WO2014201595A1 - 一种木糖苷酶Xy1_S及其编码基因与应用 - Google Patents

一种木糖苷酶Xy1_S及其编码基因与应用 Download PDF

Info

Publication number
WO2014201595A1
WO2014201595A1 PCT/CN2013/001509 CN2013001509W WO2014201595A1 WO 2014201595 A1 WO2014201595 A1 WO 2014201595A1 CN 2013001509 W CN2013001509 W CN 2013001509W WO 2014201595 A1 WO2014201595 A1 WO 2014201595A1
Authority
WO
WIPO (PCT)
Prior art keywords
xylosidase
xyl
amino acid
sequence
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2013/001509
Other languages
English (en)
French (fr)
Inventor
杨凌
窦同意
栾宏伟
刘兴宝
李世阳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian Institute of Chemical Physics of CAS
Original Assignee
Dalian Institute of Chemical Physics of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dalian Institute of Chemical Physics of CAS filed Critical Dalian Institute of Chemical Physics of CAS
Publication of WO2014201595A1 publication Critical patent/WO2014201595A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • 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
    • 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)
    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)

Definitions

  • the invention belongs to the field of bioengineering, and particularly relates to a xylosidase Xyl-S and a coding gene and application thereof.
  • the ⁇ -xylosidase is an exonuclease which hydrolyzes xylo-oligosaccharides and xylo-oligosaccharides above the xylo-oligosaccharide from the non-reducing end, and the hydrolyzed product is xylose. It is one of the key enzymes for xylan degradation and has important industrial application value. In the energy industry, xylan in industrial and agricultural waste can be converted into xylose by xylanase, and xylose can be converted into valuable fuel such as alcohol by bacteria and fungi; in the pharmaceutical industry, wood The hydrolysis of specific substrates by glycanase produces intermediate conversion products of important utility in the pharmaceutical industry.
  • Patel et al. used Mwrn / ⁇ -xylosidase to hydrolyze the 7-glycan residue of 10-deacetylpaclitaxel xyloside to obtain an important intermediate, 10-deacetylpaclitaxel, which opened up a new way for the synthesis of paclitaxel. (US005700669A; ⁇ 0668360 ⁇ 1).
  • Fibrillar microbacteria are actinomycetes that can efficiently utilize cellulose and hemicellulose, which can produce a variety of hydrolases, such as Shi-Hsiang Shen et al. (The Journal of Biological Chemistry, 1991, 266(2) : 1058-1063 ) ⁇ -1,3-glucosidase was isolated from the supernatant of the fermentation broth and cloned and exogenously expressed; Petra Tiels et al. iNature Biotechnology 2012, 30: 1225-1231 ) The 5 mannosidases produced by this strain were cloned and exogenously expressed. However, no reports of xylosidase have been obtained so far.
  • Corresponding xylosidic bonds and glucosidic bonds can hydrolyze a variety of xylosidic substrates and glucoside substrates, such as xylooligosaccharides, 4-nitrophenyl- ⁇ -D-pyridyl Xyloside (/?NP-P-Xyl), 4-nitrophenyl- ⁇ -D-glucopyranoside (pNP-P_Glu), 7-xyloside taxane, astragaloside, and ginsenoside Rbl , Rb2, Re, etc.
  • xylosidic substrates and glucoside substrates such as xylooligosaccharides, 4-nitrophenyl- ⁇ -D-pyridyl Xyloside (/?NP-P-Xyl), 4-nitrophenyl- ⁇ -D-glucopyranoside (pNP-P_Glu), 7-xyloside taxane, astragaloside, and ginsenoside Rbl
  • the object of the present invention is to provide a xylosidase Xyl-S and a coding gene thereof and application thereof, and the invention produces a large amount of substrate-specific xylosidase Xyl-S by gene expression to hydrolyze xylosidide compounds.
  • Xylose and the corresponding aglycones can also be used to hydrolyze glucoside compounds to form glucose and corresponding aglycones.
  • the present invention provides a xylosidase Xyl_S having an amino acid sequence comprising at least 1642 amino acid sequences of the sequence set forth in SEQ ID NO. 2 of the Sequence Listing; preferably the 24th amino acid residue of the sequence of SEQ ID N0.2 is At least 1128 amino acid sequences including the 1151 amino acid residue, and more preferably at least 1033 amino acid residues including the 87th amino acid residue to the 1119th amino acid residue of the sequence of SEQ ID NO.
  • the present invention provides a gene encoding a xylosidase Xyl-S having a nucleotide sequence of at least 4929 nucleotides of the nucleotide sequence set forth in SEQ ID NO. 1; preferably having SEQ ID NO. l at least 3372 nucleotides from the 72nd nucleotide to the 3453th nucleotide of the nucleotide sequence shown; further preferably having the nucleotide sequence shown in SEQ ID NO. At least 3099 nucleotides from 261 nucleotides to 3357 nucleotides.
  • the present invention provides a recombinant vector containing the coding gene, which is an Escherichia coli expression vector, a Saccharomyces cerevisiae expression vector, a Pichia expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, and a filamentous fungus expression vector. Any of them.
  • the present invention provides a recombinant cell strain comprising the recombinant vector, and the host cell of the recombinant cell strain is any one of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus subtilis, lactic acid bacteria, and filamentous fungi.
  • the recombinant cell line can express the xylosidase Xyl-S, either intracellularly or secreted extracellularly.
  • the gene of the present invention was cloned from the chromosome of Cellulosi microbium cellulans strain F16 (CCTCC M 2013201) by DNA recombination technique and overexpressed in E. coli after ligation to the pCold IV expression vector.
  • the amount of the expressed xylosidase Xyl-S can be secreted both extracellularly and intracellularly. Its molecular weight on SDS-PAGE is about 140KDa, its optimum reaction pH is 7.5, the effective pH range is 5.5 ⁇ 9.5, and it has good catalytic activity at 20 ⁇ 50 °C.
  • any gene can be altered by DNA recombination techniques to produce a variety of different mutants.
  • the proteins expressed by these mutants usually have similar properties. When a gene or protein sequence reaches a certain homology, the properties of the protein they express are more similar as the homology increases.
  • the genes and products thereof of the present invention also have the same characteristics. When the homology reaches 80% or more, the protein expressed by a similar gene will have the property of catalyzing the hydrolysis of the xyloside compound to xylose and aglycon.
  • the beneficial effects of the present invention are: using the gene of the present invention Expression, obtaining xylosidase Xyl-S or host cells containing the enzyme, can effectively catalyze the hydrolysis of xylosidic compounds and glucoside compounds, and can be applied to various industries, for example, for biomass conversion, such as for In the process of producing biomass ethanol from biomass-containing cellulose, it is used in feed compositions to improve the utilization of crude fiber by animals, or in bread making dough, and can also be used for obtaining pharmaceutical intermediates, such as hydrolysis 7 - Xylose-10-deacetylpacepol to obtain 10-deacetylpaclitaxel, while hydrolyzing the ⁇ -xylosidic bond and ⁇ -glucoside bond on the Astragalus IV IV to obtain the corresponding aglycon, hydrolyzing ginsenoside Rbl, Rb2 or Re The xyloside and glucoside on the corresponding ginsenoside
  • the xylosidase Xyl_S or the recombinant cell strain provided by the present invention is applied to the fields of medicine, biology, agriculture, energy and the like, and hydrolyzes the glycosidic bond in the xyloside compound (or glucoside compound) to obtain the corresponding aglycon.
  • Figure 1 Polyacrylamide gel electrophoresis analysis of pure xylosidase Xyl-S, wherein MUX is 4-methylumbelliferyl- ⁇ -D-xylopyranoside, the glycosidic bond is hydrolyzed Fluorescence after excitation at 365 nm excitation, so as to achieve monitoring and zymogram analysis of ⁇ -xylosidase in each component; left panel: 2% - 15% gradient Native PAGE, test stain; middle panel: 2% - 15% Gradient Native PAGE, MUX staining; Right panel: 10% SDS-PAGE, staining; Sample 1 - BSA ferrintin; 2 - fermentation broth supernatant; 3 - multi-enzyme complex; 4 ⁇ xylosidase Xyl_S ;
