WO2014166326A1 - 一种β−葡萄糖苷酶及其表达基因与应用 - Google Patents

一种β−葡萄糖苷酶及其表达基因与应用 Download PDF

Info

Publication number
WO2014166326A1
WO2014166326A1 PCT/CN2014/073502 CN2014073502W WO2014166326A1 WO 2014166326 A1 WO2014166326 A1 WO 2014166326A1 CN 2014073502 W CN2014073502 W CN 2014073502W WO 2014166326 A1 WO2014166326 A1 WO 2014166326A1
Authority
WO
WIPO (PCT)
Prior art keywords
glucosidase
expression vector
seq
reaction
gene
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/CN2014/073502
Other languages
English (en)
French (fr)
Inventor
方诩
高天龙
王明钰
刘奎美
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.)
Shandong University
Original Assignee
Shandong University
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 Shandong University filed Critical Shandong University
Publication of WO2014166326A1 publication Critical patent/WO2014166326A1/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
    • 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
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the present invention relates to a ⁇ -glucosidase and an expression gene thereof and application thereof, in particular to a ⁇ -glucosidase and an expression gene thereof, and a method for producing an oligosaccharide by using the enzyme, and belong to the field of biotechnology and biochemical technology.
  • Oligosaccharides are oligosaccharides composed of 2-10 monosaccharides, which have a very wide range of applications. For example, it is widely used in the fields of food, health care products, beverages, medicines, and feed additives. The United States, Japan, Europe and other places have scaled production. The development and application of oligosaccharides in China began in the mid-1990s and has developed rapidly in recent years. Common oligosaccharides include malto-oligosaccharide glucose ( ⁇ -1,4 glycosidic bond), gentiobiose glucose ( ⁇ -1,6-glycosidic bond), oligosaccharide xylose ( ⁇ -1,4 glycosidic bond) Combined) and so on.
  • the technical methods for manufacturing gentiobiose mainly include the following two types: 1. Extraction from natural raw materials, but this method has low extraction yield and high price, and cannot meet industrial production. 2. Enzymatic production of gentiobiose, but the current enzymatic production of gentiobiose has inconvenient factors such as low enzyme activity and high enzyme production cost.
  • the oligosaccharide is produced by enzymatic method, and a monosaccharide is used as a substrate to produce an oligosaccharide using a glycosidase.
  • Chinese Patent Document CN101492661A (Application No. 200910029055. 4) discloses cloning, expression and preparation of a ⁇ -glucosidase gene for reverse transcription synthesis of Aspergillus niger WX-07 total RNA.
  • BGL CDNA uses plasmid PPIC9K as an expression vector, and Pichia pastoris (P.
  • BGL BGL
  • the CDNA is 2,523 nucleotides in length and encodes 841 amino acids.
  • the constructed BGL/PPIC9K transforms P. PASTORIS KM71 to express the BGL enzyme.
  • the BGL enzyme has transglycoside activity and is capable of converting glucose into glycosides to form gentiooligosaccharides.
  • the enzyme disclosed in this document takes too long to produce oligosaccharides, and the yield of the product is too low to meet the production requirements.
  • the present invention is directed to the deficiencies of the prior art, and provides a method for efficiently producing ⁇ -glucosidase and its expressed gene and producing oligosaccharide using the enzyme.
  • the invention transforms the ⁇ -glucosidase produced in the T. reesei strain and finds that the yield of the modified ⁇ -glucosidase product is increased by 3.3 times. Moreover, compared to the reported similar technology, the time required to produce the same yield of oligosaccharide was shortened by 4. 8 times, and the yield of the final product was increased by 25%.
  • An expression gene of ⁇ -glucosidase the nucleotide sequence of which is shown in SEQ ID NO.
  • a recombinant expression vector obtained by inserting the nucleotide sequence shown in SEQ ID NO. 1 into an expression vector.
  • the expression vector is a PET-32A expression vector.
