WO2014107953A1 - 来源于瘤胃木聚糖酶XynA的C端结构域及提高木聚糖酶催化率的方法 - Google Patents

来源于瘤胃木聚糖酶XynA的C端结构域及提高木聚糖酶催化率的方法 Download PDF

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WO2014107953A1
WO2014107953A1 PCT/CN2013/080844 CN2013080844W WO2014107953A1 WO 2014107953 A1 WO2014107953 A1 WO 2014107953A1 CN 2013080844 W CN2013080844 W CN 2013080844W WO 2014107953 A1 WO2014107953 A1 WO 2014107953A1
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xylanase
xyna
terminal domain
seq
rumen
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PCT/CN2013/080844
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French (fr)
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姚斌
赵珩
李中媛
杨培龙
罗会颖
黄火清
石鹏君
王亚茹
孟昆
柏映国
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中国农业科学院饲料研究所
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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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of genetic engineering, and in particular, the present invention relates to a C-terminal domain derived from the rumen xylanase XynA and a method for increasing the catalytic rate of a xylanase. Background technique
  • xylan is the main component of many biomaterials hemicellulose, and it is the most abundant one in hemicellulose. Its main chain is composed of xylopyrose linked by ⁇ -1,4 glycosidic bonds. Degrees 150-200, with arabinose, glucuronic acid, diethyl ether, coumaric acid, cinnamic acid, etc. in the side chain (Collins et al. FEMS Microbiology Reviews. 2005, 29: 3-23.). The complete degradation of xylan requires the synergy of multiple enzymes, but the role of xylanase is the most important and important in the degradation of xylan.
  • the main function of the xylanase is to randomly cleave the ⁇ -1,4-linkage between the glucopyranose residues from the interior of the xylan backbone to form oligo-xylose of different lengths.
  • Xylanases are widely available and are found in bacteria, marine algae, protozoa, fungi, terrestrial plant tissues, and snails, crustaceans, and the like. The research on xylanase has been carried out for half a century. The main research focuses on xylanase suitable for feed industry, food industry, pulp and paper industry, energy industry, etc., which has been separated from microorganisms from different sources. Different types of xylanases with different functions, and various xylanase genes have been isolated, and various xylanase products have been industrially produced.
  • a xylanase XynA having a C-terminal domain
  • the excess sequence at the C-terminus has an effect of improving the degradation ability of the substrate, and provides an idea for improving the catalytic efficiency of the xylanase.
  • Another object of the present invention is to provide a nucleic acid sequence which increases the catalytic rate of a tenth family xylanase.
  • Another object of the invention is to provide a xylanase having a c-terminal domain.
  • Another object of the present invention is to provide a recombinant strain comprising the above gene.
  • Another object of the present invention is to provide the use of the above xylanase having a C-terminal domain.
  • the present invention isolates a novel xylanase having a c-terminal domain from the rumen of sheep, and its selection
  • the enzyme gene encodes 407 amino acids and a stop codon, and the first 24 amino acids are signal peptides. Therefore, the theoretical molecular weight of the mature xylanase XynA is 44 kDa.
  • the xylanase XynA screened by the present invention has an optimum pH of 6.0 and an optimum temperature of 50 °C.
  • the present invention provides a xylanase gene encoding the above-described C-terminal domain, specifically the gene:
  • a xylanase gene encoding the above C-terminal domain is selected, the gene being selected from the group consisting of:
  • the present invention provides a C-terminal domain capable of increasing the catalytic rate of a tenth family xylanase, consisting of 60 amino acids, the amino acid sequence of which is shown in SEQ ID N0.3.
  • the C-terminal domain of the xylanase XynA of the present invention can increase the substrate degradation energy of xylanase Force.
  • the present invention provides a nucleotide sequence encoding the c-terminal domain of the above xylanase. Specifically, the sequence of the gene is as shown in SEQ ID N0.4:
  • the invention provides a method of increasing the catalytic rate of a tenth family xylanase comprising the step of fusing said C-terminal domain to a xylanase.
  • the above xylanase XynA is removed from the C-terminal domain, and the results show that XynA produces more monosaccharide than XynA-Tr (polypeptide of the amino acid sequence shown in SEQ ID NO: 0.5). , with better degradation ability.
  • the above C-terminal domain of the invention is used to increase the viability of a tenth family xylanase.
  • the c-terminal domain described above is fused to other xylanases and the kinetic parameters and product analysis of the fusion protein are determined. It was found that the c-terminal domain can also improve the degradation ability of the substrate, thereby improving the degradation efficiency of the xylanase, thereby verifying that the C-terminal domain has the universal significance of improving the catalytic efficiency, and further research lays a foundation.
  • the present invention also provides an expression vector comprising the above C-terminal domain and a xylanase fusion gene.
  • the xylanase o, z is isolated and cloned by PCR, and the full sequence analysis of the DNA indicates that the gene is 1224 bp in length.
  • the amino acid sequence and the deduced amino acid sequence were determined by BLAST alignment in GenBank, which is a new xylanase.
  • the present invention also provides a recombinant vector comprising the above xylanase, designated pET22b(+)-;
  • the xylanase gene of the present invention is inserted between appropriate restriction sites of the expression vector such that the nucleotide sequence is operably linked to the expression control sequence.
  • the sequence is located downstream of and regulated by the T7 promoter, resulting in a recombinant E. coli expression plasmid pET22b(+)-;t ⁇ 3 ⁇ 44.
