protein content in the fermentation supernatant of the Pichia pastoris engineering bacteria was detected according to the above method. The results showed that the protein content of the fermentation supernatant of the Pichia pastoris engineered strain recombinantly expressing wild-type xylanase PT and its mutant was 0.04-0.1 mg/mL.
  • Specific Activity refers to the number of enzyme activity units per unit weight of protein, generally expressed in U/mg protein. Generally speaking, the higher the specific activity of the enzyme, the purer the enzyme.
  • specific activity (U/mg) enzyme activity (U/mL)/protein content (mg/mL).
  • the present invention provides xylanase mutants comprising a combination of any two or more mutation sites in M78F, V143I, R148K, F163W, I177V, and V206L, such as M78F/F163W, V143I/I177V, F163W/I177V , M78F/R148K, R148K/V206L, R148K/F163W, I177V/V206L two point mutants, M78F/V143I/F163W, M78F/F163W/I177V, M78F/R148K/F163W, V143I/R148K/V206L, R148K/F163W/V206L Three-point mutant, M78F/V143I/R148K/F163W, M78F/F163W/I177V/V206L, V143I/R148K/F163W/V206L, V143I/F163W/I177V/V206L,

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Abstract

提供一种比活力提高的木聚糖酶突变体，包含M78F、V143I、R148K、F163W、I177V、V206L中任意一个或多个突变位点，突变体的比活力得到提高，生产木聚糖酶的成本降低，可应用在饲料中。

Description

比活力提高的木聚糖酶突变体
本申请要求于2019年06月18日提交中国专利局、申请号为201910524192.9、发明名称为“比活力提高的木聚糖酶突变体”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和蛋白质改造技术领域，具体涉及一种比活力提高的木聚糖酶突变体。
背景技术
木聚糖(xylan)是一种杂合多聚五碳糖，主链由多个吡喃木糖基通过木糖苷键相连，侧链上连着多种不同大小的短的取代基。木聚糖主要存在于植物细胞的次生壁中，处于木质素及其它多聚糖之间，起着连接作用。木聚糖是植物半纤维素的重要组分，它占植物碳水化合物总量的三分之一，在自然界中是继纤维素之后含量第二丰富的再生物质资源。木聚糖在裸子植物中占干物质重量的7％-12％，在被子植物中占干物质重量的15％-30％。我国的畜禽饲料主要原料来源于植物，如玉米、小麦、大麦等，它们都含有一定数量的木聚糖，如小麦种皮中阿拉伯木聚糖占其非淀粉多糖(NSPs)总量的66％，糊粉层中阿拉伯木聚糖和β-葡聚糖分别占其NSPs总量的65％和31％，胚乳细胞中88％的NSPs是阿拉伯木聚糖，其中1/3是可溶的。在实际生产中，饲料原料中木聚糖不能有效降解，会显著降低营养物质的消化率，降低采食量，影响畜禽的生产性能。粘性粪便的排泄，给卫生控制带来困难，畜禽发病率增加；同时，还能影响禽蛋色素的沉积，使肉禽胴体色质偏白，降低胴体等级。
木聚糖酶是指能够将木聚糖降解为低聚木糖和木糖这一类酶的总称，主要包括内切β-1，4木聚糖酶、木糖苷酶、阿拉伯糖苷酶等，其中内切β-1，4木聚糖酶在这一类酶中发挥主要作用。产生木聚糖酶的微生物有很多种：包括丝状真菌、细菌、放线菌等。根据国内外研究报道，产生木聚糖酶的细菌主要有枯草芽孢杆菌、热纤维梭菌、荧光假单胞菌等； 产生木聚糖酶的放线菌主要有浅青紫链霉菌、温紫罗兰链霉菌等；产木聚糖酶的丝状真菌主要有黑曲霉、构巢曲霉、海枣曲霉、里氏木霉、康氏木霉等。目前国内对木聚糖酶的研究主要集中在丝状真菌。
