HUANG Jian-Zhong, E-mail:
SHU Zheng-Yu, E-mail:
芽胞杆菌源枯草杆菌蛋白酶(subtilisin carlsberg)、乙酰基木聚糖酯酶(acetyl xylan esterase)和头孢菌素乙酰水解酶(cephalosporin acetyl hydrolase)具有较高的过水解催化活性,有商业开发价值。
挖掘芽胞杆菌菌株中具有过水解酶催化活性的水解酶蛋白基因,为后续制备过水解酶及酶法合成过氧乙酸奠定基础。
利用定向筛选培养基,从植物根际及纳豆产品中筛选产蛋白酶芽胞杆菌候选菌株,并利用RFLP及16S rRNA基因对其进行鉴定。从蛋白酶高产芽胞杆菌菌株中克隆枯草杆菌蛋白酶、乙酰木聚糖酯酶和头孢菌素乙酰水解酶的全长基因。
从植物根际土壤及纳豆产品中共分离到85个候选菌株,RFLP及16S rRNA基因鉴定结果表明候选菌株均为芽胞杆菌,分别属于
芽胞杆菌源具过水解催化活性水解酶基因的克隆,为后续开发酶法合成过氧乙酸工艺奠定了基础。
Subtilisin carlsberg, acetyl xylan esterase and cephalosporin acetyl hydrolase from
To produce perhydrolase for enzymatic synthesis of peroxyacetic acid in the future, series of hydrolase with promising perhydrolysis activity-encoding genes were cloned from
Protease-producing thermotolerant
Total 85 protease-producing pseudo-
Cloning and expression of hydrolase with perhydrolysis activity-encoding genes from
植物根际通过植物、微生物及土壤的互作形成了一个特殊的微生态环境,植物根际土壤中已分离出真菌、细菌、古细菌、病毒等多种类型的微生物[
α/β水解酶折叠家族酶蛋白(α/β hydrolase fold enzymes)是指酶蛋白的3D分子结构中存在8个平行的β-折叠片层(β-sheet),β-折叠片层之间和周围围绕有系列α-螺旋(α-helix)结构,能催化水解反应(醇解反应或氨解反应)等的一类酶蛋白[
由于过水解催化活性属于α/β水解酶的多功能催化活性,因此挖掘并开发具有较好过水解催化活性的酶蛋白具有重要的意义。已报道的具有良好过水解催化活性(过水解活性/水解活性 > 1)的α/β水解酶有
由于枯草芽胞杆菌分泌的纳豆激酶(nattokinase)与本研究拟克隆的枯草杆菌蛋白酶具有高度的序列及结构一致性(仅少数几个氨基酸差异),因此本文除了主要从植物根际土壤中分离并鉴定芽胞杆菌外,也对部分市售纳豆产品中的芽胞杆菌进行了分离鉴定。本文从植物根际土壤及纳豆产品中共分离获得了85株产蛋白酶芽胞杆菌,基于限制性片段长度多态性(restriction fragment length polymorphism,RFLP)和16S rRNA基因序列进行了初步分类,同时结合已报道的芽胞杆菌源具较高过水解催化活性的酶蛋白资源(SCP、CAH和AXE),克隆了相应类群芽胞杆菌菌株中的SCP、CAH和AXE蛋白的编码基因,为后续开发利用上述酶蛋白催化合成过氧乙酸奠定了基础。
土壤样品分别采自山东青岛莱西、福建永泰、山西洪洞等8个地区19种植物的根际土壤。对土壤样品进行植物名称结合地域原则进行编号,如从洪洞(Hongtong)地区采集的小麦(
干酪素、三氯乙酸、福林酚、L-酪氨酸等各种化学试剂均为分析纯,购自国药集团化学试剂有限公司。
本实验所用的PCR引物
Pairs of the primers used in this study
Primers name | Primers sequence (5ʹ→3ʹ) | Annealing temperature (℃) | PCR product (bp) | |