  • the present invention also provides a recombinant strain comprising the above C-terminal domain and a xylanase gene fusion gene
  • the strain is Escherichia coli, yeast, Bacillus or Lactobacillus, preferably Escherichia coli BL21 (DE3).
  • the first technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and to provide an efficient and applicable new xylanase.
  • the xylanase of the present invention has an optimum pH of 6.0 and an optimum temperature of 50 °C.
  • the present inventors have discovered that its C-terminal domain can increase the ability to degrade substrates and produce more monosaccharides to increase the catalytic efficiency of xylanase. By fusion expression, it was found that this ability to improve degradation is equally applicable to other xylanases.
  • the substitution, deletion and/or insertion of an amino acid residue is such that after substitution, deletion and/or insertion of one or more amino acids, the enzyme activity is not changed.
  • a common strategy is to preserve amino acid substitutions by replacing amino acid residues with amino acid residues having similar side chains.
  • a family of amino acid residues having similar side chains is well defined in the art. Thus, replacement of one or more sites with another amino acid residue from the same side chain species in a xylanase protein will not substantially affect its enzymatic activity.
  • hybridization under stringent conditions is used to describe hybridization and washing conditions in which nucleotide sequences which are typically at least 60% homologous to each other can still hybridize to each other.
  • stringent conditions are those under which conditions having at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homology to each other will generally still hybridize to each other. .
  • Such stringent conditions are well known to those of ordinary skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions is: hybridization at about 45 °C in 6xSSC followed by one or more washes in 0.2xSSC, 0.1% SDS at 50-65 °C.
  • highly stringent conditions can be achieved by increasing the hybridization temperature, for example to 50 ° C, 55 ° C, 60 ° C or 65 ° C.
  • the present invention relates to a recombinant vector comprising a C-terminal domain and a fusion gene of a xylanase gene, a recombinant host cell into which the vector or the gene has been introduced (for example, Pichia pastoris) And methods of expressing the enzyme in a host cell.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked, for example, it may be a plasmid or a viral vector.
  • the expression vector of the present invention can be introduced into a host cell to produce a protein or peptide including the fusion protein.
  • the recombinant expression vector of the present invention can be designed to express xylanase eggs in prokaryotic or eukaryotic cells White.
  • the xylanase gene can be in bacterial cells such as E. coli, yeast (such as Pichia, Aspergillus niger), insect cells (such as S ⁇ cells or silkworm cells using a baculovirus expression vector) or plant cells (eg Expression in Pseudomonas mediated by Arabidopsis thaliana, Tobacco, Maize, etc.
  • the present invention relates to a host cell, preferably Pichia pastoris, into which a recombinant expression vector of the present invention has been introduced.
  • the host cell can be any prokaryotic or eukaryotic cell including, but not limited to, those host cells described above, preferably Pichia pastoris cells.
  • the present invention also relates to a fusion xylanase comprising said c-terminal domain, said fusion xylanase and a host cell producing said fusion xylanase as a feed additive.
  • the feed additive may be prepared as a dry powder or liquid formulation, and may additionally include one or more enzyme preparations.
  • the additional enzyme preparation may also be in the form of a dry or liquid preparation.
  • the feed additive of the present invention may additionally include other non-pathogenic beneficial microorganisms.
  • Figure 1 Expression and purification of recombinant xylan, wherein, 1 XynA-Tr 2 XynA 3 XynB 4 Figure 2 Optimal pH of recombinant xylan XynA and XynA-Tr.
  • E. coli expression vector pET22b (+) and strain BL21 (DE3) were preserved in the laboratory.
  • Enzymes and other biochemical reagents Endonuclease was purchased from TaKaRa, and ligase was purchased from Invitragen. Purchased from Sigma, all other domestically produced reagents (both available from common biochemical reagents).
  • E. coli medium LB 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.
  • the molecular biology experimental methods not specified in the following examples refer to the "molecules”. Guide to Cloning Experiments (Third Edition) The specific method listed in J. Sambrook's book, or in accordance with the kit and product instructions.
  • Example 1 Cloning of rumen-derived xylanase XynA gene from sheep
  • the macrogenome of the rumen environment of sheep was extracted by liquid nitrogen milling combined with SDS-CTAB chemical lysis.
  • the degenerate primers GH10-F and GH10-R were designed according to the conserved region sequence of the xylanase gene: X10-F: 5 '-CTACGACTGGGAYGTNIBSAAYGA-3 'He ⁇ X10-R : 5 ' - GTGACTCTGGAWRCCIABNCCRT-3 '.
  • PCR amplification was performed using rumen total DNA as a template. A fragment of about 260 bp was obtained, which was recovered and ligated to the pEASY-T3 vector for sequencing.
  • the design direction is the direction of the unknown region to be amplified
  • the position of sp2 is designed on the inner side of spl
  • sp3 is located on the inner side of sp2.
  • the distance between each two primers is not strictly defined.
  • the primer length is generally 22 to 30 nt
  • the annealing temperature is 60 to 65 °C.
  • TAIL-PCR-specific primers for xylanase XynA Primer name Primer sequence (5'--3') Primer length (bp) us l GGCACACCGTTCTCCTTCATGCGC 24 usp2 CTTCACCATCTCGTAGATGCGCTGGCTC 28 usp3 GGCATACTGGAAAGCCTTGGCGATGAAC 28 ds l ATGACCGATGACCCGAAGGCCGAG 23 dsp2 CCGAGGATCCCTTCCGCCAGTCG 23 Dsp3 CCGACCCCAACGCCCTCTTGTTC 23 The flanking sequence of the known gene sequence was obtained by TAIL-PCR, and the amplified product was recovered and sent to the Bommed company for sequencing.
