WO2013177834A1 - 一种海洋细菌适冷蛋白酶及其编码基因和应用 - Google Patents

一种海洋细菌适冷蛋白酶及其编码基因和应用 Download PDF

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WO2013177834A1
WO2013177834A1 PCT/CN2012/077199 CN2012077199W WO2013177834A1 WO 2013177834 A1 WO2013177834 A1 WO 2013177834A1 CN 2012077199 W CN2012077199 W CN 2012077199W WO 2013177834 A1 WO2013177834 A1 WO 2013177834A1
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cold
protease
gene
hspa2
usy
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French (fr)
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张偲
杨键
李洁
麦志茂
田新朋
尹浩
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中国科学院南海海洋研究所
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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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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  • the invention belongs to the field of biotechnology, and particularly relates to a suitable cold protease which can be used for protein hydrolysis and its coding gene and application cloned from marine bacteria HalobaciUiis sp. SCSIO 20089. Background technique:
  • Protease catalyzes the hydrolysis of protein peptide bonds, which accounts for nearly 60% of the industrial enzyme market. It is widely used in washing, leather, food, medicine and other industries. Proteases are widely found in animals, plants and microorganisms, and microbial proteases are the most studied for their large-scale production due to their high viability.
  • the protease secreted by microorganisms in marine sediments plays a key role in the hydrolysis process of marine organic large granular proteins in the nitrogen cycle of the whole ecosystem. Microorganisms that live in seabed sediments all the year round have evolved to secrete at low temperatures in order to adapt to the environment. Under the ability to efficiently hydrolyze large molecular protein enzymes in vitro, marine sediments are an excellent resource for excavating microorganisms that produce cold proteases. Summary of the invention:
  • a first object of the present invention is to provide a novel cold protease and its encoding gene.
  • the cold-stable protease of the present invention is characterized in that the amino acid sequence thereof is shown in SEQ ID N0.2.
  • a gene encoding a suitable cold protease of the present invention which is characterized by a gene encoding a cold-stable protease having an amino acid sequence as shown in SEQ ID NO:
  • the gene encoding the cold protease is preferably, and the nucleotide sequence thereof is shown in SEQ ID N0.1.
  • a second object of the present invention is to provide an expression vector comprising the above gene encoding a cold protease.
  • a third object of the present invention is to provide a microorganism comprising the above expression vector.
  • a fourth object of the invention is to provide the use of a cold protease in proteolysis.
  • the invention utilizes the degenerate primer PCR, TAIL-PCR to obtain a suitable cold protease gene hspal encoding a suitable cold protease from the marine bacterial Halobacillus sp. SCSIO 20089 genomic DNA, and the nucleotide sequence thereof is shown in SEQ ID N0.1.
  • the composition of 1590 nucleotides, blastn results show that the gene sequence has no significant similarity to the published genes in the database.
  • the encoded cold-stable protease HSPA2 is predicted to have an amino acid sequence as shown in SEQ ID N0.2 and consists of 529 amino acids.
  • GenBank database which has the highest similarity to the catalytic domain of HSPA2, is the M4 family protease BWMP (serial number BAD13318) of Bacillus vietnamensis with a consensus of 54%.
  • the component structure of the cold-stable protease HSPA2 derived from the marine bacterium Halobacillus sp. SCSIO 20089 was analyzed using the component structure research tool Pfam (http://pfam.sanger.ac.uk/search), and the results were shown from the 1st to the 25th positions of the N-terminus.
  • the amino acid is a signal peptide, and the amino acid at positions 75 to 125 from the N-terminus is the Fungalysin region of the Thermolysin protease leader peptide.
  • the amino acid at positions 136-221 from the N-terminus is the YPEB domain of the protease leader peptide, and the 225-377 from the N-terminus.
  • the amino acid is the catalytic domain sequence of the Thermolysin metalloproteinase, and the amino acid at positions 379-528 from the N-terminus is the alpha helix region of the Thermolysin metalloproteinase.
  • the cold-trypsin gene hspal was ligated to the expression vector pWB980, and then transferred into Bacillus subtilis WB600 to express the recombinant protein HSPA2 for enzyme activity determination and enzymatic properties.
  • the results showed that the expression of the cold-resistant protease gene hspal was cold.
  • the protease HSPA2 is capable of hydrolyzing casein with an enzyme specific activity of 3077 U/mg protein.
  • the enzymatic properties of the cold-stable protease HSPA2 are shown in Fig. 5 and Fig. 6. As can be seen from Fig. 5 and Fig. 6, the cold-stable protease HSPA2 of the present invention also has good enzymatic activity at low temperature, and its optimum effect. The temperature is 35 ° C and the optimum pH is 8.0.