木聚糖酶在工业上的应用十分广泛，除了饲料领域外，还可以应用于造纸、食品、纺织以及生物质能源领域。由于在不同工业中应用的环境不同，木聚糖酶也需要具有与之相适应的酶学性质。例如，饲料工业需要耐酸性的木聚糖酶，然而造纸工业则偏好适碱性的木聚糖酶。而且，除了酶学性质之外，比活力性也是限制木聚糖酶应用的一个关键指标。因木聚糖酶本身的比活力性愈高，其生产成本就愈低，酶的价格也就愈低，将更有利于促进其被广泛的应用。因此，筛选出高比活力性的木聚糖酶，也是目前本领域研究人员正在努力的目标。
发明内容
有鉴于此，本发明提供一种木聚糖酶突变体，获得突变体蛋白，提高其比活力，从而有利于木聚糖酶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：
本发明涉及一种木聚糖酶突变体，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：78，143，148，163，177，206。
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：M78F，V143I，R148K，F163W，I177V，V206L。
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：
M78F；
M78F/V143I；
M78F/R148K；
M78F/F163W；
M78F/I177V；
M78F/V206L；
M78F/V143I/R148K；
M78F/V143I/F163W；
M78F/V143I/I177V；
M78F/V143I/V206L；
M78F/R148K/F163W；
M78F/R148K/I177V；
M78F/R148K/V206L；
M78F/F163W/I177V；；
M78F/F163W/V206L；；
M78F/I177V/V206L；
M78F/V143I/R148K/F163W；
M78F/V143I/R148K/I177V；
M78F/V143I/R148K/V206L；
M78F/V143I/F163W/I177V；
M78F/V143I/F163W/V206L；
M78F/V143I/I177V/V206L；
M78F/R148K/F163W/I177V；
M78F/R148K/F163W/V206L；
M78F/R148K/I177V/V206L；
M78F/F163W/I177V/V206L；
V143I；
V143I/R148K；
V143I/F163W；
V143I/I177V；
V143I/V206L；
V143I/R148K/F163W；
V143I/R148K/I177V；
V143I/R148K/V206L；
V143I/F163W/I177V；
V143I/F163W/V206L；
V143I/I177V/V206L；
V143I/R148K/F163W/I177V；
V143I/R148K/F163W/V206L；
V143I/R148K/I177V/V206L；
R148K；
R148K/F163W；
R148K/I177V；
R148K/V206L；
R148K/F163W/I177V；
R148K/F163W/V206L；
R148K/I177V/V206L；
R148K/F163W/I177V/V206L；
F163W；
F163W/I177V；
F163W/V206L；
F163W/I177V/V206L；
I177V；
I177V/V206L；
V206L；
M78F/V143I/R148K/F163W/I177V；
M78F/V143I/R148K/F163W/V206L；
M78F/V143I/R148K/I177V/V206L；
M78F/R148K/F163W/I177V/V206L；
V143I/R148K/F163W/I177V/V206L；
M78F/V143I/F163W/I177V/V206L；
M78F/V143I/R148K/F163W/I177V/V206L。
本发明还涉及编码上述木聚糖酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达载体。
本发明还涉及一种宿主细胞，包含上述重组表达载体。
将上述的质粒转入宿主细胞中，重组表达的木聚糖酶突变体的比活力得到显著提高。
在本发明的一些实施例中，宿主细胞为毕赤酵母(Pichia pastoris)。
本发明还提供了上述木聚糖酶突变体的制备方法，包括：
步骤1：获取编码木聚糖酶突变体的DNA分子，所述木聚糖酶突变体包含与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至少一种氨基酸的取代：78，143，148，163，177，206；
步骤2：将步骤1获得的所述DNA分子与表达载体融合，构建重组表达载体，转化宿主细胞；
步骤3：诱导含重组表达载体的宿主细胞表达融合蛋白，分离纯化表达的融合蛋白。