  • the cyz gene After splicing, the cyz gene is 1224 bp in length, encoding 407 amino acids and a stop codon. The predicted molecular weight of the mature protein encoded by this gene is predicted to be 44 kDao.
  • the sequence and the deduced amino acid sequence are BLAST aligned in GenBank, and XynA has the highest agreement with the putative xylanase derived from Bacteroides intestinalis DSM 17393 ( 61%), indicating that XynA is a new xylanase.
  • the C-terminus of the xylanase After aligning and structural analysis, the C-terminus of the xylanase has one The 60 amino acid domain is rich in proline (21.2%).
  • Example 2 Preparation of recombinant xylanase
  • the expression vector pET22 ( + ) was double digested (EcoRI+X ⁇ I), and the gene encoding xylanase was simultaneously ⁇ , double-digested (EcoRI+X ⁇ I), and the mature xylanase was excised.
  • the gene fragment was ligated to the expression vector pET22(+) to obtain a recombinant plasmid pET22(+)-;o3 ⁇ 44 containing the xylanase gene and transformed into BL21(DE3).
  • the gene after XynA truncation of the C-terminal 60 amino acid sequence was named XynA-Tr, and the expression vector pET22 ( + ) and the gene encoding xylanase; ⁇ , / -7> were double-digested (EcoRI+X/).
  • ⁇ I the gene fragment encoding the mature xylanase was excised and ligated with the expression vector PET22 ( + ) to obtain a recombinant plasmid containing the xylanase gene; 0% 4-7> ⁇ 22(+)-; ⁇ %4 -7> and convert BL21 (DE3).
  • the BL21CDE3) strain containing the plasmid was inoculated into 300 mL of LB medium, and cultured at 37 ° C for 220 h with shaking for about 2 h. Then, 1 mM IPTG was added and induced at 220 ° C for 30 h, and the cells were measured after about 8 h. Internal and extracellular xylanase activity. The activity of xylanase was detected extracellularly and purified by nickel column. The results of SDS-PAGE showed that the recombinant xylanase was expressed and purified by electrophoresis (Fig. 1).
  • DNS method The specific method is as follows: Under the conditions of pH 6.0 and 37, 1 mL of the reaction system includes 100 ⁇ appropriate diluted enzyme solution, 900 ⁇ substrate, reaction for 10 min, and 1.5 mL of DNS is added to terminate the reaction, boiling water. Cook for 5 min. The OD value was measured at 540 nm after cooling. One enzyme unit (U) is defined as the amount of enzyme that releases 1 ⁇ reducing sugar per minute under the given conditions.
  • U One enzyme unit (U) is defined as the amount of enzyme that releases 1 ⁇ reducing sugar per minute under the given conditions.
  • the method for determining the optimum pH and pH stability of recombinant xylanase is as follows: The recombinant xylanase purified in Example 2 was subjected to an enzymatic reaction at different pH to determine its optimum pH. The xylanase activity of the substrate xylan was measured at 37 ° C in O. lmol / L citrate - disodium hydrogen phosphate buffer at different pH. The results showed that the optimum pH of the recombinant enzymes XynA and XynA-Tr were 6.0, and the relative enzyme activity of XynA was higher than that of XynA-Tr in the range of pH 5.0 ⁇ 7.5 (Fig. 2).
  • the xylanase was treated at 37 ° C for 60 min in the various pH buffers described above, and the enzyme activity was measured at 37 ° C in a pH 6.0 buffer system to study the pH tolerance of the enzyme.
  • the results (Fig. 3) showed that the xylanases XynA and XynA-Tr were stable at pH 5.0-9.0, and the residual enzyme activity was above 70% after 60 min treatment in this pH range, indicating that the enzyme is neutral. It has good pH stability in the range of partial alkali.
  • the optimum temperature for the xylanase is determined by enzymatic reaction in a citrate-dibasic phosphate disodium buffer (pH 6.0) buffered liquid system and at different temperatures.
  • the temperature resistance was determined by measuring the enzyme activity of the xylanase at different temperatures for different times and then at 50, 55, 60 °C.
  • the optimum temperature measurement result of the enzyme reaction indicates that the optimum temperature of XynA is 50 °C, and XynA-Tr is 45 °C.
  • the thermostability test of the enzyme showed (Fig. 5) that XynA and XynA-T have good thermal stability and can maintain more than 80% of enzyme activity when incubated at 55 °C for 1 h.
  • the enzyme activity was determined under the optimum stability in the buffer system of citric acid-dibasic sodium phosphate buffer (pH 6.0), and the optimal reaction conditions were calculated. U straight. It was determined that the values of XynA and XynA-Tr were 11 and 19 mg/mL, respectively, and the maximum reaction rates V max were 2000 and 222 ⁇ 1/ ⁇ 3 ⁇ 4, respectively.
  • the sequence of the xylanase XynB is shown in SEQ ID No. 6, and the sequence of the xylanase XynA4 is as follows.