  • a recombinant cell obtained by transforming the above recombinant expression vector into a cell.
  • the cell is Escherichia coli BL21 (DE3).
  • a method for producing oligosaccharides using ⁇ -glucosidase the steps are as follows:
  • the gene fragment having the nucleotide sequence as shown in SEQ ID NO. 2 is introduced into the PET-32A plasmid vector, and then transferred into E. coli BL21 (DE3), and purified by affinity chromatography to obtain ⁇ -glucoside.
  • the enzyme was then added to the reaction solution containing glucose as a substrate, and the reaction was carried out under the conditions of a temperature of 30 ° and a pH of 6.0 to obtain an oligosaccharide.
  • the reaction rate is significantly faster than that of the existing ⁇ -glucosidase.
  • the optimal enzyme activity temperature of the ⁇ -glucosidase according to the present invention is closer to the normal temperature, and in practical industrial production, it is advantageous to reduce the production energy consumption.
  • the ⁇ -glucosidase of the present invention has a high expression level in a recombinant strain, a simple purification process, and is easy to be industrially produced in a large scale.
  • M marker, 1, E. coli cell disruption, 2, nickel affinity chromatography column elution peak, 3, anion exchange chromatography elution peak, 4, anion exchange chromatography elution peak.
  • Trichoderma reesei QM6a was purchased from the American Standard Biological Collection, strain accession number ATCC No. 13631;
  • the Pet-32A plasmid vector was purchased from Novagen.
  • the T. reesei QM6a strain was cultured for 2 days in a sputum medium supplemented with 2 wt% of microcrystalline cellulose, and hyphae were collected by filtration through a filter paper.
  • the collected hyphae were ground in a pre-cooled mortar, in which a certain amount of liquid nitrogen was added during the grinding.
  • the ground hyphae powder was transferred to a 1.5 ml centrifuge tube, and 1 ml of RNAiso (purchased from Biotech Bioengineering Co., Ltd. B6402-1) was added and shaken evenly in an oscillator at room temperature for 5 min. It was then centrifuged at 12,000 rpm for 10 min.
  • the components of the mash medium are as follows: ammonium sulfate 3g, potassium dihydrogen phosphate 4.5g, magnesium sulfate 0.18g, calcium chloride dihydrate 0.24g urea 1.5g, 1000 X trace elements (iron sulfate heptahydrate 5g / L, sulfuric acid monohydrate Manganese 1.6g / L, zinc sulfate heptahydrate 1.4g / L, cobalt chloride 2g / L) 30 ⁇ 1, made up to 300ml with water.
  • RNA of T. reesei QM6a was synthesized by reverse transcription (purchased from takara reverse transcription kit BK1201):
  • the bgl gene was amplified by using F and R as upstream and downstream primers:
  • Primer F CCGGAATTCATGCCCGAGTCGCTAGCTCTGCCC;
  • Primer R CCCAAGCTTTGCCGCCACTTTAACCCTCTGC;
  • the PCR reaction was carried out in a 50 ⁇ 1 system: 2XPCR Buffer 25 ⁇ 1, 2mM dNTPs 10 ⁇ 1, primer F 1.5 l, primer R 1.5 ⁇ 1, template DNA 1 ⁇ 1, K0D FX polymerase 1 ⁇ 1, plus double distilled water to make up 50 ⁇ 1.
  • PCR reaction system Start the cycle after denaturation at 94 degrees for 2 minutes, then denature at 98 degrees for 10s, annealing at 60 degrees for 30s, and extend at 68 degrees for 90s. After 35 cycles, and then extended at 68 degrees for 10 minutes, the PCR fragment was amplified and tapped. The recovered fragment was ligated to the PET32A plasmid vector at a cloning site between the multiple cloning sites EcoRl and Hindll.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ , and the transformed product was plated on an LB plate containing 100 mg/L ampicillin, and cultured overnight at 37°C, colonies were selected, and LB liquid medium was added thereto, and the plasmid was extracted 10 hours later.
  • the ⁇ -glucosidase gene in the above recombinant plasmid was subjected to site-directed mutagenesis, and the mutation sites were I177S, I174S.
  • the mutation site I 177S was first mutated.
  • Site-directed mutagenesis was carried out using Fl, R1 as the reverse primer (the kit was obtained from Toyobo KOD-PLUS-Mutagenesis Kit 167300), and the sequence was as follows:
  • R 1 GGCCTGAATCCAGGGTTCGTTGATGGTG;
  • PCR reaction system The cycle was started after denaturation at 94 degrees for 2 minutes, then denaturation at 98 degrees for 10 s, annealing at 65 degrees for 30 seconds, and extension at 68 degrees for 7 minutes for 5 cycles.
  • Escherichia coli DH5 ⁇ was transformed, and the transformed product was applied to an LB plate containing 100 mg/L ampicillin, and cultured overnight at 37 °C, colonies were selected, and LB liquid medium was added thereto, and the plasmid was extracted 10 hours later. This plasmid was subjected to sequence measurement. Pick the correct mutation plasmid.
  • the plasmid with the correct mutation site I 177S was used as a template, and the I 174S site was mutated.
  • Reverse PCR primers F2 and R2 were designed, and the plasmid with the correct mutation was selected in the same manner as above.
  • the obtained ⁇ -glucosidase expression gene was verified by sequencing by Huada Gene Bioengineering Co., Ltd., and the nucleotide sequence is shown in SEQ ID NO.
  • E. coli BL21 E. coli containing the recombinant plasmid of the obtained culture expanded, grown to 0D 6. .
  • IPTG IPTG
  • induction was carried out for 16 hours at 16 degrees.
  • the cells were collected by centrifugation at 7000 rpm, and the cells were washed twice with a pH of 6.0, 50 mM PBS buffer.
  • the target protein is purified by nickel ion affinity chromatography and anion exchange chromatography, and then the liquid after separation, the elution peak solution after affinity chromatography of nickel ion and the anion exchange chromatography are respectively purified.
  • the reaction was carried out by SDS-PAGE, and the result was as shown in Fig. 1.
  • the reaction solution was reacted with the reaction solution in an amount of 0.35 mg per 1 ml of the reaction solution.
  • the reaction condition is 30 ° C
  • the pH is 6.0
  • the reaction liquid component is 10 ml, 40% glucose, 500 ⁇ l sodium azide, 1 mL of pH 1.00 50 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer Solution.
  • the sample was boiled in a water bath for 10 minutes to kill the enzyme.
  • Product concentration and species were then identified by HPLC and thin layer chromatography ( Figure 2). Its amino acid sequence is shown in SEQ ID NO.
  • the purified ⁇ -glucosidase and wild-type ⁇ -glucosidase were separately added to a 50 mM disodium hydrogen phosphate/diacid sodium dihydrogen buffer solution containing 5 mM p-nitrophenyl glucoside (; 10% was added) Glycerol), pH 7.4, temperature 30 ° C, reaction for 30 minutes.
  • the reaction was quenched with 10% sodium bicarbonate 150 ⁇ l.
  • the OD value was measured at an appropriate wavelength of the light at 420 nm to obtain its hydrolase activity.
  • the protein concentration was determined using a Bradford kit (Shanghai Shenggong Bioengineering SK3041-1).
  • the enzyme activity is defined as follows: Conversion per minute produces 1 mM pNP as an enzyme unit (U). The results are shown in Table 1.
  • Example 2 The conditions in Example 2 were optimized to achieve a higher level of transglycosylation activity. Different temperatures were set, and the pH value was optimized for the transglycosylation of the ⁇ -glucosidase of the present invention.
  • the experimental condition was that the condition was ⁇ 5. 0, when the temperature is 30 degrees, the transglycosylation activity is the highest, and reaches the highest value in about 72 hours. Under this condition, 60%, 70%, 80% glucose concentration is used as the substrate, and the highest point of 72 hours is obtained. , As shown in table 2.
  • the reaction protein was reacted with the reaction solution in an amount of 0.35 mg of enzyme per 1 ml of the reaction solution.
  • the reaction condition is 30 ° C
  • the pH is 6.0
  • the reaction liquid component is 10 ml
  • 40% glucose, 500 1 sodium azide, and 1 mL of pH 6.0 are added.
  • the reaction phase was sampled at regular intervals, and the sample was boiled in a water bath for 10 minutes to kill the enzyme. The results are shown in Table 3.