  • Figure 2 Pure enzyme catalyzed hydrolysis of ⁇ - ⁇ -D-Xyl, substrate concentration corresponding to the corresponding initial reaction rate of the Michaelis curve;
  • Figure 3 Pure enzyme catalyzed hydrolysis of ⁇ - ⁇ -D-Glu, substrate concentration corresponding to the corresponding initial reaction rate of the Michaelis curve;
  • Figure 4 Detection of PCR products by agarose electrophoresis, wherein M is a standard, ⁇ phage Hind lll degradation product; 1 is a cloned ⁇ gene;
  • Figure 5 purified recombinant enzyme Xyl-S
  • Figure 6 Xyl-S pure enzyme converts 99 DAXT results with a purity of 99%, wherein 10 DAXT is 7-xylose-10-deacetylpaclitaxel and 10 DAT is 10-deacetylpaclitaxel;
  • Figure 7 Results of Xyl_S pure enzyme conversion of a 7-xylose taxane mixture, wherein 10 DAXT is 7-xylose-10-deacetylpaclitaxel, 10 DAT is 10-deacetylpaclitaxel, 10 DAXC is 7- Xylose-10-deacetyl cephalosporin, 10 DAC is 10-deacetyl cephalosporin, 10 DAXTC is 7-xylose-10-deacetylpaclitaxel C, 10 DATC is 10-deacetylated Paclitaxel C;
  • Figure 8 Mass spectrum of the hydrolyzate, wherein A is 10-deacetyl cephalosporin and B is 10-deacetylpaclitaxel;
  • Figure 9 Pure enzyme conversion of astragaloside IV to cyclodextrin TLC.
  • the genetic resource on which the present invention is dependent is a fibrosis fiber microbacteria (Ce! luhsi microbiwn cellulans) strain F16, deposited as CCTCC M 2013201, and stored in the China Center for Type Culture Collection.
  • a fibrosis fiber microbacteria (Ce! luhsi microbiwn cellulans) strain F16, deposited as CCTCC M 2013201, and stored in the China Center for Type Culture Collection.
  • ⁇ -xylosidase activity was followed by p-nitrophenyl- ⁇ -D-xyloside ( ⁇ NP-Xyl) as a specific chromogenic substrate.
  • One enzyme unit is defined as the amount of enzyme required to catalyze the production of 1 ⁇ p-nitrophenol in lh at 30 ° C, pH 7.5, and NP-Xyl.
  • the precipitate component in the 20%-40% stage was collected by ammonium sulfate precipitation; the Toyopearl DEAE 650M ion exchange column was used to collect 0.5 ⁇ 0.7 mol/L NaCl stage elution fraction; Seph aC ryl S-200 HR gel filtration layer The column is collected, and the active component having a molecular weight of between 30 kDa and 200 kDa is collected; the active component is separated from the source 15Q column, and the enzyme activity component having the highest specific activity is collected, thereby obtaining pure xylosidase Xyl-S (Fig. 1) ).
  • Reaction system Reaction conditions:
  • the 4DS bp of the target gene CDS was amplified by PCR, and the Nde VBamU I restriction site was added to both ends of the fragment.
  • the 6*His tag was added before the stop codon TGA, and cloned into the pCoWIV expression vector.
  • the positive clone plasmids were verified by sequencing, and the results showed that the sequences were correct. That is, the corresponding recombinant vector is obtained.
  • the PCR reaction system, reaction conditions and primers used are shown in the table below:
  • Example 4 Recombinant expression of the key sequence of xylosidase XyI_S in Escherichia coli.
  • PCR amplification of the 261th nucleotide to the 3357th nucleotide of the CDS region of the target gene was 3099 bp.
  • Nde I BamH I restriction site was added to both sides of the fragment, and the 6*His tag was added before the stop codon TGA, and cloned into pCold lV expression vector.
  • Two positive clone plasmids were selected for sequencing verification, and the results showed that the sequence was correct. That is, the corresponding recombinant vector is obtained.
  • the above recombinant vector was transferred to E. coli BL21 competent cells by using 1 ⁇ , and coated with LB/antibiotic Amp (100 g/ml) plate, 50 ul of transformant, and cultured at 37 °C. Control pCold lV does the same.
  • chromogenic substrates such as xyloside, glucoside, fructoside, mannoside, fucoside, galactoside and cellobioside were prepared, and 50 mM Tris-HCl buffer was used to prepare a solution of 5 mM and pH 7.5.
  • the reaction system of pure enzyme + 150 ⁇ buffer + 10 ⁇ 20 mM CaC12 + 10 ⁇ substrate obtained in Example 1 was reacted in a 96-well plate at 30 ° C, and monitored by BioTek hyrbid Reader at 405 nm. The absorbance value.
  • the following reaction system was prepared: a total volume of 200 ⁇ l per well, wherein the complete sequence recombinase of Example 3 and the key sequence recombinase of Example 4 were both 30 ⁇ , and the amount of substrate and buffer added was The following 9 substrate concentrations were achieved: 5 ⁇ , 10 ⁇ , 20 ⁇ , 50 ⁇ , 100 ⁇ , 200 ⁇ , 500 ⁇ , 1000 ⁇ , 2000 ⁇ .
  • a parallel sample was prepared for each of the above concentrations, both substrates ⁇ - ⁇ -D-Xyl and ?? ⁇ - ⁇ -D-Glu, occupying 72 wells and making a standard curve with ⁇ standard.
  • the experimental results show that the xylosidase Xyl-S has a greater binding ability to ⁇ - ⁇ -D-Xyl than ⁇ - ⁇ -D-Glu (the former has a smaller Km value than the latter), but has the same conversion constant t for both. That is, the ratio of the maximum reaction rate to the enzyme concentration, max / [E] ). That is, xylosidase 1_8 has the same conversion ability to xylosidic compounds and ⁇ -glucoside compounds. At the same time, the enzyme's affinity for the substrate decreased slightly after only the key regions were expressed, but it still had the same activity and could hydrolyze the substrate well (Table 2).
  • the CDS region of the Xyl_S gene obtained in Example 2 was ligated to the recombinant expression plasmid pPICZa by a PCR method at its 5' and 3' ends, respectively, and ligated into the recombinant expression plasmid pPICZa (adding a secretory expression signal peptide and Histidine tag), sequencing verified the correctness of the sequence.
  • the uncorrected recombinant vector electroporation method was transferred into Pichia pastoris, and methanol induced expression. The cells were centrifuged, and the supernatant was used as a crude enzyme solution, and the recombinant protein was isolated and purified by the same affinity chromatography column as in Example 4.
  • Example 7 Recombinant expression of xylosidase Xyl_S in Bacillus subtilis
  • the CDS region of the Xyl-S gene obtained in Example 2 was ligated into the pP43NMK shuttle expression vector by adding the 2 ffl and Hindi II restriction sites at the 5' and 3' ends thereof by PCR. Acid label), the correctness of the sequencing verification sequence, will verify the correct recombinant vector Transfer to B. subtilis A s iife 1A752S.
  • the cells were centrifuged, and the supernatant was used as a crude enzyme solution, and the recombinant protein was separated and purified by the same affinity chromatography column as in Example 4.
  • the 7-xylose taxane mixture was dissolved in methanol at a final concentration of 7 mg/ml.
  • the reaction system, reaction conditions and detection methods were all the same as those of the 10 DAXT hydrolyzed with 99% purity in the above example, except that the reaction time was changed. For 30 min, the test results are shown in Figure 7.
  • the molecular weight of the molecular weight before and after hydrolysis was analyzed by UPLC-PDA-MS method, and the specific method was as follows: Kromasil C18 column (200 mm x 4.6 mm id, 5 ⁇ ). Tedia's chromatographically pure acetonitrile, Mmipore's ultrapure water. Mass spectrometry with ESI interface ion source, nitrogen for the clamp Casing and purge gas, jacket gas pressure 40 psi, auxiliary gas 20 au, source voltage 4.0 kV, capillary temperature 20 (TC, atomizer temperature 325 ° C. The test results are shown in Figure 8.
  • the pure astragaloside IV was dissolved in methanol at a final concentration of 5 mg/ml, and the hydrolysis reaction was carried out in a 200 ⁇ reaction system: wherein the pure enzyme from Example 3 was 30 ⁇ l + 150 ⁇ M Tris hydrochloric acid buffer (50 mM, pH 7). .5) +10 l CaCl 2 +10 ⁇ substrate. After reacting at 35 ° C for 15 min, the detection method was carried out by the TLC method (Fig. 9).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供了一种木糖苷酶Xyl_S及其编码基因与应用，该木糖苷酶的氨基酸序列如SEQ ID NO.2所示，其核苷酸编码序列如SEQ ID NO.1所示。该木糖苷酶具有β-木糖苷水解酶和β-葡萄糖苷酶双重活性，可特异性地水解相应的木糖苷键和葡萄糖苷键。