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Abstract

提供了一种来源于瘤胃木聚糖酶XynA的C端结构域以及提高木聚糖酶催化率的方法。提供一种高效的新的木聚糖酶。本发明的木聚糖酶最适pH为6.0，最适温度50℃。本发明发现了其C端结构域可以提高降解底物的能力，产生更多的单糖，来提高木聚糖酶的催化效率。通过融合表达，发现这种提高降解能力的作用同样适用于其它的木聚糖酶。

Description

来源于瘤胃木聚糖酶 XynA的 C端结构域及提高木聚糖酶催化率的方法 技术领域
本发明涉及基因工程领域， 具体地， 本发明涉及来源于瘤胃木聚糖酶 XynA的 C端结构域及提高第木聚糖酶催化率的方法。 背景技术
自然界中， 木聚糖是许多生物材料半纤维素的主要组成成分， 是半纤维素 中最丰富的一种， 其主链由吡喃木糖以 β-1，4糖苷键连接而成， 聚合度 150- 200， 侧链上具有阿拉伯糖、 葡萄糖醛酸、 乙醚、 香豆酸、 肉桂酸等 (Collins et al. FEMS Microbiology Reviews. 2005, 29:3-23.)。 木聚糖的完全降解需要多种酶 的协同作用， 但是在木聚糖的降解过程中， 木聚糖酶发挥的作用是最主要和最 重要的。 木聚糖酶的主要作用是从木聚糖的主链内部随机切割吡喃木糖残基之 间的 β -1，4-连接键， 而生成不同长度的寡聚木糖。 木聚糖酶来源比较广泛， 在 细菌、 海洋藻类、 原生动物、 真菌、 陆地植物组织， 以及蜗牛、 甲壳动物等中 都存在。 对木聚糖酶的研究已有半个世纪， 主要研究集中在适合于饲料工业、 食品工业、 制浆造纸工业、 能源工业等方面的木聚糖酶， 已经从不同来源的微 生物中分离到大量的不同类型不同功能的木聚糖酶， 并分离出多种木聚糖酶基 因， 已工业化生产多种木聚糖酶产品。
高效木聚糖酶的产生、 纯化、 性质、 酸性特征的结构基础及其在饲料加 工、 酿酒工业、 果汁加工、 以及能源等领域的应用正在不断深入。 获得具有高 效降解能力的木聚糖酶将会大大减少产业成本， 提高生产效率， 仍具有重大意 义。
本发明中， 我们发现了一种具有 C端结构域的木聚糖酶 XynA, 其 C端多余 的序列具有提高底物降解能力的作用， 为提高木聚糖酶的催化效率提供了一种 思路。 发明内容 本发明的目的是提供能提高第十家族木聚糖酶催化率的特殊 C端结构域。 本发明的另一目的是提供能提高第十家族木聚糖酶催化率的核酸序列。 本发明的另一目的是提供具有 c端结构域的木聚糖酶。
本发明的另一目的是提供编码上述具有 c端结构域的木聚糖酶的基因。 本发明的另一目的是提供包含上述基因的重组载体。
本发明的另一目的是提供包含上述基因的重组菌株。
本发明的另一目的是提供 c端结构域提高第十家族木聚糖酶催化率的用 本发明的另一目的是提供提一种高木聚糖酶的催化效率的方法。
本发明的另一目的提供上述具有 C端结构域的木聚糖酶的应用。
本发明从绵羊瘤胃中分离得到一种新的具有 c端结构域的木聚糖酶， 其选
(a)包含 SEQ ID N0.1所示氨基酸序列的多肽； 或者
(b) SEQ ID N0.1所示氨基酸序列经过取代、 缺失或添加一个或几个氨基酸 且具有木聚糖酶活性的由 (a)衍生的多肽。
其中， 该酶基因编码 407个氨基酸和一个终止密码子， 前 24个氨基酸是信 号肽， 因此， 成熟的木聚糖酶 XynA的理论分子量为 44 kDa。
本发明筛选到的木聚糖酶 XynA， 其最适 pH值为 6.0， 最适温度为 50°C。 本发明提供了编码上述具有 C端结构域的木聚糖酶基因， 具体地该基因：
(a)编码 SEQ ID N0.1所示氨基酸序列的多肽；
(b)编码 SEQ ID N0.1所示氨基酸序列经过取代、 缺失或添加一个或几个氨 基酸且具有木聚糖酶活性的由 (a)衍生的多肽；
优选地， 根据本发明的编码上述具有 C端结构域的木聚糖酶基因， 所述基 因选自：
(a)包含 SEQ ID N0.2所示核酸分子的 DNA; 或
(b)在严谨条件下与 SEQ ID N0.2所示 DNA序列杂交、 且编码且具有木聚 糖酶活性的多肽的 DNA分子。
本发明提供了一种能提高第十家族木聚糖酶催化率的 C端结构域， 由 60个 氨基酸组成， 其氨基酸序列如 SEQ ID N0.3所示。
SEQ ID NO. 3：
PSELNQPG
本发明的木聚糖酶 XynA的 C端结构域可以提高木聚糖酶的底物降解能 力。
本发明提供了编码上述木聚糖酶的 c端结构域的核苷酸序列。 具体地， 该 基因的序列如 SEQ ID N0.4所示：
SE ID N0.4
Figure imgf000005_0001
TCGTGCCCTCTGAGCTCAACCAGCCTGGA