Landscapes

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

Abstract

提供了一种β−葡萄糖苷酶及其表达基因与应用。该β−葡萄糖苷酶的氨基酸序列如SEQ ID NO.1所示。该β−葡萄糖苷酶的表达基因的核苷酸序列如SEQ ID NO.2所示。该β−葡萄糖苷酶用于生产寡糖。

Description

_ _ ί
一种 β -葡萄糖苷酶及其表达基因与应用 技术领域
本发明涉及一种 β -葡萄糖苷酶及其表达基因与应用，特别是一种 β -葡萄糖苷酶及其表 达基因与利用该酶生产寡糖的方法， 属于生物技术和生物化工技术领域。
背景技术
寡糖是一种由 2-10个单糖组成的低聚糖， 其有着非常广泛的应用价值。 如目前广泛应 用于食品、 保健品、 饮料、 医药、 伺料添加剂等领域。 美国、 日本、 欧洲等地均有规 模化生产， 我国低聚糖的开发和应用起于 90年代中期， 近几年发展迅猛。 常见的寡糖 包括麦芽低聚糖葡萄糖（α— 1， 4糖苷键结合）， 龙胆二糖葡萄糖 （ β— 1， 6糖苷键结 合）， 低聚糖木糖 （ β—1， 4糖苷键结合） 等。 目前制造龙胆二糖的技术方法主要包 括以下两种： 1、 从天然原料中提取， 但这种方法提取产量低， 价格昂贵， 无法满足工业化 生产。 2、 酶法生产龙胆二糖， 不过目前酶法生产龙胆二糖存在着酶活性效率低下， 酶生产 费用高昂等不便因素。
目前酶法生产寡糖， 是将单糖作为底物， 利用糖苷酶生产寡糖。 如中国专利文献 CN101492661A (申请号 200910029055. 4) 公开了一种 β _葡萄糖苷酶基因的克隆、 表达及用 于龙胆低聚糖的制备，该发明由黑曲霉 WX-07总 RNA逆转录合成 β -葡萄糖苷酶基因（BGL) SEQ ID N0 : 1 , BGL的 CDNA以质粒 PPIC9K为表达载体， 以毕赤酵母（P. PAST0RIS)为表达宿主，实 现 BGL基因在胞外的可溶性表达； BGL的 CDNA全长 2523个核苷酸， 编码 841个氨基酸， 构 建的 BGL/PPIC9K转化 P. PASTORIS KM71可表达 BGL酶。 BGL酶具有转糖苷活性， 能将葡萄 糖转糖苷生成龙胆低聚糖。 但是该文献公开的酶生产寡糖所用时间过长， 产物得率过低， 无 法满足生产要求。
发明内容
本发明针对现有技术的不足， 提供一种 β -葡萄糖苷酶及其表达基因的高效生产与利用 该酶生产寡糖的方法。
发明概述
本发明通过对里氏木霉菌株中产生的 β -葡萄糖苷酶进行相关改造，发现其改造后的 β - 葡萄糖苷酶的作用产物的产量提高了 3. 3倍。而且与报道的同类技术相比， 产生相同产量寡 糖所需的时间缩短了 4. 8倍， 最终产物得率提高了 25%。
发明详述
本发明技术方案如下：
一种 β -葡萄糖苷酶， 氨基酸序列如 SEQ ID NO. 1所示。
一种 β -葡萄糖苷酶的表达基因， 核苷酸序列如 SEQ ID NO. 2所示。 一种重组表达载体， 是将如 SEQ ID NO. 1所示核苷酸序列插入表达载体获得。
根据本发明优选的， 所述表达载体为 PET-32A表达载体。
一种重组细胞， 是将上述重组表达载体转化入细胞中获得。
根据本发明优选的， 所述细胞为大肠杆菌 BL21 (DE3)。
一种利用 β -葡萄糖苷酶生产寡糖的方法， 步骤如下：
将核苷酸序列如 SEQ ID NO. 2所示的基因片段导入到 PET-32A质粒载体中， 然后转入大 肠杆菌 BL21 (DE3)中， 通过亲和层析色谱分离纯化制得 β _葡萄糖苷酶， 然后将 β _葡萄糖苷 酶加入以葡萄糖为底物的反应液中， 在温度为 30度， ρΗ为 6. 0的条件下反应， 获得寡糖。
有益效果
1、 利用本发明所述的 β _葡萄糖苷酶生产寡糖， 与现有 β _葡萄糖苷酶相比， 反应速度 明显加快。
2、 本发明所述的 β _葡萄糖苷酶的最佳酶活温度更接近常温， 在实际工业化生产中， 有 利于降低生产耗能。
3、 本发明所述的 β _葡萄糖苷酶在重组菌株中表达量高， 纯化过程简单， 易于大规模工 业化生产。
附图说明
图 1亲和层析纯化蛋白结果 SDS-PAGE图谱；
其中： M、 marker, 1、 大肠杆菌细胞破碎液， 2、 镍亲和层析柱洗脱峰， 3、 阴离子交换 层析洗脱峰， 4、 阴离子交换层析洗脱峰。
