WO2018054244A1 - 一种木聚糖酶突变体及其应用 - Google Patents

一种木聚糖酶突变体及其应用 Download PDF

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WO2018054244A1
WO2018054244A1 PCT/CN2017/101394 CN2017101394W WO2018054244A1 WO 2018054244 A1 WO2018054244 A1 WO 2018054244A1 CN 2017101394 W CN2017101394 W CN 2017101394W WO 2018054244 A1 WO2018054244 A1 WO 2018054244A1
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xylanase
mutant
xylanase mutant
mutation
gene
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PCT/CN2017/101394
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French (fr)
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黄遵锡
苗华彪
韩楠玉
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云南师范大学
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    • 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
    • C12N9/248—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

Definitions

  • the invention belongs to the technical field of bioengineering, and in particular relates to a xylanase mutant and an application thereof.
  • Xylan is an important component of hemicellulose in plant cell tissues and is a rich biological resource. However, it is difficult to be degraded and utilized in nature, resulting in a waste of biological resources.
  • Xylanase (EC.3.2.1.8) is a generic term for a class of multifunctional enzymes that degrade xylan, which can hydrolyze xylan into reducing sugars such as xylooligosaccharides and xylose.
  • Xylanase is abundant in nature, and many fungi, plant tissues and bacteria can produce xylanase, which has received widespread attention in recent years.
  • xylanase is also an important industrial enzyme preparation, which is widely used in food processing, textile, pulp bleaching, brewing, feed processing and other fields.
  • xylanases are medium-temperature xylanases, which have poor thermal stability and are difficult to meet industrial requirements. demand. Therefore, improving the thermal stability of xylanase has become a hot spot of widespread concern today.
  • the fungus Rhizoctonia xylanase xylanase has a wide range of pH and relatively high relative enzyme activity. If the enzyme is improved to increase its heat resistance, the xylanase will have wider application value.
  • the present invention is based on the phenomenon that the C-terminus of the xylanase having good heat resistance in the GH11 family has a distinct ⁇ -folding, and is aimed at the fungus Rhizoctonia xylanase C by sequence alignment and shortcut structure analysis. The end is modified in sequence to enhance its heat resistance.
  • the technical scheme adopted by the present invention is: a xylanase mutant, and the amino acid sequence of the xylanase mutant is shown in SEQ ID No: 1.
  • the xylanase mutant is obtained by changing amino acids in the amino acid sequence of the fungus-like Rhizoctonia xylanase xylanase SEQ ID No: 3 by changing N207S, G208S, A210S as follows.
  • the xylanase mutant has an optimum pH of 6.0 for enzymatic reaction; the optimum temperature is 55 ° C; at pH 3 - pH 11 and 37 ° C, the pH is tolerated for 1 hour, and the residual activity is still 50. %, at pH4-pH9, At 37 °C, the pH was tolerated for 1 hour, and the residual activity was still above 90%.
  • the xylanase was tolerated for 120 min at 70 °C, 60 min at 80 °C, and 30 min at 90 °C, and the residual activity was 60. %.
  • the invention also provides a method for constructing a xylanase mutant, the steps comprising:
  • the fungal N. difficile xylanase gene was ligated into the ppic9k vector as a template, and the following primers were designed for mutation PCR amplification; the PCR system was prepared according to the kit instructions 50 ⁇ L;
  • the present invention also provides a gene encoding a xylanase mutant, the nucleotide sequence of which is shown in SEQ ID No: 2.
  • the present invention also provides an engineered bacterium of a gene of a xylanase mutant.
  • the present invention also provides a method for constructing an engineered strain of a xylanase mutant gene, which is exemplified by PPIC9, PPICZaA ⁇ B ⁇ C, PPICZA ⁇ B ⁇ C, PGAPZaA ⁇ B ⁇ C , PICHIAPINK-Hc, PICHIAPINK-Lc expression vector construct recombinant plasmid, transform the corresponding host strain GS115 or X33, SMD1168, PICHIAPINK, screen to obtain xylan by adding phytate on the plate or adding G418, Zeocin antibiotic to the plate
  • the enzyme mutant is genetically engineered and then a new xylanase mutant is obtained by fermentation.
  • the xylanase mutant provided by the present invention has a distinct ⁇ -sheet at the C-terminus according to the spatial structure of the heat-resistant xylanase of the GH11 family, and is analyzed by sequence alignment and crystal structure, and the fungus class is novel.
  • the C-terminus of the flagellin xylanase was sequence engineered to make it a thermostable xylanase.
  • the amino acid sequence of the fungus N. difficile xylanase amino acid sequence SEQ ID No: 3 is changed as follows: N207S, G208S, A210S.
  • the specific scheme is to use the fungus Rhizoctonia xylanase xylanase as a template to mutate the fungal Genus dinoflagellate xylanase gene, and obtain a novel xylanase gene mutant SEQ ID No: 2,
  • the mutated gene can also construct a recombinant plasmid with PPIC9, PPICZaA ⁇ B ⁇ C, PPICZA ⁇ B ⁇ C, PGAPZaA ⁇ B ⁇ C and other expression vectors, and transform the corresponding host strain (Pichia pastoris).