  • the cold-stable protease HSPA2 of the present invention is a novel protease which is also active at a low temperature, and the gene encoding a suitable cold protease gene fepW is also a novel gene.
  • the present invention provides a novel gene-resistant cold protease gene hspal encoding a cold-resistant protease HSPA2 and a cold-stable protease HSPA2 thereof, which has excellent cold-keeping properties and has wide application in protein hydrolysis. .
  • the marine bacterium Hflfobfld/to sp. SCSIO 20089 used in the present invention is isolated and preserved from marine sediments in the laboratory of the inventor, but has been disclosed in the non-patent document UNIPROT database. , Taxon identifier: 1151003, Scientific name Halobacillus sp. SCSIO 20089, Link page: A ⁇ t: ⁇ g ; 3 ⁇ 4 : ⁇ Mimx ⁇ S1Q ⁇ . The applicant guarantees that the marine bacteria Hfltobfld//i « sp. SCSIO 20089 will be provided to the public within 20 years from the date of application. BRIEF DESCRIPTION OF THE DRAWINGS:
  • Figure 1 is an electrophoresis pattern of a PCR product, wherein M is a Marker and 1 is a PCR product;
  • Fig. 2 is an electrophoresis pattern of a plasmid double-digested product, wherein Ml and M2 are Marker, and 1 is a plasmid double-cut product;
  • Fig. 3 is a plate photograph of a cold protease gene hspal expressed in Bacillus subtilis WB600;
  • Figure 4 is a purified electrophoresis map of the cold protease HSPA2, wherein 1 is Marker, 2 and 3 are purified cold protease HSPA2 pure enzyme solution;
  • Figure 5 is a measurement of the optimum temperature of the cold protease HSPA2
  • FIG. 6 shows the optimum pH of the cold protease HSPA2.
  • Materials used in the examples of the present invention include: Bacillus subtilis WB600, expression vector pWB 980 is a commercially available product, LA taq enzyme, pMD 18-T vector, restriction endonuclease was purchased from TaKaRa, and the gel recovery kit was used. Purchased from OMEGA.
  • 20089thermysin-antisense CGCGGAGTTGGCGCCRTANAR TC , with 20089thermysin-sense, 20089thermysin-antisense for bow
  • SCSIO 20089 genomic DNA was used as a template for PCR amplification.
  • the PCR reaction conditions were 94 °C, 5 min; 94 °C, lmin, 55 °C, lmin, 72 °C, 2 min, 30 cycles; 72 °C, extension for 10 min.
  • a fragment of about 400 bp was obtained, and the gel was recovered and purified, and ligated into the pMD18-T vector for sequencing.
  • the homologous alignment revealed that the 437 bp sequence was highly similar to the neutral protease of Bacillus in Genebank.
  • AD-1 5 ' -ACGATGGACTCCAGAGVNV NNGGAA-3 ';
  • fragments of about 1500 bp and 300 bp were obtained, ligated into pMD18-T vector, and spliced into the complete chilly protease gene fepW, the nucleotide sequence of the suitable cold protease gene hspal. As shown in SEQ ID N0.1.
  • suitable cold protease gene fepW encoding product cold protease HSPA2 amino acid sequence analysis suitable cold protease gene hspal encoding a protein containing 529 amino acids - a cold protease
  • HSPA2 whose amino acid sequence is shown in SEQ ID N0.2, uses Blast (http://blast.ncbi.nlm.nih.gov/) to search the GenBank database, and the highest similarity to the catalytic domain of the cold-resistant protease HSPA2 is Vietnam.
  • the M4 family protease BWMP (SEQ ID NO: BAD13318) of Bacillus vietnamensis has a consensus of 54%.
  • the component structure of the cold-stable protease HSPA2 derived from the marine bacterium Halobacillus sp. SCSIO 20089 was analyzed using the component structure research tool Pfam (http://pfam.sanger.ac.uk/search), and the results were shown from positions 1-25 of the N-terminus.
  • the amino acid is a signal peptide
  • the amino acid at positions 75-125 from the N-terminus is the sequence of the Firmalysin region of the Thermolysin protease leader peptide.
  • the amino acid at positions 136-221 from the N-terminus is the YPEB domain of the protease leader peptide, and the 225-377 from the N-terminus.
  • the amino acid is the catalytic domain sequence of the Thermolysin metalloproteinase, and the amino acid at positions 379-528 from the N-terminus is the alpha helix region of the Thermolysin metalloproteinase.