图 2薄层层析分析寡糖产物成分；
具体实施方式
下面通过实施例对本发明的技术方案做进一步的阐述， 应该说明的是， 本说明的保护范 围不仅限于此。
里氏木霉（trichoderma reesei ) QM6a购自美国标准生物品保藏中心， 菌种保藏号 ATCC No. 13631；
Pet-32A质粒载体购自 Novagen公司。
实施例 1：
(1)里氏木霉 QM6a总 RNA的提取：
里氏木霉 QM6a菌株在加有 2wt%微晶纤维素的匪培养基中培养 2天， 用滤纸过滤收集菌 丝。 将收集的菌丝放入预冷的研钵中研磨， 其中研磨时加入一定的液氮。 将研磨成的菌丝粉 末移至 1. 5ml离心管中， 并加入 1ml RNAiso (购自生工生物工程有限公司 B6402-1 ) 于振 荡器中震荡均匀， 室温放 5min。 然后 12000rpm离心 10min。 然后将上清吸到干净的 1. 5ml 离心管中。然后加入 160 μ 1氯仿，震荡 15s混匀，室温放置 5min， 12000rpm, 4度离心 5min。 然后吸上清至新的 1. 5ml离心管。然后再加入 800 μ 1异丙醇，上下颠倒 5次。室温放置 lOmin, 12000rpm, 4度离心 10min。 弃上清。 加入 lml预冷的 75%乙醇清洗 RNA， 震荡后 7500rpm离 心 5min。 加入 50 μ 1 DEPC处理过的水， 溶解 RNA。
匪培养基组分如下： 硫酸铵 3g， 磷酸二氢钾 4.5g, 硫酸镁 0.18g， 二水氯化钙 0.24g 尿素 1.5g， 1000 X微量元素（七水硫酸铁 5g/L， 一水硫酸锰 1.6g/L， 七水硫酸锌 1.4g/L， 氯化钴 2g/L) 30μ 1, 用水补足到 300ml。
(2) β-葡萄糖苷酶编码基因的克隆：
以里氏木霉 QM6a总 RNA为模板， 利用逆转录合成 cDNA (购自 takara反转录试剂盒 BK1201):
①基因组 DNA的去除反应
按以下比例配制反应液：
5XgDNA Eraser Buffer 2μ 1
gDNA Eraser 1 μ 1
Total RNA 0.5 g
RNAase Free dd¾0 up to 10 1
将上述反应液在 42度条件下反应 2min。
②反转录反应：
按以下比例配制反应液：
5XPrimeScript Buffer 2 4μ 1
PrimeScript RT Enzyme Mix I 1 1
RT Primer Mix 1 μ 1
步骤①配制的反应液 10 μ ΐ
RNase Free dd¾0 up to 20 1
将上述反应液在 37度反应 15min， 接着在 85度反应 5s。
以 F， R为上下游引物扩增出 bgl基因：
引物 F： CCGGAATTCATGCCCGAGTCGCTAGCTCTGCCC；
引物 R： CCCAAGCTTTGCCGCCACTTTAACCCTCTGC；
PCR反应在 50μ 1体系中进行： 2XPCR Buffer 25 μ 1， 2mM dNTPs 10μ 1, 引物 F 1.5 l， 引物 R 1.5μ 1, 模板 DNA 1μ 1， K0D FX聚合酶 1μ 1， 加双蒸水补足到 50 μ 1。
PCR反应体系： 在 94度变性 2min后开始循环， 然后 98度变性 10s, 60度退火 30s， 68 度延伸 90s， 共 35个循环后， 再于 68度延伸 lOmin, 扩增得到 PCR片段， 割胶回收， 回收 片段连接在 PET32A质粒载体上，连接位置在质粒载体的多克隆位点 EcoRl和 Hindlll之间。 连接产物转化大肠杆菌 DH5 α， 转化产物涂布于含 lOOmg/L氨苄青霉素的 LB平板， 经 37度 培养过夜， 挑选菌落， 接入 LB液体培养基， 10个小时后提取质粒。
(3) β_葡萄糖苷酶基因的改造
将上述重组质粒中的 β-葡萄糖苷酶基因进行定点突变， 突变位点为 I177S, I174S。 首先突变位点 I 177S。 以 Fl， R1 为反向引物进行定点突变 （试剂盒来源于东洋纺公司 KOD-PLUS-Mutagenesis Kit 167300)， 序列如下：
F 1： AGCTATGGATATGCCACCGGCAGCAACGC；
R 1： GGCCTGAATCCAGGGTTCGTTGATGGTG；
①反向 PCR
按以下比例配制 PCR反应液：