在本发明的一些实施例中，步骤1所述的木聚糖酶突变体包含下组中至少一个氨基酸的取代：M78F，V143I，R148K，F163W，I177V，V206L。
在本发明的一些实施例中，步骤2所述的宿主细胞为毕赤酵母(Pichia pastoris)。
本发明还提供了上述木聚糖酶突变体在饲料中的应用。
本发明以野生型木聚糖酶PT为基础，提供了包含M78F、V143I、R148K、F163W、I177V、V206L中至少一个突变位点的突变体。与木聚糖酶PT相比，本发明提供的单点突变体的比活力普遍提高了12.3％-71.1％，组合突变体的比活力普遍提高了84.0％-106.4％，效果显著， 从而有利于降低木聚糖酶的生产成本，促进木聚糖酶在饲料中的广泛应用。
具体实施方式
本发明公开了一种木聚糖酶突变体、其制备方法及应用、编码该木聚糖酶突变体的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，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；
下面结合实施例，进一步阐述本发明。
实施例1：高比活力木聚糖酶突变体的筛选
来源于真核生物壶菌门新美鞭菌属的野生型木聚糖酶PT的氨基酸序列为SEQ ID NO:1，其编码核苷酸序列为SEQ ID NO:2。为了提高木聚糖酶PT的比活力，申请人对其基因进行了蛋白结构分析。该蛋白是由折叠片构成的右手半握状，两个催化残基位于高度扭曲的β-折叠片形成的可以容纳木聚糖糖链的裂缝中。在不破坏蛋白二级结构与活性中心的前提下，申请人进一步对该基因进行突变。
1.1设计PCR引物PT-F1、PT-R1：
PT-F1：GGC GAATTC CAAAGTTTCTGTAGTTCAGCTTCTC(下划线为限制性内切酶EcoRI识别位点)；
PT-R1：ATA GCGGCCGC TTATCATTAATCACCAATGTAAACCT(下划线为限制性内切酶NotI识别位点)。
以PT基因(SEQ ID NO：2)为模板，利用上述引物用GeneMorph II随机突变PCR试剂盒(Stratagene)进行PCR扩增，胶回收PCR产物，EcoRI、NotI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150μL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，获得含有木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；将其中一块板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，另一块板加入150ul考马斯亮蓝溶液，静置10min，考马斯亮蓝(Bradford)结合法测定蛋白质含量，分别计算不同突变子酶活水平及蛋白含量。最终，申请人从两万多个转化子中筛选出能显著提高PT比活力，又不会影响其原有酶学性质的突变位点：M78F，V143I，R148K，F163W，I177V，V206L。
在上述野生型木聚糖酶PT的基础上，本发明提供了分别含M78F，V143I，R148K，F163W，I177V，V206L单个突变位点的突变体。
本发明还提供了包含M78F，V143I，R148K，F163W，I177V，V206L中至少2个，至少3个，至少4个，至少5个，至少6个突变位点的木聚糖酶突变体，例如：M78F/F163W、V143I/I177V、F163W/I177V、M78F/R148K、R148K/V206L、R148K/F163W、I177V/V206L两点突变体，M78F/V143I/F163W、M78F/F163W/I177V、M78F/R148K/F163W、V143I/R148K/V206L、R148K/F163W/V206L三点突变体，M78F/V143I/R148K/F163W、M78F/F163W/I177V/V206L、V143I/R148K/F163W/V206L、V143I/F163W/I177V/V206L、R148K/F163W/I177V/V206L四点突变体，M78F/V143I/R148K/F163W/V206L、M78F/V143I/F163W/I177V/V206L五点突变体，以及M78F/V143I/R148K/F163W/I177V/V206L六点突变体。
实施例2：木聚糖酶突变体在毕赤酵母中的表达
依照毕赤酵母的密码偏爱性分别对PT的基因序列SEQ ID NO：2，以及上述突变体的基因序列进行优化合成，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。
2.1表达载体的构建