  • High heat-resistant xylanase, the xylanase mutant at high temperature, the temperature tolerance of xylanase is shown in Figure 5-7, regardless of the temperature and time, the relative after the mutation The enzyme activity was higher than that before the mutation, and the relative zymosanase activity at 70 °C for 2 hours was about 59%, while the fungal Rhizoctonia xylanase only had 51% remaining.
  • the time before the mutation of xylanase relative to the remaining half of the enzyme activity is about 60 min, while the relative enzyme activity still retains 60% of the enzyme activity after 60 min of tolerance.
  • the xylanase is relatively resistant after 30 min tolerance at 95 °C.
  • the enzyme activity remains close to 58%, and the remaining 51% before the mutation, the total mutation Compared with the pre-mutation temperature tolerance, the temperature tolerance is increased by 10%; at the same time, the xylanase mutant has a more suitable temperature for the feed enzyme preparation than the original fungus Rhodotorula xylanase, and the relative fungus class is new.
  • the xylanase the optimum temperature of the mutated enzyme is close to 10 °C to the gastrointestinal tract temperature of the livestock, and the relative enzyme activity remains close to 70% in the gastrointestinal temperature environment of the livestock and poultry, and the relative enzyme activity remaining before the mutation 48% improvement.
  • the mutant works well in the gastrointestinal environment of livestock and poultry, and has desirable heat resistance characteristics, so it is particularly suitable as a feed additive.
  • FIG. 1 is a flow chart of a method for constructing a xylanase mutant according to an embodiment of the present invention
  • FIG. 3 is a graph showing an optimum temperature profile provided by an embodiment of the present invention.
  • Figure 5 is a 70 ° C tolerance curve provided by an embodiment of the present invention.
  • Figure 6 is a graph showing the 80 ° C tolerance curve provided by an embodiment of the present invention.
  • Figure 7 is a 90 °C tolerance curve provided by an embodiment of the present invention.
  • the C-terminal has a distinct ⁇ -sheet, and the fungal Hymenoptera is detected by sequence alignment and analysis of the difference in the C-terminal temperature factor in the crystal structure.
  • the C-terminus of the xylanase was sequence-modified, and the sequence was as shown in SEQ ID No: 1, which improved the heat resistance of the xylanase.
  • the amino acid sequence of the fungus N. difficile xylanase amino acid sequence SEQ ID No: 3 is changed as follows: N207S, G208S, A210S.
  • the specific scheme is to use the fungus Rhizoctonia xylanase xylanase as a template to mutate the fungal Genus dinoflagellate xylanase gene, and obtain a novel xylanase gene mutant SEQ ID No: 2,
  • the mutated gene can also construct a recombinant plasmid with PPIC9, PPICZaA ⁇ B ⁇ C, PPICZA ⁇ B ⁇ C, PGAPZaA ⁇ B ⁇ C and other expression vectors, and transform the corresponding host strain (Pichia pastoris).
  • the method for constructing a xylanase mutant comprises the following steps:
  • S101 a recombinant plasmid of the fungus Rhizoctonia xylanase xylanase gene linked to the ppic9k vector is used as a template for mutation PCR amplification; the PCR system is prepared according to the kit instructions 50 ⁇ L;
  • S104 verify positive clones, positive clones from each locus are sent for sequencing, sequencing results and original sequences To find out the correct recombinant plasmid, and then use the mutant plasmid as a template to carry out the second mutation to the all-mutation site, and then transfer the mutated plasmid into yeast expression to measure the enzyme activity.
  • Mutation PCR reaction parameters were: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 5 min for 30 cycles; extension at 72 °C for 10 min;
  • Escherichia coli DMT competent form was purchased from Beijing Quanjin Biotechnology Co., Ltd., expression vector PPIC9K or PPIC9, PPICZaA ⁇ B ⁇ C, PPICZA ⁇ B ⁇ C, PGAPZaA ⁇ B ⁇ C, PPINK Hc ⁇ Lc Etc.
  • Pichia GS115 or (X33, SMD1168, PICHIAPINK) from INVITROGEN.
  • DNA polymerase nuclease endonuclease and dNTP were purchased from TaKaRa; xylan was purchased from Sigma; Fast Mutagenesis System kit was purchased from TRANSGEN BIOTECH, all other domestically produced reagents (all available from common biochemical reagents) ).
  • the solid medium was supplemented with 2.0% (w/v) agar.
  • YEPD medium Peptone 20g, Yeast extract 10g, glucose 20g (single extinction), distilled water to 1000ml, pH natural (7).
  • the solid medium was supplemented with 2.0% (w/v) agar.
  • Yeast fermentation media FA and FB were purchased from INVITROGEN.
  • the sequence of Table 1 is a primer, and the recombinant plasmid of the fungus Rhizoctonia xylanase ligated to the ppic9k vector is used as a template for mutation PCR amplification.