10 X Buffer 5 μ 1
2mM dNTPs 5 μ 1
Fl 1. 5 μ 1
Rl 1. 5 μ 1
重组质粒 （PET32A-BGL) 1 μ 1
KOD-PLUS-Mutagenesis Kit 1 μ 1
dd¾0 up to 50 1
PCR反应体系： 在 94度变性 2min后开始循环， 然后 98度变性 10s， 65度退火 30s， 68 度延伸 7min， 共 5个循环。
② DPN I对模板质粒的消化
在上述反应后的反应液中加入 1 μ 1DPN I， 在 37°C条件下反应 1个小时；
③ PCR产物的自身环化
按以下比例配制反应液
Ligation high 5 μ 1
dd¾0 7 μ 1
②中的反应液 2 μ 1
Τ4 Kinase 1 μ 1
将上述反应液在 16度条件下反应 1小时
反应后转化大肠杆菌 DH5 α， 转化产物涂布于含 lOOmg/L氨苄青霉素的 LB平板， 经 37 度培养过夜， 挑选菌落， 接入 LB液体培养基， 10个小时后提取质粒。 将此质粒进行序列测 定。 挑选出突变正确的质粒。
以上述突变位点 I 177S突变正确的质粒为模板， 突变 I 174S位点， 设计反向 PCR引物 F2和 R2， 按上述同样的方法挑选出突变正确的质粒。
F2： AGTCAGGCCAGCTATGGATATGCCACCG
R2： CCAGGGTTCGTTGATGGTGATCCAGTTCT
制得的 β _葡萄糖苷酶表达基因经华大基因生物工程公司测序验证， 核苷酸序列如 SEQ ID NO. 2所示。
接着将重组质粒导入大肠杆菌 BL21 (DE3)中。 将得到的含有重组质粒的大肠杆菌扩大培 养，培养至 0D6。。为 0. 6时加入千分之一含量为 100 mM的 IPTG, 16度条件下诱导 16个小时。 然后在 7000rpm条件下离心， 收集菌体， 用 pH为 6. 0， 50mM PBS缓冲液洗涤菌体两遍。 通 过超声破碎后， 用镍离子亲和层析和阴离子交换色谱的方式纯化出目的蛋白， 然后分别将破 碎后的液体、 镍离子亲和层析后的洗脱峰溶液及阴离子交换色谱纯化后的洗脱峰溶液经 SDS-PAGE电泳后， 结果如图 1所示， 将其与反应液按每 1ml反应液添加 0. 35mg的酶量进行 反应。 反应条件为 30°C， pH为 6. 0， 反应液成分为 10ml体系中加入 40%葡萄糖， 500 μ 1叠 氮化钠， lmL的 pH6. 0的 50mM磷酸氢二钠 /磷酸二氢钠缓冲溶液。 反应 10小时后， 样品沸 水浴 lOmin灭酶活。 然后通过 HPLC和薄层层析（图 2)鉴定产物浓度和种类。 其氨基酸序列 如 SEQ ID NO. 1所示。
实施例 2：
将纯化出来的 β -葡萄糖苷酶和野生型 β -葡萄糖苷酶分别取 ΙΟΟμΙ加入到含 5mM 的 p-nitrophenyl glucoside的 50mM的磷酸氢二钠 /憐酸二氢钠缓冲溶液中 (；加入 10%甘油）， pH 为 7.4，温度为 30°C，反应 30分钟。用 10%碳酸氢钠 150μ1终止反应。取适量的体积在 420nm 光波长处测定 OD值，得出其水解酶活。使用 Bradford试剂盒（上海生工生物工程 SK3041-1 ) 测定其蛋白质浓度。酶活定义如下： 每分钟转化产生 ImM pNP为一个酶活单位 (U)。 结果如 表 1所示。
表 1
Figure imgf000006_0001
实施例 3：
将实例 2中的条件进行优化，使转糖基活性达到较高水平.分别设定不同的温度， pH值优 化本发明 β -葡萄糖苷酶的转糖基作用.通过实验得出在条件为 ΡΗ5. 0, 温度为 30度时， 转 糖基活性最高，并在 72小时左右达到最高值.在此条件下分别以 60%， 70%, 80%葡萄糖浓度 为底物， 得到 72小时的最高点， 如表 2所示。
表 2
Figure imgf000006_0002
实施例 4：
将纯化后目的蛋白与反应液按每 1ml反应液添加 0. 35mg的酶量进行反应。 反应条件为 30°C， pH为 6. 0，反应液成分为 10ml体系中加入 40%葡萄糖， 500 1叠氮化钠， lmL的 pH6. 0 的 50mM磷酸氢二钠 /磷酸二氢钠缓冲溶液。 反应阶段定时取样， 样品沸水浴 lOmin灭酶活。 结果如表 3。
表 3
Figure imgf000007_0001