  • Hspai- Xhol GCGC CTCGAG GGAAAAAGGAAC AGATGTCC AAAAG , hspai- Pstl: GCGC CTGCAG ATATACTCCTACATTATCCCAA,
  • the cold-protease gene fe/3 ⁇ 4 was amplified by PCR using Halobacillus sp. SCSIO 20089 genomic DNA as template.
  • the PCR reaction procedure was: 95 °C for 5 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1.5 min, 30 cycles; 72 °C 10 min, the PCR product was subjected to agarose electrophoresis.
  • the electrophoresis pattern is shown in Figure 1.
  • the size of the PCR product is basically the same as the size of the suitable cold protease gene fepW.
  • the PCR product is recovered by tapping. Cold protease gene hspal PCR product.
  • the cold-trypsin gene hspal PCR product was digested with the restriction enzymes Pst I and Xho I, and ligated with the expression vector PWB980 digested with Pst I and Xho I.
  • the ligation product is transformed into Bacillus subtilis WB 600 and coated to contain 100 ⁇ ⁇ / ⁇ 1 ampicillin and LB medium plates containing 1% skim milk powder were screened for positive clones on LB medium containing 10 (g/ml ampicillin and 1% skim milk powder)
  • the growing colonies further extract the plasmid DNA, and then double-digested with the endonucleases Pst I and Xho I, and the digested products are subjected to agarose gel electrophoresis.
  • the electrophoresis pattern is shown in Fig. 2, as can be seen from Fig. 2,
  • the size of the double-cut product was basically the same as that of the suitable cold protease gene fepW. Further sequencing analysis showed that the sequence was consistent with the sequence of the suitable cold protease gene hspal, and the correct recombinant plasmid was pWB9m-hspal.
  • Bacillus subtilis WB 600 containing the recombinant plasmid pWB980-fe/3 ⁇ 4 was inoculated onto a LB solid medium plate containing 1% skim milk powder, and cultured at 37 ° C for 1 day to transfer to the empty plasmid pWB980.
  • WB 600 was used as a control.
  • the growth pattern is shown in Fig. 3. It is shown in Fig. 3 that Bdcillus subtilis WB 600 (left panel of Fig. 3) containing the recombinant plasmid pWB980-fe/3 ⁇ 4 is capable of hydrolyzing milk in LB solid medium containing 1% skim milk powder.
  • the protein component forms a clear hydrolyzed circle, but Bacillus subtilis WB 600 (pictured right in Figure 3) transferred to the empty plasmid pWB980 does not.
  • Bacillus subtilis WB 600 pictured right in Figure 3
  • the cold-stable protease HSPA2 encoded by the cold-resistant protease gene hspal can hydrolyze milk. Protein ingredient.
  • a single colony of Bacillus subtilis WB 600 containing the recombinant plasmid pWB980-fe/ ⁇ was inoculated into 5 ml of LB medium containing kanamycin, and cultured overnight at 37 °C with shaking. The overnight culture was transferred to superrich medium at 2% inoculation, and cultured at 30 ° C, shaking at 200 rpm for 40 h. Centrifugation at 9000 rpm for 10 minutes, the supernatant is the crude enzyme solution containing the cold protease HSPA2.
  • the crude enzyme solution was purified by ion exchange chromatography and molecular sieve gel to obtain the electrophoresis cold protease HSPA2 pure enzyme solution.
  • the purification process was referred to the "Protein Technology Manual", and the electrophoresis of the pure enzyme solution is shown in Fig. 4.
  • the ⁇ supernatant was mixed with 500 ⁇ 0.4 mol/1 sodium carbonate solution, then 100 ⁇ Folin reagent was added, and the absorbance at 660 nm was measured after 20 min in a 40 °C water bath. The absorbance value was compared with different levels of tyrosine absorbance standard curve to calculate the enzyme activity.
  • the enzyme specific activity of the cold-stable protease HSPA2 was determined to be 3077 U/mg protein (1 U is defined as the amount of enzyme required to convert 1 ⁇ ⁇ tyrosine per minute under the above conditions).
  • step 7 for the method of measuring enzyme activity, using casein with different pH buffer (pH 3.0-6.5 sodium citrate buffer; pH 7.0-8.0 phosphate buffer; pH 9.0-10.0 glycine-NaOH buffer).
  • the enzyme activity at different pH was determined to determine the optimum pH.
  • the enzyme activity of the cold protease HSPA2 changes with pH as shown in Fig. 6. As can be seen from Fig. 6, the optimum pH of the cold-stable protease HSPA2 of the present invention is 8.0.