  • the PCR system was prepared according to the kit instructions. The PCR reaction parameters were: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 5 min for 30 cycles, and extension at 72 °C for 10 min. 10 ⁇ l of PCR product was taken and detected by 1% agarose gel electrophoresis. After the band was correctly added, 1 ⁇ l of DMT enzyme was added to the PCR product, mixed, and incubated at 37 ° C for 1 h and then transformed.
  • Pichia expression vectors and Pichia pastoris transformation were carried out according to the corresponding Pichia yeast vector and host strain instructions (from INVITROGEN).
  • Constant temperature water bath analytical balance, constant temperature culture oscillator, pH meter, etc., microplate reader and ELISA plate.
  • Mutant xylanase strain fermentation enzyme solution (mutation), original template xylanase strain fermentation enzyme solution (fungi fungi)
  • Tris-Hcl buffer pH 8.0-9.0
  • the reactants are placed at different temperatures under the above optimum pH conditions.
  • the enzyme solution of the control group was an untolerated enzyme solution.
  • the enzyme solution was diluted to the corresponding multiple, and then placed in different temperatures: 70 ° C, 80 ° C, 95 ° C tolerated 1 min, 3 min, 5 min, 10 min, 15 min, 20 min, 30 min, 60 min, 90 min, 120 min, 150 min.
  • the reaction is then carried out at an optimum pH and an optimum temperature according to the method of 4.3.
  • the control experiment group was an enzyme solution that was not temperature-tolerant.
  • the optimum pH value of the xylanase enzymatic reaction is shown in Figure 2.
  • the optimum pH of the mutated and fungal Xanthomonas xylanase was 6.0; the difference between the two was not obvious before and after the mutation.
  • the optimum temperature values for the xylanase enzymatic reaction are shown in Figure 3.
  • the optimum temperature for the xylanases of the mutant and fungi were 55 °C and 65 °C, respectively;
  • the optimum temperature of the mutated xylanase was 10 °C lower than that before the mutation, and it was closer to the intestinal temperature of the livestock, which retained the enzyme activity at 37-40 °C.