Claims

权 利 要 求 书
1、 一种 β _葡萄糖苷酶， 氨基酸序列如 SEQ ID NO. 1所示。
2、 一种 β _葡萄糖苷酶的表达基因， 核苷酸序列如 SEQ ID NO. 2所示。
3、 一种重组表达载体， 是将如 SEQ ID NO. 1所示核苷酸序列插入表达载体获得。
4、 如权利要求 3所述的重组表达载体， 其特征在于， 所述表达载体为 PET-32A表达载 体。
5、 一种重组细胞， 是将权利要求 3或 4所述的重组表达载体转化入细胞中获得。
6、 如权利要求 5所述的重组细胞， 其特征在于， 所述细胞为大肠杆菌 BL21 (DE3)。
7、 一种利用 β -葡萄糖苷酶生产寡糖的方法， 其特征在于， 步骤如下：
将核苷酸序列如 SEQ ID NO. 2所示的基因片段导入到 PET-32A质粒载体中， 然后转入大 肠杆菌 BL21 (DE3)中， 通过亲和层析色谱分离纯化制得 β _葡萄糖苷酶， 然后将 β _葡萄糖苷 酶加入以葡萄糖为底物的反应液中， 在温度为 30度， pH为 6. 0的条件下反应， 获得寡糖。
PCT/CN2014/073502 2013-04-11 2014-03-17 一种β−葡萄糖苷酶及其表达基因与应用 Ceased WO2014166326A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310125106.XA CN103160483B (zh) 2013-04-11 2013-04-11 一种β-葡萄糖苷酶及其表达基因与应用
CN201310125106.X 2013-04-11

Publications (1)

Publication Number Publication Date
WO2014166326A1 true WO2014166326A1 (zh) 2014-10-16

Family

ID=48584018

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/073502 Ceased WO2014166326A1 (zh) 2013-04-11 2014-03-17 一种β−葡萄糖苷酶及其表达基因与应用

Country Status (2)

Country Link
CN (1) CN103160483B (zh)
WO (1) WO2014166326A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11371032B2 (en) 2018-06-05 2022-06-28 Teknologian Tutkimuskeskus Vtt Oy Beta glucosidase with high glucose tolerance, high thermal stability and broad PH activity spectrum