  • the cold-stable protease HSPA2 of the present invention is a novel protease, and the gene encoding the cold-proteinase gene hspal is also a novel gene.
  • V3V333IVII JjmVi) ⁇ ⁇ ) VIVW3333V vmvmi) ⁇ 3VVI3V33I3:) : WmV
  • VV3VV3V3IV VVVV3II3I3 ⁇ : W ⁇ VI333I33VV V3VVI33IIV

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Abstract

本发明公开了一种海洋细菌适冷蛋白酶及其编码基因和应用。本发明的适冷蛋白酶，其氨基酸序列如SEQ ID NO.2所示。编码所述的适冷蛋白酶的基因，其核苷酸序列如SEQ ID NO.1所示。本发明提供了一种新的编码适冷蛋白酶HSPA2的基因适冷蛋白酶基因hspa1及其编码的适冷蛋白酶HSPA2，该适冷蛋白酶HSPA2具有优良的适冷性能，在蛋白质的水解中具有广泛的用途。

Description

一种海洋细菌适冷蛋白酶及其编码基因和应用 技术领域：
本发明属于生物技术领域， 具体涉及一种克隆自海洋细菌 HalobaciUiis sp. SCSIO 20089的、 能用于蛋白质水解的适冷蛋白酶及其编码基因和应用。 背景技术：
2010年世界工业酶的市场销售额为 33亿美元， 据估计在未来五年内会以平 均每年 6%的速度增长， 即到 2015年可达到 44亿美元。 适冷酶因其在低温条件 下比中温高温酶具有更高的催化活力，在当今提倡增效节能的背景下越来越受到 研究者的关注。海洋占据了地球表面的 70%，海洋拥有着与陆地迥然不同的生态 环境， 如低温、 高压、 特殊的光照条件等。 据报道在任何纬度下， 距海水表面 1000m 以下的温度不会超过 5 °C ， 这就意味着深海或者海底沉积物环境中的微 生物是产适冷酶的重要来源。
蛋白酶催化蛋白质肽键的水解， 占据了工业酶市场份额的近 60%， 在洗涤、 皮革、食品、医药等行业都有广泛的应用。蛋白酶广泛存在于动植物及微生物中， 其中微生物蛋白酶因活力高易于规模生产被研究得最多。海洋沉积物中的微生物 分泌的蛋白酶在整个生态系统氮循环中对海洋有机大颗粒蛋白的水解过程起着 关键的作用， 常年生活在海底沉积物中的微生物为了适应环境， 进化出能分泌在 低温下高效水解体外大分子蛋白质酶类的能力，因而海洋沉积物是挖掘产适冷蛋 白酶微生物的优良资源。 发明内容：
本发明的第一个的目的是提供一种新的适冷蛋白酶及其编码基因。
本发明的适冷蛋白酶， 其特征在于， 其氨基酸序列如 SEQ ID N0.2所示。 本发明的编码适冷蛋白酶的基因， 其特征在于， 编码氨基酸序列如 SEQ ID N0.2所示的适冷蛋白酶的基因：
所述的编码适冷蛋白酶的基因优选， 其核苷酸序列如 SEQ ID N0.1所示。 本发明的第二个目的是提供一种表达载体，其特征在于， 含有上述编码适冷 蛋白酶的基因。 本发明的第三个目的是提供一种含有上述表达载体的微生物。
本发明的第四个目的是提供适冷蛋白酶在蛋白质水解中的应用。
本发明利用兼并引物 PCR， TAIL-PCR从海洋细菌 Halobacillus sp. SCSIO 20089基因组 DNA中扩增获得编码适冷蛋白酶的适冷蛋白酶基因 hspal ,其核苷 酸序列如 SEQ ID N0.1所示， 共有 1590个核苷酸组成， blastn结果显示该基因 序列与数据库中公布基因无显著相似性。按照密码子原则推测其编码的适冷蛋白 酶 HSPA2,其氨基酸序列如 SEQ ID N0.2所示，由 529个氨基酸组成，使用 Blast (http://blast.ncbi.nlm.nih.gov/)搜索 GenBank数据库，与适冷蛋白酶 HSPA2催化功 能域相似性最高的为越南芽孢杆菌 ( Bacillus vietnamensis ) 的 M4 家族蛋白酶 BWMP (序列号为 BAD13318 ) , 其一致性为 54%。 用组件结构研究工具 Pfam (http://pfam.sanger.ac.uk/search)分析来源于海洋细菌 Halobacillus sp. SCSIO 20089的适冷蛋白酶 HSPA2的组件结构，结果显示自 N端的第 1〜25位氨基酸为 信号肽，自 N端的第 75〜125位氨基酸为 Thermolysin蛋白酶前导肽的 Fungalysin 区序列， 自 N端第 136-221位氨基酸为蛋白酶前导肽的 YPEB功能域， 自 N端 的第 225-377位氨基酸为 Thermolysin金属蛋白酶的催化域序列，自 N端 379-528 位氨基酸为 Thermolysin金属蛋白酶的 alpha螺旋区序列。