  • the temperature tolerance of xylanase at high temperature is shown in Figure 5-7. As the temperature increases, the relative enzyme activity decreases, and the relative enzyme activity decreases with time. At any temperature and time, the relative enzyme activity after mutation was slightly higher than that before the mutation, and the relative zymase activity at 70 °C for 2 hours was about 59%, while the fungus was fresh before the mutation. The flagellate xylanase only had 51% remaining, and the mutant xylanase was tolerated for 3 hours before it fell below half-life. After mutation at 80 ° C, the xylanase was tolerated for 2 hours relative to the remaining 51% of the enzyme activity, and the half-life was reached after 90 min of tolerance. After the 60min tolerance at 60°C, the relative enzyme activity of xylanase remained close to 55%, and only 48% remained before the mutation. After the mutation, the temperature tolerance was increased by about 10% compared with that before the mutation.

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Abstract

一种木聚糖酶突变体及其应用，木聚糖酶突变体的氨基酸序列如SEQ ID No：1所示，木聚糖酶突变体由编码真菌类新丽鞭毛菌木聚糖酶氨基酸序列SEQ ID No：3中的氨基酸发生如下改变N207S，G208S，A210S而获得。该突变体能在畜禽胃肠环境中很好发生作用，并且具有较理想耐热特性，因此特别适合于作为饲料添加剂。

Description

一种木聚糖酶突变体及其应用 技术领域
本发明属于生物工程技术领域，尤其涉及一种木聚糖酶突变体及其应用。
背景技术
木聚糖是植物细胞组织中半纤维素的重要成分，是一种含量丰富的生物资源。然而在自然界中很难被降解利用，造成了大量生物资源的浪费。木聚糖酶(EC.3.2.1.8)是一类降解木聚糖的多功能酶类的总称，可以将木聚糖水解成低聚木糖、木糖等还原性糖。木聚糖酶在自然界中含量丰富，许多真菌、植物组织、细菌都可产生木聚糖酶，近年来得到了人们的广泛关注。
同时，木聚糖酶也是一种重要的工业酶制剂，广泛应用于食品加工、纺织、纸浆漂白、酿造、饲料加工等多个领域。但不同领域需要的木聚糖酶的特性不同，如纸浆漂白、饲料加工等都需要经过高温处理，而大多数木聚糖酶属于中温木聚糖酶，热稳定性较差，难以满足工业的需求。因此，提高木聚糖酶热稳定性已成为当今人们广泛关注的热点。
真菌类新丽鞭毛菌木聚糖酶具有广泛的pH作用范围，且相对酶活较高，如果改进该酶使其耐热性增强，将使该木聚糖酶有更广泛的应用价值。本发明根据GH11家族中耐热性好的木聚糖酶的空间结构C端有明显的β折叠这一现象，通过序列比对和捷径结构分析，针对真菌类新丽鞭毛菌木聚糖酶C端进行序列改进，使其耐热性增强。
发明内容
本发明的目的在于提供一种木聚糖酶突变体及其应用，旨在提高木聚糖酶的热稳定性。
为实现上述目的，本发明采用的技术方案是：一种木聚糖酶突变体，所述木聚糖酶突变体的氨基酸序列如SEQ ID No：1所示。
进一步的，所述木聚糖酶突变体由编码真菌类新丽鞭毛菌木聚糖酶氨基酸序列SEQ ID No：3中的氨基酸发生如下改变N207S，G208S，A210S而获得。
进一步的，所述木聚糖酶突变体的酶促反应最适pH值为6.0；最适温度为55℃；在pH3-pH11、37℃条件下，pH耐受1小时，残活还在50％，在pH4-pH9、 37℃条件下，pH耐受1小时，残活还在90％以上，木聚糖酶在70℃耐受120min、80℃时耐受60min、高温90℃时耐受30min，残活都在60％。
本发明还提供一种木聚糖酶突变体的构建方法，步骤包括：
真菌类新丽鞭毛菌木聚糖酶基因连接到ppic9k载体上的重组质粒为模板，分别设计下列引物进行突变PCR扩增；PCR体系按照试剂盒说明书配制50μL；
取10μl PCR产物，1％琼脂糖凝胶电泳检测，条带正确后加1μl DMT酶于PCR产物中，混匀，37℃孵育1h；
然后转化，加入5μl突变后的产物于50μl DMT感受态细胞中，轻弹混匀，冰浴30min；42℃准确热激45s，立即置于冰上10min；加500μlLB培养基，200转，37℃培养1h；7000rpm离心3min，弃掉上清，保留100-150μl，轻弹悬浮菌体，取全部菌液涂板，培养过夜；
验证阳性克隆子，每个位点的阳性克隆送出测序，测序结果与原序列比对，找出突变正确的重组质粒，再以突变一次质粒作为模板，进行第二位点突变至止突变位点全部突变掉，突变后的质粒转入毕赤酵母GS115或X33、SMD1168、PICHIAPINK中进行表达，发酵测酶活，研究酶学及应用特性。
本发明还提供一种编码木聚糖酶突变体的基因，所述基因核苷酸序列如SEQ ID No：2所示。
本发明还提供一种木聚糖酶突变体的基因的工程菌。