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103160483B (zh) * 2013-04-11 2015-03-18 山东大学 一种β-葡萄糖苷酶及其表达基因与应用
CN104232606B (zh) * 2014-08-29 2017-12-05 山东大学 一种改造的β‑葡萄糖苷酶及其表达基因与应用
CN107988186A (zh) * 2017-12-06 2018-05-04 南京林业大学 一种冷适应性内切β-1,4-葡聚糖酶及其表达基因和应用
CN115772476A (zh) * 2021-09-08 2023-03-10 山东大学 一种丝状真菌重组菌株在纤维素酶生产领域的应用
CN115976074B (zh) * 2022-10-18 2024-09-10 内蒙古农业大学 AsBgluc 1基因在提高植物耐旱性中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7727754B2 (en) * 2001-12-18 2010-06-01 Danisco Us Inc. β-glucosidase 5 (BGL5) compositions
CN102154344A (zh) * 2011-02-09 2011-08-17 天津大学 编码β-葡萄糖苷酶的基因及重组表达载体及重组酿酒酵母表达菌株及应用
CN102220369A (zh) * 2011-05-11 2011-10-19 天津大学 里氏木霉β-葡萄糖苷酶基因BGL1的重组载体、重组菌及在重组菌中的表达
CN103160483A (zh) * 2013-04-11 2013-06-19 山东大学 一种β-葡萄糖苷酶及其表达基因与应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102220302B (zh) * 2011-05-20 2013-08-07 安徽大学 一种β-葡萄糖苷酶突变体、其重组表达质粒及转化的工程菌株
CN102517307B (zh) * 2011-12-29 2013-01-30 山东大学 β-葡萄糖苷酶Mut1b及其表达基因与应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7727754B2 (en) * 2001-12-18 2010-06-01 Danisco Us Inc. β-glucosidase 5 (BGL5) compositions
CN102154344A (zh) * 2011-02-09 2011-08-17 天津大学 编码β-葡萄糖苷酶的基因及重组表达载体及重组酿酒酵母表达菌株及应用
CN102220369A (zh) * 2011-05-11 2011-10-19 天津大学 里氏木霉β-葡萄糖苷酶基因BGL1的重组载体、重组菌及在重组菌中的表达
CN103160483A (zh) * 2013-04-11 2013-06-19 山东大学 一种β-葡萄糖苷酶及其表达基因与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FOREMAN P K ET AL.: "Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 278, no. 34, 22 August 2003 (2003-08-22), pages 31988 - 31997 *
ZHANG, LIANG ET AL.: "Cloning and expression of bgll gene from Trichoderma reesei in Saccharomyces cerevisiae", LIQUOR-MAKING SCIENCE & TECHNOLOGY, 18 September 2006 (2006-09-18), pages 17 - 20 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11371032B2 (en) 2018-06-05 2022-06-28 Teknologian Tutkimuskeskus Vtt Oy Beta glucosidase with high glucose tolerance, high thermal stability and broad PH activity spectrum

Also Published As

Publication number Publication date
CN103160483A (zh) 2013-06-19
CN103160483B (zh) 2015-03-18

Similar Documents

Publication Publication Date Title
CN106414728B (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
CN107177612B (zh) 一种外切型褐藻胶裂解酶、基因及其应用
WO2014166326A1 (zh) 一种β−葡萄糖苷酶及其表达基因与应用
CN106884025B (zh) 一种酶法水解定向制备褐藻胶寡糖的方法
CN109312375B (zh) 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶
CN108342374B (zh) 一种甲壳素酶及其应用
CN108949852B (zh) 一种利用全细胞催化制备木糖醇的方法
CN106282211A (zh) 一种重组大肠杆菌全细胞转化合成d‑阿洛酮糖的方法
CN104232606B (zh) 一种改造的β‑葡萄糖苷酶及其表达基因与应用
CN107828806A (zh) 一种新型耐葡萄糖的β‑葡萄糖苷酶基因及其应用
CN114410611B (zh) 昆布多糖降解酶OUC-BsLam26及其应用
CN104789539B (zh) 一种海藻糖合酶的突变体及其制备方法和应用
CN102864160A (zh) 一种木聚糖酶基因及表达蛋白和应用
CN104726435A (zh) 一种β-葡萄糖苷酶突变体、其重组表达质粒及转化的工程菌株
CN107142254A (zh) 高葡萄糖耐受性β‑葡萄糖苷酶突变体及其基因和应用
CN103114099A (zh) 编码糖基水解酶家族1的β-葡萄糖苷酶基因及其应用
CN105695552A (zh) 一种酶法制备稀有人参皂苷Rh1的方法
CN114164224A (zh) 一种低温脱苦酶的制备方法
CN114317509A (zh) 一种纤维二糖差向异构酶突变体及其应用
CN102392036A (zh) β-1,4-内切甘露聚糖酶(Tvi Man5A)基因的克隆及重组酶的制备
CN104087604A (zh) 一种菊糖果糖转移酶基因表达序列
CN117187216A (zh) 一种耐热α-L-鼠李糖苷酶及其耐糖突变体和应用
CN112442488A (zh) 一种溶解性多糖单加氧酶突变体及其应用
CN104651337A (zh) 一种木聚糖酶、其编码基因xyn-lxy及其应用
CN103290039B (zh) 一种来自动物粪便宏基因组的α-淀粉酶及其基因

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: 14783298

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: 14783298

Country of ref document: EP

Kind code of ref document: A1