根据本发明的具体实施方式， 本发明提供了提高第十家族木聚糖酶催化率 的方法， 包括将上述 C端结构域与木聚糖酶融合的步骤。 例如， 根据本发明的 具体实施例， 将上述木聚糖酶 XynA去除 C端结构域， 结果表明， XynA比 XynA-Tr ( SEQ ID N0.5所示氨基酸序列的多肽） 产生更多的单糖， 具有更好的 降解能力。 优选地， 本发明的上述 C端结构域用于提高第十家族木聚糖酶的活 力。
根据本发明的具体实施方式， 将上述 c端结构域与其它木聚糖酶融合， 并 且测定融合蛋白的动力学参数和产物分析。 结果发现， 该 c端结构域同样可以 提高底物降解能力， 从而提高木聚糖酶的降解效率， 从而验证该 C端结构域具 有提高催化效率的普遍性意义， 进一步的研究打下一定的基础。 本发明还提供了包含上述 C端结构域与木聚糖酶融合基因的表达载体。 例 如， 根据本发明的具体实施方式， 通过 PCR的方法分离克隆了木聚糖酶 o，z ， DNA全序列分析结果表明， 该基因全长 1224 bp。 将 的氨基酸序列及推导 出的氨基酸序列在 GenBank中进行 BLAST比对后确定 ， 是一种新的木聚糖 酶。
本发明还提供了包含上述木聚糖酶 的重组载体， 命名为 pET22b(+)- ;^ζ 。 将本发明的木聚糖酶基因插入到表达载体合适的限制性酶切位点之间， 使其核苷酸序列可操作的与表达调控序列相连接。 作为本发明的一个最优选的 实施方案， 优选为将本发明的木聚糖酶基因插入到质粒 pET22b(+)上的 EcoR I 和 Xho I限制性酶切位点之间， 使该核苷酸序列位于 T7启动子的下游并受其调 控， 得到重组大肠杆菌表达质粒 pET22b(+)-;t}¾4。
本发明还提供了包含上述 C端结构域与木聚糖酶基因融合基因的重组菌 株， 优选所述菌株为大肠杆菌、 酵母菌、 芽孢杆菌或乳酸杆菌， 优选为大肠杆 菌 BL21(DE3)。
本发明首先所要解决的技术问题是题是克服现有技术的不足， 提供一种高 效的可应用新的木聚糖酶。 本发明的木聚糖酶最适 pH为 6.0， 最适温度 50°C。 本发明发现了其 C端结构域可以提高降解底物的能力， 产生更多的单糖， 来提 高木聚糖酶的催化效率。 通过融合表达， 发现这种提高降解能力的作用同样适 用于其它的木聚糖酶。
根据本发明的技术方案， 氨基酸残基的取代、 缺失和 /或插入是经取代、 缺失和 /或插一个或多个氨基酸后， 不改变酶活性。 例如， 一个常见的策略是保 守氨基酸取代， 即将氨基酸残基用具有相似侧链的氨基酸残基替换。 具有相似 侧链的氨基酸残基的家族在本领域已有明确定义。 因此， 在木聚糖酶蛋白中用 来自同一侧链类的另一氨基酸残基替换一个或几个位点， 将不会在实质上影响 其酶活性。
根据本发明的技术方案， 术语"在严谨条件下杂交"是用来描述典型地相互 间至少 60%同源的核苷酸序列仍可相互杂交的杂交和清洗条件。 优选地， 严谨 条件为这样的条件， 在此条件下相互间具有至少约 65%、 更优选地至少约 70%、 且甚至更优选地至少约 75%或更高同源性的序列一般仍可相互杂交。 此 严谨条件为本领域普通技术人员所公知， 可在 Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6中找至 lj。 严谨杂交条件的一个 优选、 非限制性实例为： 在 6xSSC中于约 45 °C杂交， 然后在 0.2xSSC、 0.1% SDS 中于 50-65 °C洗涤一次或多次。 本领域技术人员能够理解， 高度严谨条件可通过 提高杂交温度， 例如至 50°C、 55 °C、 60°C或 65 °C来实现。
又在一个方面， 本发明涉及含有 C端结构域以及木聚糖酶基因的融合基因 的重组载体、 已导入所述载体或所述基因的重组宿主细胞 （例如， 巴斯德毕赤 酵母 Pichia Pastoris) 、 以及在宿主细胞中表达酶的方法。 术语 "载体"指能运 送已与其连接的另一核酸的核酸分子， 例如其可以为质粒或病毒载体。 本领域 普通技术人员将理解表达载体的设计可取决于如欲转化的宿主细胞的选择、 所 需的蛋白质表达水平等因素。 可以将本发明的表达载体导入宿主细胞， 以产生 包括融合蛋白的蛋白或肽。
本发明的重组表达载体可设计用于在原核或真核细胞中表达木聚糖酶蛋 白。 例如， 木聚糖酶基因可在如大肠杆菌的细菌细胞、 酵母 （如毕赤酵母、 黑 曲霉） 、 昆虫细胞 （如使用杆状病毒表达载体的 S©细胞或家蚕细胞） 或植物 细胞 （如脓杆菌介导的拟南芥、 烟草、 玉米等） 中表达。 从而， 本发明涉及已 导入本发明的重组表达载体的宿主细胞、 优选毕赤酵母。 宿主细胞可为任何原 核或真核细胞， 其包括但不限于上述的那些宿主细胞， 优选毕赤酵母细胞。