将适冷蛋白酶基因 hspal 与表达载体 pWB980连接， 再转入枯草芽孢杆菌 WB600中表达重组产物适冷蛋白酶 HSPA2进行酶活的测定和酶学性质测定， 结 果表明适冷蛋白酶基因 hspal的表达产物适冷蛋白酶 HSPA2能够水解酪蛋白， 其酶比活为 3077 U/mg蛋白。 该适冷蛋白酶 HSPA2的酶学性质如图 5和图 6所 示， 从图 5和图 6可以看出， 本发明的适冷蛋白酶 HSPA2在低温下也具有较好 的酶活性， 其最适作用温度为 35 °C， 最适作用 pH值为 8.0。
综上所示， 本发明的适冷蛋白酶 HSPA2是一种在低温下也具有较好活性的 新的蛋白酶， 其编码基因一适冷蛋白酶基因 fepW也是一种新的基因。
本发明提供了一种新的编码适冷蛋白酶 HSPA2 的基因适冷蛋白酶基因 hspal及其编码的适冷蛋白酶 HSPA2, 该适冷蛋白酶 HSPA2具有优良的适冷性 能， 在蛋白质的水解中具有广泛的用途。
用于本发明的海洋细菌 Hflfobfld/to sp. SCSIO 20089为本发明人所在实验室 从海洋沉积物中分离得到并保藏， 但已经公开于非专利文献一 UNIPROT数据库 中， 分类识别号 （Taxon identifier) : 1151003 , 学名 （ Scientific name ) Halobacillus sp. SCSIO 20089, 链接网页〗 : A^ t:^g;¾ :^Mimx 丄 S1Q ≤。 本申请人 保证自申请日起 20年内向公众提供该海洋细菌 Hfltobfld//i« sp. SCSIO 20089。 附图说明：
图 1为 PCR产物电泳图， 其中 M为 Marker, 1为 PCR产物；
图 2为质粒双酶切产物电泳图，其中 Ml和 M2为 Marker, 1为质粒双酶切产物； 图 3为适冷蛋白酶基因 hspal 在枯草杆菌 WB600中表达的平板照；
图 4为适冷蛋白酶 HSPA2的纯化电泳图， 其中 1为 Marker, 2和 3为纯化后的 适冷蛋白酶 HSPA2纯酶液；
图 5为适冷蛋白酶 HSPA2的最适作用温度的测定；
图 6为适冷蛋白酶 HSPA2的最适作用 pH的测定。 具体实施方式：
以下实施例是对本发明的进一步说明， 而不是对本发明的限制。
在本发明的实施例中所用到的材料包括： Bacillus subtilis WB600、 表达载体 pWB 980为市售产品， LA taq酶， pMD 18-T载体， 限制性内切酶购自 TaKaRa 公司， 胶回收试剂盒购自 OMEGA公司。
实施例 1 :
1、 HfltobfldZ/ s sp. SCSIO 20089基因组 DNA的提取
参考 《分子克隆实验指南》
( 1 ) 培养 5ml海洋细菌 Halobacillus sp. SCSIO 20089的培养物， 取 1ml 的培养物 12000rpm离心 2min， 收集菌体；
( 2 ) 菌体沉淀物中加入 567μ1 TE buffer中，反复吹打使之重悬。加入 30 μΐ 20mg/ml溶菌酶， 37°C水浴 lh;
( 3 ) 加入 300 μΐ 10% SDS和 30 μΐ 20mg/ml 蛋白酶 K， 混匀， 37°C水浴 lh;
( 4 ) 加入 100 μΐ 5Μ NaCl, 充分混匀， 再加入 80 μΐ CTAB/NaCl溶液， 混 匀， 65度水浴 lOmin; ( 5 ) 分别加入等体积酚 /氯仿， 氯仿溶液抽提一次；
( 6) 加入 0.6倍体积异丙醇， 轻轻混合， 12000 rpm离心 5 min， 弃上清， 70%乙醇洗涤两次， 超净台风干；
( 7) 加入 50 μΐ ΤΕ 缓冲液， 4°C过夜溶解 DNA， 由此得到海洋细菌 Halobacillus sp. SCSIO 20089的基因组 DNA， 保存备用。
2、 兼并引物 PCR克隆适冷蛋白酶基因 hspal的部分序列
根据芽孢杆菌属 M4家族蛋白酶保守氨基酸设计两条兼并引物
20089thermysin-sense: C AGATGGTGTACGGCGAYGGNGAYGG；
20089thermysin-antisense： CGCGGAGTTGGCGCCRTANAR TC , 以 20089thermysin-sense， 20089thermysin-antisense为弓 |物， Halobacillus sp.