本发明还提供一种木聚糖酶突变体的基因的工程菌的构建方法，所述木聚糖酶基因突变体与PPIC9、PPICZaA\B\C、PPICZA\B\C、PGAPZaA\B\C、PICHIAPINK-Hc、PICHIAPINK-Lc表达载体构建重组质粒，转化相应宿主菌GS115或X33、SMD1168、PICHIAPINK中，通过在平板上加入植酸钙或在平板中加入G418、Zeocin抗生素，筛选获得木聚糖酶突变体基因工程菌，然后通过发酵获得新的木聚糖酶突变体。
本发明提供的木聚糖酶突变体，根据GH11家族耐热性强的木聚糖酶的空间结构，其C端有明显的β折叠，通过序列比对和结晶结构分析，对真菌类新丽鞭毛菌木聚糖酶C端进行序列改造，使其成为耐热性高的木聚糖酶。真菌类新丽鞭毛菌木聚糖酶氨基酸序列SEQ ID No：3中的氨基酸发生如下改变N207S,G208S,A210S。具体方案为以真菌类新丽鞭毛菌木聚糖酶作为模版，使真菌类新丽鞭毛菌木聚糖酶基因发生突变，获得了新的木聚糖酶基因的突变体SEQ ID No：2， 该突变后的基因除与PPIC9K构建重组质粒外，还可以与PPIC9、PPICZaA\B\C、PPICZA\B\C、PGAPZaA\B\C等表达载体构建重组质粒，转化相应宿主菌(毕赤酵母GS115或(X33、SMD1168、PICHIAPINK)，通过在在平板中加入G418、Zeocin等抗生素，筛选获得木聚糖酶突变体基因工程菌，然后通过发酵获得新的木聚糖酶突变体。使其成为耐热性高的木聚糖酶，该木聚糖酶突变体在高温下，木聚糖酶的温度耐受情况如图5-7所示，无论在任何温度和时间下，突变后的相对酶活都高于突变前的，在70℃耐受2小时突变木聚糖酶相对酶活大约还有59％，而真菌类新丽鞭毛菌木聚糖酶仅仅剩余51％。在80℃时突变前木聚糖酶相对酶活剩余一半时的时间大约为60min，而突变后耐受60min其相对酶活仍然保留60％的酶活。高温95℃时耐受30min突变后木聚糖酶相对酶活还剩余接近58％，突变前剩余51％，总之突变后与突变前相比温度耐受相对提高了10％；同时木聚糖酶突变体较原真菌类新丽鞭毛菌木聚糖酶具有更适合饲用酶制剂的作用温度，相对真菌类新丽鞭毛菌木聚糖酶，突变后的酶的最适温度向畜禽胃肠道温度靠近10℃，并且在畜禽胃肠温度环境下相对酶活剩余接近70％左右，与突变之前相对酶活剩余48％有改进。该突变体能在畜禽胃肠环境中很好发生作用，并且具有较理想耐热特性，因此特别适合于作为饲料添加剂。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的木聚糖酶突变体的构建方法流程图；
图2是本发明实施例提供的最适pH的测定曲线图；
图3是本发明实施例提供的最适温度曲线图；
图4是本发明实施例提供的pH耐受曲线图；
图5是本发明实施例提供的70℃耐受曲线图；
图6是本发明实施例提供的80℃耐受曲线图；
图7是本发明实施例提供的90℃耐受曲线图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本发明根据GH11家族耐热性强的木聚糖酶的空间结构，其C端有明显的β折叠，通过序列比对和分析结晶结构中C端温度因子的差异，对真菌类新丽鞭毛菌木聚糖酶C端进行序列改造，序列如SEQ ID No：1所示，使木聚糖酶的耐热性提高。真菌类新丽鞭毛菌木聚糖酶氨基酸序列SEQ ID No：3中的氨基酸发生如下改变N207S,G208S,A210S。具体方案为以真菌类新丽鞭毛菌木聚糖酶作为模版，使真菌类新丽鞭毛菌木聚糖酶基因发生突变，获得了新的木聚糖酶基因的突变体SEQ ID No：2，该突变后的基因除与PPIC9K构建重组质粒外，还可以与PPIC9、PPICZaA\B\C、PPICZA\B\C、PGAPZaA\B\C等表达载体构建重组质粒，转化相应宿主菌(毕赤酵母GS115或(X33、SMD1168、PICHIAPINK)，通过在在平板中加入G418、Zeocin等抗生素，筛选获得木聚糖酶突变体基因工程菌，然后通过发酵获得新的木聚糖酶突变体。该突变体能在畜禽胃肠环境中很好发生作用，并且具有较理想耐热特性，适合耐高温制粒，因此特别适合于饲料添加剂。
实施例2
如图1所示，木聚糖酶突变体的构建方法包括以下步骤：
S101：真菌类新丽鞭毛菌木聚糖酶基因连接到ppic9k载体上的重组质粒为模板，进行突变PCR扩增；PCR体系按照试剂盒说明书配制50μL；
S102：取10μl PCR产物，1％琼脂糖凝胶电泳检测。条带正确后加1μlDMT酶于PCR产物中，混匀，37℃孵育1h；
S103：然后转化，加入5μl突变后的产物于50μl DMT感受态细胞中(在感受态细胞刚刚解冻时加入产物)，轻弹混匀，冰浴30分钟；42℃准确热激45秒，立即置于冰上10min；加500μlLB培养基，200转，37℃培养1小时；7000rpm离心3min，弃掉部分上清，保留100-150μl，轻弹悬浮菌体，取全部菌液涂板，培养过夜；
S104：验证阳性克隆子，每个位点的阳性克隆送出测序，测序结果与原序列 比对，找出突变正确的重组质粒，再以突变一次质粒作为模板，进行第二位点突变至止突变位点全部突变掉，然后将突变后的质粒转入酵母表达，发酵测酶活。
突变PCR反应参数为：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸5min，共30个循环；72℃扩增后延伸10min；
实施例3
酶活定义为：样品在温度为50℃、pH＝6.0条件下，每分钟从浓度为5.0mg/mL木聚糖溶液中释放1μmol还原糖所需要的酶量为一个酶活性单位，以U表示(QB/T 4483-2013)。
1、试验材料和试剂