Description

一种木糖苷酶 Xyl— S及其编码基因与应用
技术领域
本发明属于生物工程领域， 具体涉及一种木糖苷酶 Xyl一 S及其编码基 因与应用。
背景技术
β-木糖苷酶是一种外切酶， 以外切方式从非还原性末端水解木二糖及 木二糖以上的低聚木糖， 水解产物为木糖。它是木聚糖降解的关键酶之一， 有着重要的工业应用价值。 在能源工业中， 工农业废弃物中的木聚糖可被 木聚糖酶系转化为木糖， 而木糖又可被细菌及真菌转化成酒精等有价值的 燃料； 在医药行业中， 木聚糖酶系水解特定底物可产生在医药行业具有重 要应用价值的中间转化产物。 例如， Patel等人利用 Mwrn / β-木糖苷 酶水解 10-去乙酰紫杉醇木糖苷的 7位木糖残基， 得到重要的中间产物 10- 去乙酰紫杉醇， 为紫杉醇的合成开辟了一条崭新的途径（US005700669A; ΕΡ0668360Β1)。
纤维化纤维微细菌是一种能够高效利用纤维素、 半纤维素的放线菌， 其可以产生多种水解酶， 如 Shi-Hsiang Shen等人（ The Journal of Biological Chemistry, 1991, 266(2): 1058-1063 ) 从此菌株发酵液上清中分离得到了 β-1，3-葡萄糖苷酶， 并对其进行了克隆和外源表达； Petra Tiels等人 iNature Biotechnology 2012, 30: 1225-1231 )对此菌株所产生的 5个甘露糖苷酶进行 了克隆和外源表达。 但目前为止， 并没有获得其中木糖苷酶的报道。 我们 在前期工作中分离到一株纤维化纤维微细菌 （ Celluhsimicmbium cellulans ) 菌株 F16 (CCTCC M 2013201 ), 通过深入研宄发现其培养液上清能够分泌 一种木糖苷酶，该酶具有 β-木糖苷水解酶和 β-葡萄糖苷酶水解酶双重活性， 可以特异性水解相应的木糖苷键和葡萄糖苷键， 同时具有广泛的底物特异 性，可水解多种木糖苷底物和葡萄糖苷底物，例如木寡糖、 4-硝基苯基 -β-D- 吡喃木糖苷 （/?NP-P-Xyl)、 4-硝基苯基 -β-D-吡喃葡萄糖苷 （pNP-P_Glu)、 7-木糖苷紫杉烷、 黄芪甲苷， 以及人参皂苷 Rbl、 Rb2、 Re等。 这些优良 的水解性质使其有可能具有非常高的工业利用价值。 因此， 如何大量获得 该酶或含有该酶的菌已经成为制约其工业利用的关键问题。
发明内容
本发明的目的是提供一种木糖苷酶 Xyl— S及其编码基因与应用， 本发 明通过基因表达后大量产生具有广泛底物特异性的木糖苷酶 Xyl—S， 用以 水解木糖苷化合物生成木糖及相应的苷元， 亦可用于水解葡萄糖苷化合物 生成葡萄糖及相应的苷元。
本发明提供了一种木糖苷酶 Xyl_S，其氨基酸序列包含如序列表中 SEQ ID NO.2所示序列的至少 1642个氨基酸序列； 优选 SEQ ID N0.2示序列的 第 24个氨基酸残基到第 1151个氨基酸残基在内的至少 1128个氨基酸序 列，进一步优选包括 SEQ ID NO.2所示序列的第 87个氨基酸残基到第 1119 个氨基酸残基在内的至少 1033个氨基酸序列。
本发明提供了一种木糖苷酶 Xyl— S的编码基因， 该基因的核苷酸序列 具有 SEQ ID NO.l所示的核苷酸序列的至少 4929个核苷酸;优选具有 SEQ ID NO.l所示的核苷酸序列的第 72个核苷酸到第 3453个核苷酸在内的至 少 3372个核苷酸； 进一步优选具有 SEQ ID NO.l所示的核苷酸序列的第 261个核苷酸到第 3357个核苷酸在内的至少 3099个核苷酸。
本发明提供了一种含有所述编码基因的重组载体， 该重组载体是大肠 杆菌表达载体、 酿酒酵母表达载体、 毕赤酵母表达载体、 枯草芽孢杆菌表 达载体、 乳酸菌表达载体、 丝状真菌表达载体中的任意一种。
本发明提供了一种含有所述重组载体的重组细胞株， 该重组细胞株的 宿主细胞是大肠杆菌、 酿酒酵母、 毕赤酵母、 枯草芽抱杆菌、 乳酸菌、 丝 状真菌的任意一种。 该重组细胞株可以表达木糖苷酶 Xyl一 S， 既可以胞内 表达， 也可以分泌到细胞外。
本发明所涉及的基因是通过 DNA 重组技术从纤维化纤维微细菌 (Cellulosimicrobium cellulans) 菌株 F16 (CCTCC M 2013201 ) 的染色体 中克隆， 并经连接在 pCold IV表达载体后在大肠杆菌中过量表达。 经过量 表达的木糖苷酶 Xyl一 S既可以分泌到细胞外， 也可以在细胞内表达。 其在 SDS-PAGE上呈现的分子量约为 140KDa, 其最佳反应 pH为 7.5， 有效作 用的 pH范围为 5.5〜9.5， 并且在 20〜50°C均具备良好的催化活性。
由于密码子的简并性， 所有与 SEQ ID NO.l同源性低至约 60%的简并 序列也能编码出 SEQ ID NO.2所述的序列。 另外， 任何基因通过 DNA重 组技术可以改变其序列， 从而产生各种不同的突变体。 这些突变体所表达 的蛋白质通常具有类似的性质。当基因或蛋白质序列达到一定的同源性时， 它们所表达的蛋白质的性质随同源性增加而更为相似。 本发明涉及的基因 及其产物也具有相同的特点。 当同源性达到 80%以上时， 类似基因所表达 的蛋白质将会具有催化木糖苷化合物水解为木糖和苷元的性质。