SCSIO 20089基因组 DNA为模板， PCR扩增。 PCR反应条件为， 94 °C， 5 min; 94 °C， lmin, 55 °C， lmin, 72 °C , 2 min, 30个循环； 72 °C， 延伸 10 min。 获得约 400bp的片段， 胶回收纯化后连接 pMD18-T载体进行测序， 同源比对发 现获得 437bp序列与 Genebank中芽孢杆菌属的中性蛋白酶有较高的相似性。
3、 利用 TAIL-PCR克隆适冷蛋白酶基因 hspal的全基因
根据已获得的 437bp的基因序列分别设计 3条特异性的引物以扩增 5'端序列 (20089M4-5-1： 5 ' -GGCGTTAAATAAAC AGTTAGTGC ACGATAGTA-3 '； 20089M4-5-2： 5 '-TGGTATGGACGCCACCCCAGTC；
20089M4-5-3, 5 '-GGTAGATTCTGATACTCATCCATAGAATCCGG-3 ' ) 和两条 3'端特异性引物
(20089M4-3-1： 5'-GACTGGGGTGGCGTCCATACCA-3'；
20089M4-3-2, 5 '-TACTATCGTGCACTAACTGTTTATTTAACGCC-3 ' )； TAIL-PCR所用的通用引物为：
AD- 1： 5 ' -ACGATGGACTCCAGAGVNV NNGGAA-3 '；
AC1 : 5 '-ACGATGGACTCCAGAG-3 '
经过两组 TAIL-PCR后，分别获得约 1500bp和 300bp的片段，连接 pMD18-T 载体测序， 与前面获得的 437bp片段拼接成完整的适冷蛋白酶基因 fepW， 适冷 蛋白酶基因 hspal的核苷酸序列如 SEQ ID N0.1所示。
4、 适冷蛋白酶基因 fepW的核苷酸序列分析 用 NCBI(National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/)上的软件如 ORF finder, Blast对 DNA序列进行分析。 适冷蛋白酶基因 fepW的开放阅读框由 1590个脱氧核苷酸组成，序列如 SEQ ID NO.l所示， 其中起始密码子为 ATG， 终止密码子为 TAA。 blastn结果显示该基 因序列与数据库中公布基因无显著相似性。
5、适冷蛋白酶基因 fepW的编码产物适冷蛋白酶 HSPA2的氨基酸序列分析 适冷蛋白酶基因 hspal 编码一个含 529 个氨基酸的蛋白质一适冷蛋白酶
HSPA2 ， 其 氨 基 酸 序 列 如 SEQ ID N0.2 所 示 ， 使 用 Blast (http://blast.ncbi.nlm.nih.gov/)搜索 GenBank数据库，与适冷蛋白酶 HSPA2催化功 能域相似性最高的为越南芽孢杆菌 ( Bacillus vietnamensis ) 的 M4 家族蛋白酶 BWMP (序列号为 BAD13318 ), 其一致性为 54%。
用组件结构研究工具 Pfam (http://pfam.sanger.ac.uk/search)分析来源于海洋细 菌 Halobacillus sp. SCSIO 20089的适冷蛋白酶 HSPA2的组件结构， 结果显示自 N端的第 1-25位氨基酸为信号肽， 自 N端的第 75-125位氨基酸为 Thermolysin 蛋白酶前导肽的 Fimgalysin区序列， 自 N端第 136-221位氨基酸为蛋白酶前导肽 的 YPEB功能域， 自 N端的第 225-377位氨基酸为 Thermolysin金属蛋白酶的催 化域序列， 自 N端 379-528位氨基酸为 Thermolysin金属蛋白酶的 alpha螺旋区 序列。
6、 适冷蛋白酶基因 fepW的克隆和表达
使用上下游引物
hspai- Xhol: GCGC CTCGAG GGAAAAAGGAAC AGATGTCC AAAAG , hspai- Pstl: GCGC CTGCAG ATATACTCCTACATTATCCCAA,