将合成的PT及其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 (反应体系：模板挑取的单克隆，rTaqDNA聚合酶0.5μL，10×Buffer 2.0μL，dNTPs(2.5mM)2.0μL，5’AOX引物(10M)：0.5μL，3’AOX引物：0.5μl，ddH 2O 14.5μL，反应程序：95℃预变性5min，30cycles：94℃30sec，55℃30sec，72℃2min，72℃10min)验证阳性克隆子，经测序验证后获得了正确的重组表达质粒。
2.2毕赤酵母工程菌株的构建
2.2.1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6mL YPD液体培养基中，30℃、220rpm，培养约12h后转接菌液于装有30mL YPD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。
2.2.2转化和筛选
分别将2.1构建得到的表达质粒用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木聚糖酶PT和木聚糖酶突变体的发酵上清液。
按照上述方法，申请人分别构建得到重组表达野生型木聚糖酶PT和上述木聚糖酶突变体的毕赤酵母工程菌株。
2.3木聚糖酶酶活测定
(1)酶活单位定义
在37℃、pH值为5.5的条件下，每分钟从浓度为5mg/ml的木聚糖溶液中释放1μmol还原糖所需要的酶量即为一个酶活力单位U。
(2)酶活测定方法
取2ml浓度为1％的木聚糖底物(pH5.5乙酸-乙酸钠缓冲液配制)，加入到比色管中，37℃平衡10min，再加入2ml经pH5.5乙酸-乙酸钠缓冲液适当稀释并经37℃平衡好的酸性木聚糖酶酶液，混匀于37℃精确保温反应30min。反应结束后，加入5ml DNS试剂，混匀以终止反应。然后沸水浴煮沸5min，用自来水冷却至室温，加蒸馏水定容至25ml，混匀后，以标准空白样为空白对照，在540nm处测定吸光值AE。
酶活计算公式：
Figure PCTCN2020090456-appb-000001
式中：X D为稀释酶液中木聚糖酶的活力，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，150.2g/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
按照上述方法分别检测上述毕赤酵母工程菌的发酵上清液中木聚糖酶酶活。结果显示，重组表达野生型木聚糖酶PT及其突变体的毕赤酵母工程菌株发酵上清液的酶活为180-400U/mL。
2.4蛋白含量测定
(1)测定方法：
考马斯亮蓝(Bradford)结合法测定蛋白质含量是比色法与色素法结合的复合方法。考马斯亮兰G-250在酸性溶液时呈棕红色，当与蛋白质结合后变为蓝色，且在蛋白质一定浓度范围内符合比尔定律，可在595nm处比色测定。在3～5分钟即成大量吸收，至少稳定1小时。在10～1000μg/mL范围内，吸光值与蛋白质浓度成正比。
按照酶液和考马斯亮蓝溶液体积比1：5的比例进行混合，静置10mim， 考马斯亮蓝(Bradford)结合法测定蛋白质含量。
(2)蛋白含量测定结果
按照上述方法分别检测上述毕赤酵母工程菌的发酵上清液中木聚糖酶蛋白含量。结果显示：重组表达野生型木聚糖酶PT及其突变体的毕赤酵母工程菌株发酵上清液的蛋白含量为0.04-0.1mg/mL。
2.5比活力的计算
“比活力(Specific Activity)”是指：单位重量的蛋白质中所具有酶的活力单位数，一般用U/mg蛋白质来表示。一般来说，酶的比活力越高，酶越纯。
比活力计算公式：比活力(U/mg)＝酶活(U/mL)/蛋白含量(mg/mL)。
分别计算重组表达野生型木聚糖酶PT及其突变体的毕赤酵母工程菌株发酵上清液的木聚糖酶比活力。
结果显示，与野生型木聚糖酶PT相比，本发明提供的分别含M78F、V143I、R148K、F163W、I177V、V206L单个突变位点的木聚糖酶突变体的比活力分别提高了24.8％，25.6％，66.6％，12.3％，63.1％，71.1％。从而说明，本发明在野生型木聚糖酶PT基础上提供的单点突变体的比活力得到显著提高。
此外，本发明提供的包含M78F、V143I、R148K、F163W、I177V、V206L中任意2个或2个以上突变位点组合的木聚糖酶突变体，例如M78F/F163W、V143I/I177V、F163W/I177V、M78F/R148K、R148K/V206L、R148K/F163W、I177V/V206L两点突变体，M78F/V143I/F163W、M78F/F163W/I177V、M78F/R148K/F163W、V143I/R148K/V206L、R148K/F163W/V206L三点突变体，M78F/V143I/R148K/F163W、M78F/F163W/I177V/V206L、V143I/R148K/F163W/V206L、V143I/F163W/I177V/V206L、R148K/F163W/I177V/V206L四点突变体，M78F/V143I/R148K/F163W/V206L、M78F/V143I/F163W/I177V/V206L五点突变体，以及M78F/V143I/R148K/F163W/I177V/V206L六点突变体， 其比活力普遍提高了84.0％-106.4％，均显著高于上述单点突变体的比活力水平，取得了意料不到的技术效果。