由于上述技术方案的使用， 本发明的有益效果是： 利用本发明的基因 进行表达， 获得木糖苷酶 Xyl— S或含该酶的宿主细胞， 能有效地催化木糖 苷化合物以及葡萄糖苷化合物的水解， 可应用于各种工业， 例如用于生物 质转化， 如用于由包含生物质的纤维素生产燃料乙醇的过程中， 用于饲料 组合物中， 提高动物对粗纤维的利用率， 或用于面包制造面团中， 还可以 用于医药中间体的获得， 如水解 7-木糖 -10-去乙酰紫杉醇获得 10-去乙酰紫 杉醇， 同时水解黄芪甲苷 IV上的 β -木糖苷键和 β -葡萄糖苷键从而获得相 应苷元， 水解掉人参皂苷 Rbl、 Rb2或 Re上的木糖苷和葡萄糖苷得到相应 人参皂苷 CK、 Compound Y PPD等等。
本发明提供的木糖苷酶 Xyl_S或重组细胞株应用于医药， 生物，农业， 能源等领域， 将木糖苷化合物 （或葡萄糖苷化合物） 中的糖苷键水解， 获 得相应的苷元。
附图说明
图 1 : 聚丙烯酰胺凝胶电泳分析分离出纯的木糖苷酶 Xyl—S， 其中， MUX为 4-甲基伞形酮酰 -β-D-吡喃木糖苷， 其糖苷键经水解后经 365nm激 发光激发后出现荧光， 从而实现对各个组分中 β-木糖苷酶的监测和酶谱分 析；左图： 2%- 15%梯度 Native PAGE，考染；中图： 2%- 15%梯度 Native PAGE， MUX染色； 右图： 10% SDS-PAGE, 考染； 样品 1一 BSA ferrintin; 2—发 酵液上清； 3—多酶复合物； 4~木糖苷酶 Xyl_S;
图 2: 纯酶催化 ρΝΡ-β-D-Xyl水解， 底物浓度对应相应初始反应速率的 米氏曲线；
图 3: 纯酶催化 ρΝΡ-β-D-Glu水解， 底物浓度对应相应初始反应速率的 米氏曲线； 图 4: 琼脂糖电泳检测 PCR产物， 其中， M为标准品， λ噬菌体 Hind lll 降解物； 1为克隆出的^ 基因；
图 5 : 纯化后的重组酶 Xyl— S;
图 6: Xyl— S纯酶转化纯度为 99%的 10 DAXT结果， 其中， 10 DAXT为 7-木糖 -10-去乙酰基紫杉醇， 10 DAT为 10-去乙酰基紫杉醇；
图 7: Xyl_S纯酶转化 7-木糖紫杉烷混合物的结果， 其中， 10 DAXT为 7-木糖 -10-去乙酰基紫杉醇， 10 DAT为 10-去乙酰基紫杉醇， 10 DAXC为 7- 木糖基 -10-去乙酰三尖杉宁碱， 10 DAC为 10-去乙酰三尖杉宁碱， 10 DAXTC 为 7-木糖 -10-去乙酰基紫杉醇 C, 10 DATC为 10-去乙酰基紫杉醇 C;
图 8: 水解产物的质谱图， 其中， A为 10-去乙酰三尖杉宁碱， B为 10- 去乙酰基紫杉醇；
图 9: 纯酶转化黄芪甲苷 IV生成环黄芪醇 TLC图。
具体实施方式
下面的实施例将对本发明予以进一步的说明， 但并不因此而限制本发 明。
下述实施例中， 如无特殊说明， 均为常规方法。
本发明所依赖的遗传资源是纤维化纤维微细菌 ( Ce!luhsimicrobiwn cellulans) 菌株 F16， 保藏号为 CCTCC M 2013201， 保存于中国典型培养 物保藏中心。
实施例 1: 糖苷醵的纯化及其活性亚基的分离
1、 纤维化纤维微细菌 i Cellulosimicrobium ceUulcms 菌株 F16的培养 从培养好的菌种斜面（木聚糖培养基， 含 1.5 %琼脂）挑取约 1cm2见 方的菌苔， 接种到 100ml无菌的麦麸液体培养基中 （木聚糖培养基成分： 木聚糖 2%， 酵母膏 0.2%， 蛋白胨 0.2%， K2HPO4 0.1 % ), 30°C、 160rpm 摇瓶培养 2d。
2、 木糖苷酶 XylJS的分离纯化
lOOOOg/min, 离心 2min后收集上清即为粗酶液。 以对硝基苯基 -β-D- 木糖苷 (^NP-Xyl)作为特异性生色底物对有 β-木糖苷酶活性的蛋白进行跟 踪。 一个酶单位定义为在 30°C， pH 7.5 , 以; ?NP-Xyl为底物， lh内催化产 生 1 μιηοΐ对硝基酚所需要的酶量。依次经过硫酸铵沉淀， 收集 20%-40%阶 段的沉淀组分； Toyopearl DEAE 650M离子交换柱， 收集 0.5〜0.7 mol/L NaCl阶段洗脱组分； SephaCryl S-200 HR凝胶过滤层析柱， 收集分子量在 30kDa〜200kDa之间的活性组分； 将上述活性组分过 Source 15Q柱分离， 收集比活性最高的酶活组分， 即得纯的木糖苷酶 Xyl— S (图 1 )。
实施例 2: 木糖苷醵 Xyl— S编码基因的克隆
以纤维化纤维微细菌 ( Cellu simicrobium cellulans) 菌株 F16基因组 DNA为模板， PCR扩增木糖苷酶 Xyl— S的编码基因 4929bp (见图 4)， 克 隆至 pMD-T载体上， 测序验证结果显示， 与 SEQ ID N0.1所示序列一致。
PCR反应体系以及反应条件见下表：
反应体系: 反应条件:
Cel lulononas C«l lulans DNA i μ'
98 10 sec
INF (20 (»»Ι/μΙ) 0.5 μ(
55C 15 sec L 30 cycles
IN (20 (ΜΚ>Ι/μΙ) 0.5 μΙ
721C 3 min
dNTP Mixture (各 2.5 di) 4 μΙ
4"C oo
5 X PrimoSTAR GXL Buffer (M ^ Plus) 10 μΙ
PrimeSTAR 8XL DNA Polymrase (1.25 ϋ/μΙ) 0.5 μΙ dH>0 Up to 50 μΙ 实施例 3: 木糖苷醇 Xyl_S在大肠杆菌中的重组表达
以实施例 2所得载体为模板， PCR扩增目的基因 CDS区 4929bp， 片 段两侧添加 Nde VBamU I酶切位点， 终止密码子 TGA前添加 6*His标签， 克隆至 pCoWIV表达载体中， 挑选 2个阳性克隆质粒进行测序验证， 结果 显示序列无误。 即得相应重组载体。 PCR反应体系， 反应条件及所用引物 见下表：