菌株及载体：大肠杆菌Escherichia coli DMT感受态购于北京全式金生物技术有限公司，表达载体PPIC9K或PPIC9、PPICZaA\B\C、PPICZA\B\C、PGAPZaA\B\C、PPINK Hc\Lc等及毕赤酵母GS115或(X33、SMD1168、PICHIAPINK)(来源于INVITROGEN公司)。
2、酶类及其他生化试剂
DNA聚合酶,核酸酶内切酶和dNTP购自TaKaRa公司；木聚糖购自Sigma公司；Fast Mutagenesis System试剂盒购自TRANSGEN BIOTECH公司，其它都为国产试剂(均可从普通生化试剂公司购买得到)。
培养基：LB培养基：Peptone10g，Yeast extract5g，NaCl10g，加蒸馏水至1000ml，pH＝7。固体培养基在此基础上加2.0％(w/v)琼脂。
YEPD培养基：Peptone20g，Yeast extract10g，glucose20g(单灭)，加蒸馏水至1000ml，pH自然(为7)。固体培养基在此基础上加2.0％(w/v)琼脂。
酵母发酵培养基FA和FB购自INVITROGEN公司。
3、木聚糖酶突变体的构建
表1的序列为引物，真菌类新丽鞭毛菌木聚糖酶连接到ppic9k载体上的重组质粒为模板，进行突变PCR扩增。PCR体系按照试剂盒说明书配制50μL，PCR反应参数为：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸5min，共30个循环；72℃扩增后延伸10min。取10μl PCR产物，1％琼脂糖凝胶电泳检测。条带正确后加1μl DMT酶于PCR产物中，混匀，37℃孵育1h然后转化。
a.加入5μl突变后的产物于50μl DMT感受态细胞中(在感受态细胞刚刚解冻时加入产物)，轻弹混匀，冰浴30分钟；
b.42℃准确热激45s，立即置于冰上10min；
c.加500μlLB培养基，200转，37℃培养1h；
d. 7000rpm离心3min，弃掉部分上清，保留100-150μl，轻弹悬浮菌体，取全部菌液涂板，培养过夜。
验证阳性克隆子，每个位点的阳性克隆送出测序，测序结果与原序列比对，找出突变正确的重组质粒，再以突变一次质粒作为模板，进行第二位点突变至止突变位点全部突变掉。突变后的质粒转入毕赤酵母GS115来源于INVITROGEN公司表达，发酵测酶活。
其它毕赤酵母表达载体构建及毕赤酵母转化、筛选与发酵按相应的毕赤酵母载体与宿主菌株说明书(来源于INVITROGEN公司)进行。
表1、真菌类新丽鞭毛菌木聚糖酶基因突变所用引物
Figure PCTCN2017101394-appb-000001
4、木聚糖酶酶学性质的测定法
4.1、仪器与设备
恒温水浴锅、分析天平、恒温培养振荡器、pH仪等、酶标仪和酶标板等。
4.2、实验材料
4.2.1、酶液
突变木聚糖酶菌株发酵酶液(突变)、原模板木聚糖酶菌株发酵酶液(真菌类新丽鞭毛菌)
4.2.2、溶液配制
4.2.2.1、缓冲液：具体参见2002年黄培德译分子克隆实验指南第三版
0.1mol/L柠檬酸-磷酸氢二钠缓冲液(pH2.0-8.0)
0.1mol/L Tris-Hcl缓冲液(pH8.0-9.0)
0.1mol/L甘氨酸-NaOH缓冲液(pH9.0-12.0)
4.2.2.2、底物溶液：10mg/mL木聚糖溶液
称取1.00g木聚糖(Sigma)和0.32g氢氧化钠于100ml容量瓶中，在加90mL水,磁力搅拌，同时缓慢加热，直至木聚糖完全溶解。然后停止加热，继续搅拌30min至室温，测定其pH。如果pH为6.0，用柠檬酸-磷酸氢二钠缓冲液定容至100ml，如果偏离Ph 6.0,再用柠檬酸或者磷酸氢二钠调节至6.0，然后再用柠檬酸-磷酸氢二钠缓冲液定容至100ml。4℃避光保存，有效期为12h。
4.2.2.3、DNS终止液配制
取80.0g NaOH加入3.5L中，待全部溶解并冷却后，50.0g 3,5-二硝基水杨酸全部加入上述溶液，搅拌溶解完全，再边搅拌边加入少量酒石酸钾钠，共计1500.0g，用不超45℃的水浴锅加热搅拌溶解，冷却后，加ddH2O定容至5.0L，并于棕色瓶中储存，室温保存备用(室温静置一周后使用)。
4.3、酶活测定方法
取10ml试管，加入450μl缓冲液(4.2.2.1)，和450μl底物木聚糖(4.2.2.2)，混合后预热5min，加入0.1ml待测酶液，，加入酶液时间间隔一致，混合后反应10分钟，之后依次加入终止液DNS(4.2.2.3)，加入时间间隔和加入酶液的时间间隔一致，混合后，与沸水中煮沸5min，对照组为先加入终止液后再加入酶液溶液。冷却后在540nm波长下测定吸光值。实验为一个对照组，三个平行试验。
4.4、木聚糖酶pH适性和温度适性的测定
4.4.1、木聚糖酶pH最适性测定
将缓冲液(4.2.2.1)调成不同pH：2到12。柠檬酸-磷酸氢二钠缓冲液(pH2.0-8.0)Tris-Hcl(8.0-9.0)甘氨酸-NaOH缓冲液(pH9.0-12.0)不同pH的缓冲液溶解底物成不同pH，将酶液稀释到合适的倍数，依照4.3的试验方法在37℃测出最适pH，之后在最大值两侧补半点继续检测最适pH值(例如最适pH为5，则再取pH4、pH4.5、pH5、pH5.5和pH6按照4.3的方法检测)。
4.4.2、木聚糖酶温度最适性的测定
依照4.3的方法测定，在上述最适pH的条件下，将反应物放在不同温度下 反应：10℃、20℃、30℃、40℃、50℃、60℃、70℃、80℃，测出最适温度后，在最大值两侧补半点(例如最适温度是40℃，则补充30℃、35℃、40℃、45℃、50℃按照4.3的方法检测)。
4.4.3、木聚糖酶pH耐受测定
将缓冲液(4.2.2.1)调节到不同pH：2-12，用这些不同pH的缓冲液稀释酶液，从放入酶液之时开始计时，稀释好的酶液放入37℃水浴锅中耐受1小时放在冰上，之后立马按照4.3的方法在最适pH和最适温度下进行反应。对照组的酶液是未耐受过的酶液。