以 Halobacillus sp. SCSIO 20089基因组 DNA为模板进行 PCR扩增适冷蛋白 酶基因 fe/¾ ， PCR反应程序为： 95 °C 5 min; 95 °C 30 s, 55 °C 30s, 72 °C 1.5 min, 30个循环； 72 °C 10 min, 将 PCR产物进行琼脂糖电泳， 电泳图如图 1所示， PCR产物的大小与适冷蛋白酶基因 fepW的大小基本一致，割胶回收 PCR 产物，由此得到适冷蛋白酶基因 hspal PCR产物。用限制性内切酶 Pst I和 Xho I 酶切适冷蛋白酶基因 hspal PCR产物后， 与经 Pst I和 Xho I酶切的表达载体 PWB980 进行连接。 将连接产物转化至枯草芽孢杆菌 WB 600 中， 涂布到含 100μ§/ηι1氨苄青霉素和含质量分数 1%脱脂奶粉的 LB培养基平板上筛选阳性克 隆， 将能在含 10(^g/ml氨苄青霉素和含质量分数 1%脱脂奶粉的的 LB培养基上 生长的菌落进一步提取质粒 DNA， 然后再用内切酶 Pst I和 Xho I进行双酶切， 酶切产物进行琼脂糖凝胶电泳， 其电泳图如图 2所示， 由图 2可以看出， 双酶切 产物的大小与适冷蛋白酶基因 fepW的大小基本一致， 进一步测序分析表明， 序 列与适冷蛋白酶基因 hspal 的序列一致， 验证正确后命名重组质粒为 pWB9m-hspal。
将含有重组质粒 pWB980-fe/¾ 的 Bacillus subtilis WB 600点接种到含 1%脱 脂奶粉的 LB固体培养基平板上， 在 37 °C下培养 1天， 以转入空质粒 pWB980 的
Figure imgf000008_0001
WB 600作为对照。 其生长图如图 3所示， 由图 3表明， 含有 重组质粒 pWB980-fe/¾ 的 Bdcillus subtilis WB 600 (图 3的左图） 能够水解含 1%脱脂奶粉的 LB固体培养基中的牛奶中的蛋白成份，形成清晰的水解圈，而转 入空质粒 pWB980的 Bacillus subtilis WB 600 (图 3的右图） 则没有， 由此可见 适冷蛋白酶基因 hspal编码的适冷蛋白酶 HSPA2能够水解牛奶中的蛋白成份。
将含有重组质粒 pWB980-fe/^ 的 Bacillus subtilis WB 600单菌落接种至 5ml 含卡纳霉素的 LB培养基中， 37 °C振荡培养过夜。 以 2%的接种量将过夜培养物 转接至 superrich培养基中， 30°C， 200rpm振荡培养 40h。 9000转 /分钟离心 10 分钟， 上清即为含有适冷蛋白酶 HSPA2的粗酶液。
7、 适冷蛋白酶 HSPA2的酶活测定
粗酶液经离子交换层析与分子筛凝胶纯化后得到电泳适冷蛋白酶 HSPA2纯 酶液， 纯化过程参照 《蛋白质技术手册》， 纯酶液的电泳如图 4所示。 取 300μ1 稀释一定倍数的适冷蛋白酶 HSPA2纯酶液， 加入到 300μ1 2%的酪蛋白溶液中， 35 °C水浴 10分钟后，加入 600μ1 0.4mol/l三氯乙酸溶液，室温静置 20 min, 12000 r/min离心 10 min。取 ΙΟΟμΙ上清与 500 μΐ 0.4 mol/1碳酸钠溶液混合后再加入 100 μΐ Folin试剂， 40 °C水浴 20 min后测定在 660 nm的吸光值。 吸光值测定值与不 同含量的酪氨酸吸光度标准曲线图做比较计算酶活。 测得适冷蛋白酶 HSPA2的 酶比活为 3077 U/mg蛋白 （ 1U定义为在上述条件下每分钟转化生成 1 μ§酪氨酸 所需的酶量）。
8、 蛋白酶 HSPA2酶学性质的测定 参照步骤 7测定酶活的方法， 测定适冷蛋白酶 HSPA2纯酶液在 0〜90°C (间 隔 5 °C ) 下的酶活， 以确定最适作用温度。 适冷蛋白酶 HSPA2的酶活随温度变 化如图 5所示， 从图 5可以看出， 本发明的适冷蛋白酶 HSPA2在低温条件下也 具有活性， 其最适作用温度为 35 °C。
参照步骤 7测定酶活的方法， 以不同 pH的缓冲液 （pH 3.0-6.5柠檬酸钠缓 冲液； pH 7.0-8.0磷酸缓冲液； pH 9.0-10.0 甘氨酸 -NaOH缓冲液）溶解的酪蛋 白为底物， 测定在不同 pH下的酶活， 以确定最适作用 pH。 适冷蛋白酶 HSPA2 的酶活随 pH变化如图 6所示， 从图 6可以看出， 本发明的适冷蛋白酶 HSPA2 的最适作用 pH为 8.0。
综上所述， 本发明的适冷蛋白酶 HSPA2是一种新的蛋白酶， 其编码基因一 适冷蛋白酶基因 hspal也是一种新的基因。