反应体系： 反应
貭敉（50«««液） 1 ul 98¾ 10 sec
5* PrimeSTAR Buffer iMg:' plus) 10 ul 55 10 sec 30cycles dNTP Mixture ί各 2.5'ΓΠΡΛ> 4 ul 72*C 5 min
Figure imgf000009_0001
F01 {20pmot u(j 0.5 ut 72"C 5 min 1 cycle
R01 (20pmol.'ul) 0.5 ul
PrimeSTAR HS DNA Pofymerase(2.5 ϋ !ul) 0.5 ut
dH20 Up \ 50 ul 引物名称 5 '-序列 -. ^'^
m rs
( TGC.AGGTCGACGATTATGG GGCGGCGACGTCGGCGA S
I\ ^TCTCTAGAGGATCCTCAGGAGCAGGCCCGGGCGGCGTA •10
AGAGGTAATACCATATGATGGCGGCGGCGACGTCGGC
GCTTGA.ATT GGArCCT AGTGArGATGATGATGATGGGA
R01
GCAGGCC GGOCGUC G l
Γ02 ACC GTC CTGTGC GGTGCACAC GTACAC GGAGAACG 35
ΓΛ(Κ TCGATCCGGGCGTTCTACTCGTTGACCTCGrrCGGGA
RO:
GCAGGCCC'GGGCGGCG丁 S 将上述重组载体取 1 μΐ转入大肠杆菌 BL21感受态细胞中， 使用 LB/ 抗生素 Amp( 100 g/ml)平板， 50 ul转化液涂布， 37'C 0/ 培养。 Control pCold IV进行同样操作。
分别挑取单菌落至 50 ml LB/Amp(100 g/ml)培养基中， 37°C O/N培 养，至 ODeoo值约为 0.6, 15°C 15 min，添加 100 mM IPTG 50 ul( final 1 mM IPTG)进行诱导， 15°C培养 22 hr。 收集菌体细胞， 加入 10 ml的 PBS重悬 后进行超声波破碎， 对菌体破碎液进行离心分离 （12000rpm， 5min)。
His-Trap Ni亲和层析柱纯化目标蛋白. · 以磷酸缓冲液（pH7.5, 50mM, 含 20mM咪唑）， l ml/min平衡层析柱， 随后以 0.5 ml/min将菌体破碎液上 清上样，同样的平衡液继续洗柱，至 OD280降到基线，以磷酸缓冲液 (pH7.5， 50mM, 含 150mM咪唑）， 1 ml/min回收目标蛋白， 结果见图 5。
实施例 4: 木糖苷酶 XyI_S的关键序列在大肠杆菌中的重组表达 以实施例 2所得载体为模板， PCR扩增目的基因 CDS区的第 261个核 苷酸到第 3357个核苷酸共 3099bp，片段两侧添加 Nde I BamH I酶切位点， 终止密码子 TGA前添加 6*His标签， 克隆至 pCold lV表达载体中， 挑选 2 个阳性克隆质粒进行测序验证， 结果显示序列无误。 即得相应重组载体。 将上述重组载体取 1 μΐ转入大肠杆菌 BL21感受态细胞中， 使用 LB/ 抗生素 Amp( 100 g/ml)平板， 50 ul转化液涂布， 37°C 0/ 培养。 Control pCold lV进行同样操作。
分别挑取单菌落至 50 ml LB/Amp(100 g/ml)培养基中， 37°C O/N培 养，至 ODeoo值约为 0.6， 15°C 15 min，添加 100 mM IPTG 50 ul( final 1 mM IPTG)进行诱导， 15°C培养 22 hr。 收集菌体细胞， 加入 10 ml的 PBS重悬 后进行超声波破碎， 对菌体破碎液进行离心分离 （12000rpm， 5min)。
His-Trap Ni亲和层析柱纯化目标蛋白： 以磷酸缓冲液（pH7.5， 50mM, 含 20mM咪唑）， l ml/min平衡层析柱， 随后以 0.5 ml/min将菌体破碎液上 清上样，同样的平衡液继续洗柱,至 OD280降到基线，以磷酸缓冲液 (pH7.5， 50mM, 含 150mM咪唑）， 1 ml/min回收目标蛋白， 结果见图 5。
实施例 5: 纯酶水解不同糖苷类底物的特异性实验
1、 底物特异性检测
选取木糖苷、 葡萄糖苷、 果糖苷、 甘露糖苷、 岩藻糖苷、 半乳糖苷和 纤维二糖苷等多种生色底物， 均用 50mM的 Tris-HCl缓冲液配制成 5mM、 pH7.5的溶液， 以 30 μΐ实施例 1 所得纯酶 +150 μΐ buffer+10 μΐ 20 mM CaC12+10 ^底物的反应体系， 30°C条件下在 96孔板中反应， BioTek hyrbid Reader酶标仪监测 405nm下的吸光值。
结果显示， 纯的木糖苷酶 Xyl一 S仍具有高的 β-木糖苷和 β-葡萄糖苷的 水解活力， 同时也有一定的 β-纤维二糖苷的水解能力 （表 1 )。 表 1 底物与酶活力
晦活U/mg
底物 发酵液 上 木糖苷懿
清 Xyl— S
jCjNP-a-D-Xyiopyranosidc 一 ― jcNP-p-D-Xylop ranoside 0.810 0.191 jcNP-a-D-Glucopyranoside 0.150 ― iiP- -D-Glucopyranosidc 0,810 0.294
jcNP-a-D-Mannopyranosidc 0.118
jc P-p-D- aanop ranosidc 0.038 ―
JNP- a-D-Galactop ranoside 一 ― iCiiP-p-D-Galactop ranoside 一 ―