此外， 本发明还涉及含所述 c端结构域的融合木聚糖酶， 所述融合木聚糖 酶以及产生上述融合木聚糖酶的宿主细胞可以作为饲料添加剂中。
所述饲料添加剂可制备为干粉或液体制剂， 并且可额外地包括一种或多种 酶制剂。 所述额外的酶制剂也可以为干燥的或液体制剂形式。
除了融合木聚糖酶和 /或产融合木聚糖酶微生物， 本发明的饲料添加剂还可 额外包括其它非致病性的有益微生物。
附图说明
图 1重组木聚糖的表达和纯化图， 其中， 1 XynA-Tr 2 XynA 3 XynB 4 图 2重组木聚糖 XynA和 XynA-Tr的最适 pH。
图 3重组木聚糖 XynA和 XynA-Tr的 pH稳定性。
图 4重组木聚糖 XynA和 XynA-Tr的最适温度。
图 5重组木聚糖 XynA和 XynA-Tr的热稳定性。 具体实施方式
试验材料和试剂
1、 菌株及载体： 大肠杆菌表达载体 pET22b(+)及菌株 BL21(DE3)为实验室 保存。
2、 酶类及其它生化试剂： 内切酶购自 TaKaRa 公司， 连接酶购自 Invitragen公司。 购自 Sigma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)。
3、 培养基：
大肠杆菌培养基 LB (1%蛋白胨、 0.5%酵母提取物、 l% NaCl，pH 7.0)。 说明： 以下实施例中未作具体说明的分子生物学实验方法， 均参照 《分子 克隆实验指南》 （第三版） J.萨姆布鲁克一书中所列的具体方法进行， 或者按 照试剂盒和产品说明书进行。 实施例 1 绵羊瘤胃来源的木聚糖酶 XynA基因的克隆
采用液氮研磨法结合 SDS-CTAB化学裂解法来提取绵羊瘤胃环境的宏基因 组。 根据木聚糖酶基因的保守区序列设计合成了简并引物 GH10-F和 GH10- R ： X10-F： 5 ' -CTACGACTGGGAYGTNIBSAAYGA-3 ' 禾卩 X10-R : 5 ' - GTGACTCTGGAWRCCIABNCCRT-3 ' 。 以瘤胃总 DNA为模板进行 PCR扩 增。 得到一约 260bp片段， 将该片段回收后与 pEASY-T3载体相连测序。
根据测序得到的核苷酸序列， 设计上游和下游各三条 TAIL-PCR特异性引 物： 设计方向为需要扩增的未知区域方向， sp2的位置设计在 spl 的内侧， sp3 位于 sp2的内侧。 每两个引物之间的距离没有严格规定， 引物长度一般 22〜30 nt， 退火温度在 60〜65 °C。 并将它们分别命名为 uspl， usp2, usp3 (；上游特异性引 物)， dspl , dsp2, dsp3 (下游特异性引物)见表 1。
表 1.木聚糖酶 XynA的 TAIL-PCR特异性引物 引物名称 引物序列 (5' ---3' ) 引物长度 (bp) us l GGCACACCGTTCTCCTTCATGCGC 24 usp2 CTTCACCATCTCGTAGATGCGCTGGCTC 28 usp3 GGCATACTGGAAAGCCTTGGCGATGAAC 28 ds l ATGACCGATGACCCGAAGGCCGAG 23 dsp2 CCGAGGATCCCTTCCGCCAGTCG 23 dsp3 CCGACCCCAACGCCCTCTTGTTC 23 通过 TAIL-PCR得到已知基因序列的侧翼序列， 扩增得到产物回收后送博 迈德公司测序。 拼接后 cyz 基因全长 1224 bp, 编码 407个氨基酸和一个终止 密码子。 预测该基因所编码的成熟蛋白的理论分子量为 44 kDao 将 序列 及推导出的氨基酸序列在 GenBank 中进行 BLAST 比对， XynA与来源于 Bacteroides intestinalis DSM 17393的假定木聚糖酶的具有最高一致性 (61% ),说 明 XynA是一种新的木聚糖酶。 经比对和结构分析， 该木聚糖酶的 C端具有一 段 60个氨基酸的结构域， 富含脯氨酸 (21.2%)。 实施例 2重组木聚糖酶的制备
XynA的重组与纯化：
将表达载体 pET22 ( + ) 进行双酶切 (EcoRI+X^I), 同时将编码木聚糖酶的 基因；ο， 双酶切 (EcoRI+X^I), 切出编码成熟木聚糖酶的基因片段与表达载体 pET22 ( + ) 连接， 获得含有木聚糖酶基因; 的重组质粒 pET22(+)-;o¾4并转 化 BL21(DE3)。
取含有质粒的 BL21(DE3)菌株， 接种于 300 mL LB培养液中， 37 °C 220 rpm 振荡培养约 2 h后， 加入 1 mM IPTG禾卩 1 mM CaCl2， 置于 30°C 220 rpmiS行诱 导， 约 8 h后测定胞内和胞外的木聚糖酶活力。 在胞外检测到木聚糖酶的活性 经过镍柱纯化， SDS-PAGE结果表明， 重组木聚糖酶得到了表达并达到电泳纯 (图 1)。