序列表
<110> 中国科学院南海海洋研究所
<120>—种海洋细菌适冷蛋白酶及其编码基因和应用
<160> 2
<210> 1
<211> 1590
<212> DNA
<213> Halobacillus sp. SCSIO 20089
<220>
<221> CDS
<222> ( 1 ) . ..(1590)
<400> 1
ATGAAATGGA AATCGTTTGT TGCATCAGTT GTTTTAGGAA CTAGTTTGGT ' ATCAGGTGGT 60
TCTTTAGTTC ATGCGGAAAA AGGAACAGAT GTCCAAAAGG TGTCAGTAGA AACACCTAAG 120
TTTGTCAAAA AGAATTTAAA AGCATCGAAG GTTCTTTCTG AACAATCTGT GAAAAAGTTT 180
CTGAAAGACA ATGAGTCAAC GCTTAAAGTG AACCCATACT CTCATCTTTC CTTTGTTAAA 240
AAAGAAACAG ATGATTTAGG AATGACCCAT TTTATATTTC AACCAGTCGT ACAAAATGTT 300
CCCATCGCTA ACTCGAAAGT TGTCGTACAT ACGGACAAGA GTGGGAAAGT AACTTCCGTA 360
AATGGTGAGC TGCATCAGGA TGCACCACAA AAAATAAAAC AGAAAAAGAA CGTTAAGAAG 420
AAAGAATCTC TTCAGCTTGC ATGGGATTAC ATTGGCGTGG ATCGTGCAAA AGCTGATAAA 480
AAACAGAAGA GCTTAGAAGG AGAAACATTC AACACTCTAA GTGAAAATAC GGATCTTGTC 540
GTCTATGATG ACAATGGTGA TTATAAGCTT GCCTACCATG TTGAACTGCA ATTTGCAGAA 600
CCGTATCCAG CGAACTGGCA AGTGTATGTA AATGCTGAGA GTGGTAAAAT TATCAAAGCA 660
AATAACCTAG TTGAAGAAGC AACGGGGACA GGAACTGGAG TTCTGGGAGA TACAAAATCT 720
CTTAATACCT ACTATTCTGG TGGCACGTAT TACTTATATG ACATTACGAA ACCAATGAAT 780
GGTGTTATTG AAACGTTTGA TAACAAACAC AATGGCTCTT ATAATCTCCC TGGAACATAT 840
GTAACAGATA CTGGAGATAC TTTCTATAGT GAAAATCAAA AAGCAGCGGT CGATGCTCAT 900
TACTATGCAG GCAAAGTTTA TGACTTCTAT TATAATAATT TTGGCCGGAA TAGTTATGAT 960
GATCAAGGGT CTTCAATCCG TTCAACTGTG CACTATGGAT CTAATTATAA CAACGCTGCG 1020 J3S usv T3JV 3Π usv ujg χτ3Λ ΙΤ3Λ "ΐθ 3qa an 3qa
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Claims

权 利 要 求
、 一种适冷蛋白酶， 其特征在于， 其氨基酸序列如 SEQ ID N0.2所示。
、 一种编码权利要求 1所述的适冷蛋白酶的基因。
、 根据权利要求 2所述的编码适冷蛋白酶的基因， 其特征在于， 其核苷酸序列 如 SEQ ID N0.1所示。
、 一种表达载体， 其特征在于， 含有权利要求 2或 3所述的编码适冷蛋白酶的 、 一种含有权利要求 4所述的表达载体的微生物。
、 权利要求 1所述的适冷蛋白酶在蛋白质水解中的应用,
PCT/CN2012/077199 2012-06-01 2012-06-20 一种海洋细菌适冷蛋白酶及其编码基因和应用 WO2013177834A1 (zh)

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