¾P-p-D-Cellobioside 0.286 0.018
―
Figure imgf000012_0001
jDNP-p-D-Gl curoside ―
iiP-a-L-Aiabino 一
ζίίΡ-β-L-Fucopyranoside 一 ― jcNP-a-L-Fucopyranoside 一 一
WP-a-L-Rhamnop ranos ― ― 注. · 一代表未检测到。
2、木糖苷酶 Xyl_S催化 ?NP-p-D-Xyl与 NP D-Glu水解的反应动力 学分析
在一个 96孔板内， 配制以下反应体系： 每孔总体积 200μ1， 其中实施 例 3的完整序列重组酶和实施例 4的关键序列重组酶均为 30 μΐ, 底物与缓 冲液的加入量为达到以下 9个底物浓度： 5μΜ, 10μΜ, 20 μΜ, 50 μΜ, 100 μΜ, 200 μΜ, 500 μΜ, 1000 μΜ, 2000 μΜ。 以上每个浓度下做一个 平行样， 两底物 ρΝΡ-β-D-Xyl与；? ΝΡ-β-D-Glu均是如此， 共占用 72个孔， 并以 ΝΡ标准品制作标准曲线。
使用 BioTek hyrbid Reader酶标仪的实时监测功能， 设定每 1 min测定 一次每个孔内反应体系在 405nm下的吸光值， 共测定 61次， 以此数据计 算出每个底物浓度下的初始反应速率， 做出底物浓度 -反应速率的米氏曲 线， 求出对应的^和 max值 （图 2， 图 3 )。
实验结果表明， 木糖苷酶 Xyl— S 对 ρΝΡ-β-D-Xyl 的结合能力大于 ΝΡ-β-D-Glu (前者 Km值小于后者)，但对二者却有着相同的转化常数 t (即最大反应速率与酶浓度的比值， max/[E] )。也即，木糖苷酶 1_8对 木糖苷化合物和 β-葡萄糖苷化合物有着相同的转化能力。 同时， 只表达关 键区域后， 酶对底物的亲和力略有下降， 但仍具有相同活力， 能够很好地 水解底物 （表 2 )。
表 2 酶促反应动力学参数
u
底物
完整表达 关键表达 完整表达 关键表达
pXP-p-D- .9- = 0. U 4.92 =0.05
xylopyraiioside
ρΧΡ-β-D-
6 二一 J 92.3 = 4.5 ?.02 =0.06
glucop\Tanoside
I I 实施例 6： 木糖苷 H Xyl一 S在毕赤酵母中的重组表达
将实施例 2所得 Xyl_S基因的 CDS区域通过 PCR方法在其 5'端、 3' 端分别加上^ 0 I、 Xba I 酶切位点， 连接到重组表达质粒 pPICZa中 （加 入分泌表达信号肽和组氨酸标签）， 测序验证序列的正确性。将验证无误的 重组载体电转化法转入巴斯德毕赤酵母中， 甲醇诱导表达。 离心去菌体， 上清作为粗酶液， 采用与实施例 4同样的亲和层析柱分离纯化重组蛋白。
实施例 7: 木糖苷酶 Xyl_S在枯草芽孢杆菌中的重组表达
将实施例 2所得 Xyl—S基因的 CDS区域通过 PCR方法在其 5'端、 3' 端分别加上^ 2 ffl、 Hindi I I酶切位点， 连接到 pP43NMK穿梭表达载体 中（添加了组氨酸标签）， 测序验证序列的正确性， 将验证无误的重组载体 转入枯草芽孢杆菌 A s iife 1A752S中。 离心去菌体， 上清作为粗酶液， 采用与实施例 4同样的亲和层析柱分离纯化重组蛋白。
实施例 8: 木糖苷酶 Xyl—S的应用
1、 纯酶水解 7-木糖 -10-去乙酰紫杉醇 （10 DAXT)
将纯度为 99%的 10 DAXT以 10 mg/ml的终浓度溶解于甲醇之中， 以 200 μΐ的反应体系进行水解反应：其中来自实施例 3的纯酶 30 μ1+150 μΐ Tris 盐酸缓冲液（50mM， pH7.5 ) +10 μΐ CaCl2+10 μΐ底物。在 35°C下反应 15min 后， 加入 200 μΐ甲醇终止反应， HPLC-UV方法检测转化率， 检测结果见图 6 ο
具体检测方法：色谱柱: Kromasil C18色谱柱（200mmx4.6mm ΐ.ά., 5μιη); 流动相：乙腈:水（V V)，具体梯度： 0-15min, 30—70; 15-22min, 10%→10%； 22-30min, 70%→70% ； 柱温 40°C ; 检测波长： 227 nm; 流速： l.Oml/min; 进样量： 20 μ1 ο
若想将体系中全部的 10 DAXT均转化为 10 DAT,可继续反应 ~105min 即可。
2、 纯酶水解 7-木糖紫杉烷混合物
以 7mg/ml的终浓度溶解 7-木糖紫杉烷混合物于甲醇之中， 反应体系， 反应条件及检测方法均与上例中水解 99%纯度的 10 DAXT基本相同，不同 的是反应时间改为 30min， 检测结果见图 7。
此外，通过 UPLC-PDA-MS法对其水解前后的分子量进行了质谱分析, 具体方法如下： Kromasil C18色谱柱 （200mmx4.6mm i.d.， 5μιη)。 Tedia公 司色谱纯乙腈， Mmipore公司超纯水。 质谱用 ESI接口离子源， 氮气为夹 套气和吹扫气， 夹套气压力 40psi， 辅助气 20 a.u.， 源电压 4.0kV, 毛细管 温度 20(TC， 雾化器温度 325°C。 检测结果见图 8。
3、 纯酶水解黄芪甲苷 IV
以 5mg/ml的终浓度溶解黄芪甲苷 IV纯品于甲醇之中， 以 200 μΐ的反应体 系进行水解反应： 其中来自实施例 3的纯酶 30 μ1+150 μΐ Tris盐酸缓冲液 (50mM, pH7.5) +10 l CaCl2+10 ^底物。 在 35°C下反应 15min后， 检测 方法采用 TLC法进行 （图 9)。