4.4.4、木聚糖酶温度耐受测定
将酶液稀释到相应倍数，然后放入不同温度：70℃、80℃、95℃耐受1min、3min、5min、10min、15min、20min、30min、60min、90min、120min、150min。之后按照4.3的方法在最适pH和最适温度下反应。对照实验组酶液是未温度耐受过的酶液。
5、结果与分析
5.1、真菌类新丽鞭毛菌木聚糖酶基因突变，按上实验方法进行突变后送华大基因公司测序，结果如SEQ ID NO：2，对应木聚糖酶氨基酸序列如SEQ ID NO：1，转化的酵母菌株具有木聚糖酶活性，选取一株发酵酶活性单位高的菌株进行发酵获得酶液进行酶学性质测定。
5.2、木聚糖酶最适pH测定
木聚糖酶酶促反应最适pH值结果如图2所示。突变和真菌类新丽鞭毛菌木聚糖酶最适pH为6.0；突变前后二者差异不明显。
5.3、木聚糖酶最适温度测定
木聚糖酶酶促反应最适温度值如图3所示，突变和真菌类新丽鞭毛菌木聚糖酶最适温度分别为55℃和65℃；突变前后二者差异较明显。突变后的木聚糖酶的最适温度较突变前降低10℃，向畜禽肠道温度更靠近，其在37-40℃保留酶活力更高。
5.4、木聚糖酶酶促反应中pH耐受
由图4可知，两种木聚糖酶的pH耐受曲线趋势是相同的，在pH3～pH11之间37℃耐受1小时相对酶活并没有太大变化，基本维持在50％以上，具有较广的耐 受范围。
5.5、木聚糖酶温度耐受
高温下木聚糖酶的温度耐受情况如图5-图7所示，随着温度的升高，相对酶活不断降低，随着时间的增加，相对酶活也逐渐降低。无论在任何温度和时间下，突变后的相对酶活都略高于突变前的，在70℃耐受2小时突变木聚糖酶相对酶活大约还有59％，而突变之前真菌类新丽鞭毛菌木聚糖酶仅仅剩余51％，突变木聚糖酶耐受3小时才降到半衰期以下。在80℃时突变后木聚糖酶耐受2小时相对酶活剩余51％，而突变前耐受90min就达到半衰期了。高温95℃时耐受60min突变后木聚糖酶相对酶活还剩余接近55％，突变前仅仅剩余48％，总之突变后与突变前相比温度耐受相对提高了10％左右；
本技术领域技术人员可以理解，除非另外定义，这里使用的所有术语(包括技术术语和科学术语)具有与本发明所属领域中的普通技术人员的一般理解相同的意义。还应该理解的是，诸如通用字典中定义的那些术语应该被理解为具有与现有技术的上下文中的意义一致的意义，并且除非像这里一样定义，不会用理想化或过于正式的含义来解释。
最后所应说明的是：以上实施例仅用以说明而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明，本领域的普通技术人员应该理解：依然可以对本发明进行修改或者等同替换，而不脱离本发明的精神和范围的任何修改或局部替换，其均应涵盖在本发明的权利要求范围当中。

Claims (8)

  1. 一种木聚糖酶突变体，其特征在于，所述木聚糖酶突变体的氨基酸序列如SEQ ID No：1所示。
  2. 根据权利要求1所述木聚糖酶突变体，其特征在于，所述木聚糖酶突变体由编码真菌类新丽鞭毛菌木聚糖酶氨基酸序列SEQ ID No：3中的氨基酸发生如下改变N207S，G208S，A210S而获得。
  3. 根据权利要求1所述木聚糖酶突变体，其特征在于，所述木聚糖酶突变体的酶促反应最适pH值为6.0；最适温度为55℃；在pH3-pH11、37℃条件下，pH耐受1小时，残活还在50％，在pH4-pH9、37℃条件下，pH耐受1小时，残活还在90％以上，木聚糖酶在70℃耐受120min、80℃时耐受60min、高温90℃时耐受30min，残活都在60％。
  4. 一种含有权利要求1-3任一项所述木聚糖酶突变体的构建方法，其特征在于，步骤包括：
    真菌类新丽鞭毛菌木聚糖酶基因连接到ppic9k载体上的重组质粒为模板，分别设计下列引物进行突变PCR扩增；PCR体系按照试剂盒说明书配制50μL；
    取10μl PCR产物，1％琼脂糖凝胶电泳检测，条带正确后加1μl DMT酶于PCR产物中，混匀，37℃孵育1h；
    然后转化，加入5μl突变后的产物于50μl DMT感受态细胞中，轻弹混匀，冰浴30min；42℃准确热激45s，立即置于冰上10min；加500μlLB培养基，200转，37℃培养1h；7000rpm离心3min，弃掉上清，保留100-150μl，轻弹悬浮菌体，取全部菌液涂板，培养过夜；
    验证阳性克隆子，每个位点的阳性克隆送出测序，测序结果与原序列比对，找出突变正确的重组质粒，再以突变一次质粒作为模板，进行第二位点突变至止突变位点全部突变掉，突变后的质粒转入毕赤酵母GS115或X33、SMD1168、PICHIAPINK中进行表达，发酵测酶活，研究酶学及应用特性。
  5. 一种编码权利要求1所述木聚糖酶突变体的基因，其特征在于，所述基因核苷酸序列如SEQ ID No：2所示。
  6. 一种含有权利要求5所述木聚糖酶突变体的基因的工程菌。
  7. 根据权利要求6所述所述木聚糖酶突变体的基因的工程菌的构建方法，其特征在于，所述木聚糖酶基因突变体与PPIC9、PPICZaA\B\C、PPICZA\B\C、PGAPZaA\B\C、PICHIAPINK-Hc、PICHIAPINK-Lc表达载体构建重组质粒，转化相应宿主菌GS115或X33、SMD1168、PICHIAPINK中，通过在平板上加入植酸钙或在平板中加入G418、Zeocin抗生素，筛选获得木聚糖酶突变体基因工程菌，然后通过发酵获得新的木聚糖酶突变体。