XynA-Tr的重组与纯化：
将 XynA截短 C端 60个氨基酸序列后的基因命名为 XynA-Tr,将表达载体 pET22 ( + ) 和编码木聚糖酶的基因 ；ο，/ -7>进行双酶切 (EcoRI+X/^I) , 切出编 码成熟木聚糖酶的基因片段与表达载体 PET22 ( + ) 连接， 获得含有木聚糖酶基 因；0%4-7>的重组质粒 ρΕΤ22(+)-;ο%4-7>并转化 BL21(DE3)。
取含有质粒的 BL21CDE3)菌株， 接种于 300 mL LB培养液中， 37°C 220 rpm振荡培养约 2 h后， 加入 1 mM IPTG, 置于 30°C 220 rpm进行诱导， 约 8 h 后测定胞内和胞外的木聚糖酶活力。 在胞外检测到木聚糖酶的活性经过镍柱纯 化， SDS-PAGE结果表明， 重组木聚糖酶得到了表达并达到电泳纯（图 1)。 实施例 3重组木聚糖酶的活性分析
DNS法： 具体方法如下： 在 pH6.0和 37下 条件下， 1 mL的反应体系包 括 100 μΐ^适当的稀释酶液， 900 μ∑底物， 反应 10 min, 加入 1.5 mL DNS终止 反应， 沸水煮 5 min。 冷却后 540 nm测定 OD值。 1个酶活单位 (U)定义为在给 定的条件下每分钟释放出 1 μηιοΐ还原糖的酶量。 实施例 4重组木聚糖酶 XynA和 XynA-Tr的性质测定
1、 重组木聚糖酶最适 pH和 pH稳定性的测定方法如下： 将实施例 2纯化的重组木聚糖酶在不同的 pH下进行酶促反应以测定其最 适 pH。 底物木聚糖用不同 pH的 O. lmol/L柠檬酸-磷酸氢二钠缓冲液中 37°C下 进行木聚糖酶活力测定。 结果表明， 重组酶 XynA和 XynA-Tr的最适 pH均为 6.0， 在 pH5.0〜7.5范围内， XynA的相对酶活性高于 XynA-Tr (图 2) 。 木聚糖 酶于上述各种不同 pH的缓冲液中 37°C处理 60min， 再在 pH6.0缓冲液体系中 37°C下测定酶活性， 以研究酶的 pH耐性。 结果 (图 3)表明木聚糖酶 XynA和 XynA-Tr在 pH 5.0-9.0之间均很稳定， 在此 pH范围内处理 60min后剩余酶活性 在 70%以上， 而这说明此酶在中性偏碱的范围内具有较好的 pH稳定性。
2、 木聚糖酶的最适温度及热稳定性测定方法如下：
木聚糖酶的最适温度的测定为在柠檬酸 -磷酸氢二钠缓冲液 (PH6.0)缓冲液体 系及不同温度下进行酶促反应。 耐温性测定为木聚糖酶在不同温度下处理不同 时间， 再在 50， 55， 60°C下进行酶活性测定。 酶反应最适温度测定结果 (图 4)表 明其 XynA的最适温度为 50°C， 而 XynA-Tr为 45 °C。 酶的热稳定性性试验表 明（图 5)， XynA和 XynA-T有良好的热稳定性， 在 55 °C下温育 lh， 能保持 80%以上的酶活。
3、 木聚糖酶的 Km值测定方法如下：
用不同浓度的木聚糖为底物， 在柠檬酸 -磷酸氢二钠缓冲液 (pH6.0)缓冲液体 系中， 各自最适稳定下测定酶活性， 计算出其在最适反应条件下的 U直。 经测 定， XynA和 XynA-Tr以榉木木聚糖为底物时的 ^值分别为 11和 19mg/mL， 最 大反应速度 Vmax分别为 2000和 222 μηιο1/ηώι·η¾。
4、 重组木聚糖酶的产物分析
以 ρΗ6.0的 0.5%榉木木聚糖和小麦阿拉伯木聚糖为底物， 加入 10U的纯化 酶 XynA或 XynA-Tr， 37°C反应 12 h。 用 3 kDa的超滤管除去酶蛋白和未降解 的大分子， 然后将滤液用 HPLC分析其产物， 以葡萄糖、 纤维二糖、 三糖和四 糖作为标准。 结果表明， XynA比 XynA-Tr产生更多的单糖， 具有更好的降解 能力。
5、 融合蛋白的构建及 C端结构域的广泛应用
我们将 C端结构域与另一个木聚糖酶 XynB ( SEQ ID No.6) 进行融合表达
(图 1 ) ， 并且测定融合蛋白的动力学参数和产物分析。 结果发现， 该 C端结 构域同样可以提高底物降解能力 （表 2) ， 从而提高木聚糖酶的降解效率 （表 3 ) ， 从而验证该 C端结构域具有提高催化效率的普遍性意义， 进一步的研究
三单二其四
着梦梦梦唐唐唐唐E - 寡
糖
打下一定的基础。
表 2 XynA和 XynA-Tr及融合表达蛋白的动力学参数
V (μηιοΐ min— mg—
Km (mg mL- !) l) ^cat (S—】) kc Km (mL s- img- !)