Claims

权 利 要 求 书
1、 一种木糖苷酶 XyI_S, 其特征在于： 其氨基酸序列包含如序列表中 SEQ ID NO.2所示序列的至少 1642个氨基酸序列。
2、 按照权利要求 1所述木糖苷酶 Xyl— S， 其特征在于： 其氨基酸序列 包含 SEQ ID NO.2所示序列的第 24个氨基酸残基到第 1151个氨基酸残基 在内的至少 1128个氨基酸序列。
3、 按照权利要求 2所述木糖苷酶 Xyl_S， 其特征在于： 其氨基酸序列 包含 SEQ ID NO.2所示序列的第 87个氨基酸残基到第 1119个氨基酸残基 在内的至少 1033个氨基酸序列。
4、 一种木糖苷酶 Xyl_S的编码基因， 其特征在于： 该基因的核苷酸序 列具有 SEQ ID N0.1所示的核苷酸序列的至少 4929个核苷酸。
5、按照权利要求 4所述木糖苷酶 Xyl一 S的编码基因， 其特征在于： 所 述基因的核苷酸序列具有 SEQ ID NO.l所示的核苷酸序列的第 72个核苷酸 到第 3453个核苷酸在内的至少 3372个核苷酸。
6、按照权利要求 5所述木糖苷酶 Xyl— S的编码基因， 其特征在于： 所 述基因的核苷酸序列具有 SEQ ID NO.l所示的核苷酸序列的第 261个核苷 酸到第 3357个核苷酸在内的至少 3099个核苷酸。
7、 一种含有权利要求 4所述编码基因的重组载体。
8、 按照权利要求 7所述的重组载体， 其特征在于： 该重组载体是大肠 杆菌表达载体、 酿酒酵母表达载体、 毕赤酵母表达载体、 枯草芽孢杆菌表 达载体、 乳酸菌表达载体、 丝状真菌表达载体中的任意一种。
9、 一种含有权利要求 7所述重组载体的重组细胞株。
10、 按照权利要求 9所述的重组细胞株， 其特征在于： 该重组细胞株 的宿主细胞是大肠杆菌、 酿酒酵母、 毕赤酵母、 枯草芽孢杆菌、 乳酸菌、 丝状真菌的任意一种。
11、 按照权利要求 9所述的重组细胞株， 其特征在于： 该重组细胞株 表达木糖苷酶 Xyl_S， 既能够胞内表达， 也能够分泌到细胞外。
12、 一种应用， 其特征在于： 权利要求 1所述木糖苷酶 Xyl— S或权利 要求 9所述重组细胞株应用于医药， 生物， 农业， 能源领域， 将木糖苷化 合物中的糖苷键水解， 获得相应的苷元。
13、 一种应用， 其特征在于： 权利要求 1所述木糖苷酶 Xyl_S或权利 要求 9所述重组细胞株应用于医药， 生物， 农业， 能源领域， 将葡萄糖苷 化合物中的糖苷键水解， 获得相应的苷元。
PCT/CN2013/001509 2013-06-21 2013-12-06 一种木糖苷酶Xy1_S及其编码基因与应用 Ceased WO2014201595A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310251300.2 2013-06-21
CN201310251300.2A CN104232605B (zh) 2013-06-21 2013-06-21 一种木糖苷酶Xyl_S及其编码基因与应用