Claims (10)

  1. 一种木聚糖酶突变体，其特征在于，所述的木聚糖酶突变体包含与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78，143，148，163，177，206中至少一个位置上发生了氨基酸的取代。
  2. 如权利要求1所述的突变体，其特征在于，所述的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，所述的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所述的突变体，其特征在于，所述的突变体包含下组中至少一个氨基酸的取代：M78F，V143I，R148K，F163W，I177V，V206L。
  5. 如权利要求1所述的突变体，其特征在于，所述的突变体包含有下述取代或取代的组合：
    M78F；
    M78F/V143I；
    M78F/R148K；
    M78F/F163W；
    M78F/I177V；
    M78F/V206L；
    M78F/V143I/R148K；
    M78F/V143I/F163W；
    M78F/V143I/I177V；
    M78F/V143I/V206L；
    M78F/R148K/F163W；
    M78F/R148K/I177V；
    M78F/R148K/V206L；
    M78F/F163W/I177V；；
    M78F/F163W/V206L；；
    M78F/I177V/V206L；
    M78F/V143I/R148K/F163W；
    M78F/V143I/R148K/I177V；
    M78F/V143I/R148K/V206L；
    M78F/V143I/F163W/I177V；
    M78F/V143I/F163W/V206L；
    M78F/V143I/I177V/V206L；
    M78F/R148K/F163W/I177V；
    M78F/R148K/F163W/V206L；
    M78F/R148K/I177V/V206L；
    M78F/F163W/I177V/V206L；
    V143I；
    V143I/R148K；
    V143I/F163W；
    V143I/I177V；
    V143I/V206L；
    V143I/R148K/F163W；
    V143I/R148K/I177V；
    V143I/R148K/V206L；
    V143I/F163W/I177V；
    V143I/F163W/V206L；
    V143I/I177V/V206L；
    V143I/R148K/F163W/I177V；
    V143I/R148K/F163W/V206L；
    V143I/R148K/I177V/V206L；
    R148K；
    R148K/F163W；
    R148K/I177V；
    R148K/V206L；
    R148K/F163W/I177V；
    R148K/F163W/V206L；
    R148K/I177V/V206L；
    R148K/F163W/I177V/V206L；
    F163W；
    F163W/I177V；
    F163W/V206L；
    F163W/I177V/V206L；
    I177V；
    I177V/V206L；
    V206L；
    M78F/V143I/R148K/F163W/I177V；
    M78F/V143I/R148K/F163W/V206L；
    M78F/V143I/R148K/I177V/V206L；
    M78F/R148K/F163W/I177V/V206L；
    V143I/R148K/F163W/I177V/V206L；
    M78F/V143I/F163W/I177V/V206L；
    M78F/V143I/R148K/F163W/I177V/V206L。
  6. 编码权利要求1-5任一所述木聚糖酶突变体的DNA分子。
  7. 一种重组表达载体，其特征在于，所述的重组表达载体中包含有权利要求6所述的DNA分子。
  8. 一种宿主细胞，其特征在于，所述的宿主细胞转入有权利要求7所述的重组表达载体。
  9. 如权利要求8所述的宿主细胞，其特征在于，所述的宿主细胞为毕赤酵母(Pichia pastoris)。
  10. 权利要求1-5任一项所述的木聚糖酶突变体在饲料领域中的应用。
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