XynA 11.0 ± 0.5 2000.0 ± 23.5 1468.7 ± 15.9 133.5
XynA-Tr
(融合蛋
白） 19.0 ± 1.2 222.0 ± 12.9 140.1 ± 8.6 7.3
XynB-Fu
(融合蛋
白） 1.9 ± 0.2 833.3 ± 5.3 621.0 ± 3.5 324.0
XynB 5.4 ± 0.4 102.0 ± 2.8 64.6 ± 3.1 12.0
XynA4-Fu
( 融 合 蛋
白） 1.1 ± 0.03 1590.3 ± 13.2 1298.5 ± 18.2 1159.4
XynA4 1.6 ± 0.02 335.8 ± 5.2 237.9 ± 6.2 152.5
XynAS9-Fu
( 融 合 蛋
白） 1.7 ± 0.03 2089.4 ± 3.8 1810.5 ± 12.2 1090.6
XynAS9 2.4 ± 0.2 490.9 ± 14.2 376.3 ± 18.2 154.9
其中木聚糖酶 XynB的序列如 SEQ ID No.6所示， 木聚糖酶 XynA4的序列如
SEQ ID No.7所示， 木聚糖酶 XynAS9的序列如 SEQ ID No.8所示。 表 3 以榉木木聚糖为底物 XynA和 XynA-Tr及融合表达蛋白的产物分析 水解产物 榉木木聚糖
XynA XynA-Tr XynB-Fu XynB XynA4-Fu XynA4 XynAS9-Fu XynAS9
9.45 24.21 4.00 60.74 51.50 33.44 20.43
55.62 62.40 53.89 38.19 34.30 61.24 60.01
16.81 1.99 23.21 1.07 7.53 5.32 19.56
1.32 6.65
18.12 11.40 17.58
表 4以小麦阿拉伯木聚糖为底物 XynA和 XynA-Tr及融合表达蛋白的产物分析 三单二其四
梦梦梦梦^. -唐唐唐唐E - 寡
糖
水解广物 小麦阿拉伯木聚糖
XynA XynA-Tr XynB-Fu XynB XynA4- XynA4 XynAS9-Fu XynA
Fu S9
25.62 9.67 26.33 19.42 37.07 7.06 22.43 15.32 30.61 16.69 34.25 23.11 58.43 28.56 77.34 78.73
6.62 1.22 11.48 2.53 25.57 0.23 5.95
1.97 38.81
43.77 67.02 38.20 45.99

Claims

权利要求
1、 来源于瘤胃木聚糖酶 XynA的 C端结构域， 其特征在于， 其选自：
(a) SEQ ID N0.3所示氨基酸序列的多肽； 或者
(b) SEQ ID N0.3所示氨基酸序列经过取代、 缺失或添加一个或几个氨基酸 且具有提高木聚糖的催化效率的功能； 或者
(c) 由 SEQ ID N0.4所示核苷酸序列编码的多肽； 或者
(d) 由在严谨条件下与 SEQ ID N0.4所示 DNA序列杂交 DNA分子编码 的多肽， 且所述多肽具有提高木聚糖的催化效率的功能。
2、 根据权利要求 1所述的来源于瘤胃木聚糖酶 XynA的 C端结构域， 其特 征在于， 该多肽具有提高木聚糖的催化效率的特性并且具有与 SEQ ID NO. 3多 肽具有至少 60-90%同一性的氨基酸序列。
3、 一种提高木聚糖酶催化率的方法， 其特征在于， 所述方法包括将源于瘤 胃木聚糖酶 XynA的 C端结构域与木聚糖酶的 C端融合的步骤， 其中， 所述源 于瘤胃木聚糖酶 XynA的 C端结构域选自：
(a) SEQ ID N0.3所示氨基酸序列的多肽； 或者
(b) SEQ ID N0.3所示氨基酸序列经过取代、 缺失或添加一个或几个氨基酸 且具有提高木聚糖的催化效率的功能； 或者
(c) 由 SEQ ID N0.4所示核苷酸序列编码的多肽； 或者
(d) 由在严谨条件下与 SEQ ID N0.4所示 DNA序列杂交 DNA分子编码 的多肽， 且所述多肽具有提高木聚糖的催化效率的功能。
4、 根据权利要求 3所述的提高木聚糖酶催化率的方法， 其特征在于， 与源 于瘤胃木聚糖酶 XynA的 C端结构域融合的木聚糖酶为第十家族的木聚糖酶。
5、 根据权利要求 3所述的提高木聚糖酶催化率的方法， 其特征在于， 与源 于瘤胃木聚糖酶 XynA的 C端结构域融合的木聚糖酶的氨基酸序列如 SEQ ID N0.5所示。
6、 融合木聚糖酶， 其特征在于， 木聚糖酶的 C端连接有来源于瘤胃木聚 糖酶 XynA的 C端结构域， 其选自：
(a) SEQ ID N0.3所示氨基酸序列的多肽； 或者
(b) SEQ ID N0.3所示氨基酸序列经过取代、 缺失或添加一个或几个氨基酸 且具有提高木聚糖的催化效率的功能； 或者
(c) 由 SEQ ID N0.4所示核苷酸序列编码的多肽； 或者 (d) 由在严谨条件下与 SEQ ID N0.4所示 DNA序列杂交 DNA分子编码 的多肽， 且所述多肽具有提高木聚糖的催化效率的功能。
7、 融合木聚糖酶基因， 其特征在于， 所述木聚糖酶基因的 3'端连接有源于 瘤胃木聚糖酶 XynA的 C端结构域的编码序列， 或者在严谨条件下与所述源于 瘤胃木聚糖酶 XynA的 C端结构域的编码序列杂交的核苷酸序列， 所述 C端结 构域选自：
(a) SEQ ID N0.3所示氨基酸序列的多肽； 或者
(b) SEQ ID N0.3所示氨基酸序列经过取代、 缺失或添加一个或几个氨基酸 且具有提高木聚糖的催化效率的功能。
8、 包含源于瘤胃木聚糖酶 XynA的 C端结构域编码序列与木聚糖酶基因的 融合基因的表达载体。
9、 包含源于瘤胃木聚糖酶 XynA的 C端结构域编码序列与木聚糖酶基因的 融合基因的宿主细胞。
10、 根据权利要求 9所述的宿主细胞， 其特征在于， 所述宿主细胞为细菌 或真菌细胞。
11、 权利要求 1所述来源于瘤胃木聚糖酶 XynA的 C端结构域用于提高木 聚糖酶催化率的应用。
12、 权利要求 6所述融合木聚糖酶作为饲料添加剂的应用。
13、 权利要求 9所述宿主细胞用于制备饲料添加剂的应用。
PCT/CN2013/080844 2013-01-09 2013-08-05 来源于瘤胃木聚糖酶XynA的C端结构域及提高木聚糖酶催化率的方法 WO2014107953A1 (zh)

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