  8. 一种如权利要求1所述木聚糖酶突变体在饲料添加剂中的应用。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109750015A (zh) * 2019-03-27 2019-05-14 云南师范大学 一种热稳性提高的木聚糖酶突变体及其应用
CN109750016A (zh) * 2019-03-27 2019-05-14 云南师范大学 热稳性提高的木聚糖酶突变体及其制备方法和应用
CN115851671A (zh) * 2022-11-30 2023-03-28 山东龙昌动物保健品有限公司 木聚糖酶突变体xynH及其与胆汁酸复合的酶制剂和应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106282141B (zh) * 2016-09-26 2018-11-13 云南师范大学 一种木聚糖酶突变体及其应用
CN107142253B (zh) * 2017-03-23 2020-01-21 中国农业科学院饲料研究所 一种木聚糖酶突变体及其制备方法和应用
CN109161538B (zh) * 2018-09-29 2021-10-15 云南师范大学 一种热稳性提高的脂肪酶突变体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101392266A (zh) * 2008-09-18 2009-03-25 复旦大学 一种耐高温耐强碱的木聚糖酶改良基因及其基因工程菌株及其制备方法
CN102757947A (zh) * 2011-04-25 2012-10-31 武汉新华扬生物股份有限公司 一种热稳定性改良的木聚糖酶xyn-CDBFV-m及其基因和应用
CN104109660A (zh) * 2013-04-17 2014-10-22 东莞泛亚太生物科技有限公司 酶活性提高的木聚糖酶
CN106282141A (zh) * 2016-09-26 2017-01-04 云南师范大学 一种木聚糖酶突变体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101392266A (zh) * 2008-09-18 2009-03-25 复旦大学 一种耐高温耐强碱的木聚糖酶改良基因及其基因工程菌株及其制备方法
CN102757947A (zh) * 2011-04-25 2012-10-31 武汉新华扬生物股份有限公司 一种热稳定性改良的木聚糖酶xyn-CDBFV-m及其基因和应用
CN104109660A (zh) * 2013-04-17 2014-10-22 东莞泛亚太生物科技有限公司 酶活性提高的木聚糖酶
CN106282141A (zh) * 2016-09-26 2017-01-04 云南师范大学 一种木聚糖酶突变体及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 12 March 2015 (2015-03-12), "PREDOCTED: Homo sapiens receptor-interacting serine-threonine kinase 2 (RIPK2), transcript variant X2, mRNA", XP055603193, retrieved from NCBI Database accession no. XM_011517357. 1 *
DATABASE Nucleotide 12 March 2015 (2015-03-12), "PREDOCTED: Homo sapiens receptor-interacting serine-threonine kinase 2 (RIPK2), transcript variant XI, mRNA", XP055603194, retrieved from NCBI Database accession no. XM_005251092. 2 *
HAN, N.Y. ET AL.: "Improving the thermostability of a fungal GH11 xylanase via site-directed mutagenesis guided by sequence and structural analysis", BIOTECHNOL BIOFUELS., vol. 10, no. 133, 23 May 2017 (2017-05-23), pages 1 - 12, XP055603187, DOI: 10.1186/s13068-017-0824-y *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109750015A (zh) * 2019-03-27 2019-05-14 云南师范大学 一种热稳性提高的木聚糖酶突变体及其应用
CN109750016A (zh) * 2019-03-27 2019-05-14 云南师范大学 热稳性提高的木聚糖酶突变体及其制备方法和应用
CN109750016B (zh) * 2019-03-27 2023-04-28 云南师范大学 热稳性提高的木聚糖酶突变体及其制备方法和应用
CN109750015B (zh) * 2019-03-27 2023-05-23 云南师范大学 一种热稳性提高的木聚糖酶突变体及其应用
CN115851671A (zh) * 2022-11-30 2023-03-28 山东龙昌动物保健品有限公司 木聚糖酶突变体xynH及其与胆汁酸复合的酶制剂和应用

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