Publications (1)

Publication Number Publication Date
WO2014201595A1 true WO2014201595A1 (zh) 2014-12-24

Family

ID=52103771

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/001509 Ceased WO2014201595A1 (zh) 2013-06-21 2013-12-06 一种木糖苷酶Xy1_S及其编码基因与应用

Country Status (2)

Country Link
CN (1) CN104232605B (zh)
WO (1) WO2014201595A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106011106B (zh) * 2016-06-20 2017-05-10 云南与诺生物工程有限责任公司 一种酶及其编码基因和它们的应用以及制备人参皂苷化合物k的方法
CN107189994B (zh) * 2017-07-05 2021-01-08 中国科学院青岛生物能源与过程研究所 β-木糖苷酶及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101870966A (zh) * 2009-04-27 2010-10-27 复旦大学 葡萄糖苷酶/木糖苷酶双功能纤维素降解酶及其制备方法和应用
WO2011160484A1 (zh) * 2010-06-25 2011-12-29 中国医学科学院药物研究所 具有β-木糖苷酶和β-葡萄糖苷酶活性的新的糖基水解酶及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101381708A (zh) * 2007-09-04 2009-03-11 中国科学院大连化学物理研究所 纤维化纤维单胞菌、水解酶及其在紫杉烷转化方面的用途
CN101429517A (zh) * 2008-11-14 2009-05-13 南开大学 耐高温木糖苷酶XynB1和编码该酶的基因与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101870966A (zh) * 2009-04-27 2010-10-27 复旦大学 葡萄糖苷酶/木糖苷酶双功能纤维素降解酶及其制备方法和应用
WO2011160484A1 (zh) * 2010-06-25 2011-12-29 中国医学科学院药物研究所 具有β-木糖苷酶和β-葡萄糖苷酶活性的新的糖基水解酶及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, TIANJIAO ET AL.: "Catalytic Property of Glycoside Hydrolases for 7-Xylosyltaxanes Lxyl-pl-1 and Lxyl-pl-2", JOURNAL OF FUNGAL RESEARCH, vol. 11, no. 2, May 2013 (2013-05-01), pages 151 *
ZHOU, J. ET AL.: "Beta-Xylosidase Activity of A GH3 Glucosidase/Xylosidase from Yak Rumen Metagenome Promotes the Enzymatic Degradation of Hemicellulosic Xylans", LETTERS IN APPLIED MICROBIOLOGY, vol. 54, no. 2, February 2012 (2012-02-01), pages 79 - 87 *

Also Published As

Publication number Publication date
CN104232605B (zh) 2016-07-06
CN104232605A (zh) 2014-12-24

Similar Documents

Publication Publication Date Title
Mao et al. Purification, characterization, and heterologous expression of a thermostable β-1, 3-1, 4-glucanase from Bacillus altitudinis YC-9
Sharma et al. Cloning and expression of acidstable, high maltose-forming, Ca2+-independent α-amylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis
Ghatge et al. Characterization of modular bifunctional processive endoglucanase Cel5 from Hahella chejuensis KCTC 2396
Zhang et al. Synergistic mechanism of GH11 xylanases with different action modes from Aspergillus niger An76
US8822179B2 (en) Nucleic acid compositions and the encoding proteins
CN106414728A (zh) 琼胶寡糖水解酶及通过使用该琼胶寡糖水解酶从琼脂糖中生成3,6-脱水-l-半乳糖和半乳糖的方法
CN108660145A (zh) 耐热型普鲁兰酶的编码基因及其重组表达和应用
CN102796751A (zh) 一种长野芽孢杆菌普鲁兰酶突变体Pul324及其应用
Yang et al. Biochemical properties of a novel glycoside hydrolase family 1 β-glucosidase (PtBglu1) from Paecilomyces thermophila expressed in Pichia pastoris
Huang et al. Improved expression and characterization of a multidomain xylanase from Thermoanaerobacterium aotearoense SCUT27 in Bacillus subtilis
de Lima Damásio et al. Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application
Ko et al. A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium
CN102827820A (zh) 一种β-葡糖苷酶及其应用
CN102719414A (zh) 一种新型阿魏酸酯酶及其应用
Wang et al. Efficient secretion of xylanase in Escherichia coli for production of prebiotic xylooligosaccharides
Pang et al. Production of a thermostable pullulanase in Bacillus subtilis by optimization of the expression elements
CN103352031B (zh) 一种糖基转移酶基因及应用
Ali et al. Thermostable xylanase10B from Clostridium acetobutylicum ATCC824
WO2014201595A1 (zh) 一种木糖苷酶Xy1_S及其编码基因与应用
Tran et al. Data on gene cloning, expression, purification, and characterization of the glycoside hydrolase family 11 from Bacillus velezensis
Kalinina et al. Expression of the xylanase gene from pyromyces finnis in Pichia pastoris and characterization of the recombinant protein
Li et al. Production, purification, and characterization of a cellulase-free thermostable endo-xylanase from Thermoanaerobacterium thermosaccharolyticum DSM 571
CN109182301B (zh) 一种二糖降解酶基因及其应用
Ng et al. Cloning and expression of Cel8A from Klebsiella pneumoniae in Escherichia coli and comparison to cel gene of Cellulomonas uda
Kalinina et al. Expression of the xylanase gene from Paenibacillus brasilensis X1 in Pichia pastoris and characteristics of the recombinant enzyme

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13887398

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13887398

Country of ref document: EP

Kind code of ref document: A1