27f | AGAGTTTGATCCTGGCTCAG | 54.2 | 51 | 1 514 |
1492r | GGTTACCTTGTTACGACTT | 52.8 | ||
BSS(F) | TGCTTGTGAAGATTTTCAGAGGCAGC | 59.9 | 60 | 1 443 |
BSS(R) | CGAGTCTACGGAAATAGCGAGAGA | 59.4 | ||
AXE(F) | GTCGGTCGTCCTCCTTTATTCGTTTCAT | 60.4 | 60 | 1 341 |
AXE(R) | CGATTCTCGGATTCATCCTTCACATCAT | 58.3 | ||
CAH(F) | ATGGAACTAAGCCGGGAAAGTCTTAAACA | 59.5 | 60 | 1 328 |
CAH(R) | GCACATAAATAAAGCGGACGTCGTAATCA | 58.8 |
蛋白酶定性筛选培养基(g/L):干酪素15.0,琼脂20.0,氯化钠10.0,蛋白胨10.0,牛肉膏3.0,pH 7.0−7.5。
蛋白酶发酵培养基(g/L):豆粕粉3.0,乳糖1.0,MgSO4
芽胞杆菌菌株的分离采用热处理法[
将上述定性筛选获得的产蛋白酶芽胞杆菌菌株接种到LB液体培养基,37 ℃、220 r/min培养8 h。活化后的菌悬液按照5% (体积比)的接种量接入蛋白酶发酵培养基中。装液量为50 mL (250 mL),37 ℃、200 r/min培养56 h。于4 ℃、8 000 r/min离心20 min收集发酵上清液,以备测定蛋白酶活性。
蛋白酶活性的测定方法采用Folin-酚法,参照Huang等[
1个酶活单位(U)定义为:在上述条件下,每分钟释放出1 μmol酪氨酸所需要的酶量。
芽胞杆菌基因组DNA的提取方法参照孙明等[
芽胞杆菌菌株16S rRNA基因的PCR扩增参照Ouattara等[
经电泳检测并纯化后的16S rRNA基因PCR扩增产物分别用3种限制性内切酶
从前述分类获得的每个类群中分别挑选产胞外蛋白酶酶活最高的2个菌株,测定其16S rRNA基因序列,并提交NCBI核苷酸数据库。根据16S rRNA基因序列,利用Mega 5.1 (Neighbor-joining method)对从土壤中分离的芽胞杆菌进行聚类分析。
根据已知
从NCBI数据库中分别检索出不同
根据克隆基因编码的多肽链氨基酸序列,利用Swiss-model进行同源建模;获得的PDB文件利用YASARA软件可视化,并评估前述基于序列比对预测的催化三联体氨基酸残基之间的氢键网络。
由于芽胞杆菌产生的芽胞具有较好的热耐受性[
产蛋白酶菌株在筛选培养基上的菌落形态
Colony morphology of protease-producing bacterial strains
各种根际细菌在产蛋白酶诱导培养基中均可分泌产生胞外蛋白酶(
不同根际细菌菌株产蛋白酶能力的比较
Protease activity of the fermentation broth from bacteria strains isolated from rhizosphere soils
Strains | Protease activity (U/mL) |
BNYT-1 | 2.9±0.2 |
BNYT-2 | 10.5±0.0 |
AGYT-1 | 9.7±1.1 |
FSYT-1 | 9.0±0.2 |
FSYT-2 | 3.2±0.1 |
FSYT-3 | 5.9±1.1 |
TAHD-1 | 12.2±0.6 |
TAHD-2 | 7.2±0.4 |
PSHD-1 | 5.6±0.1 |
PSHD-2 | 0.3±0.1 |
TALX-1 | 2.4±0.0 |
TALX-2 | 3.4±0.1 |
ZMLX-1 | 6.3±0.0 |
ZMLX-2 | 1.7±0.2 |
PPYL-1 | 5.2±6.6 |
PPYL-2 | 1.0±0.2 |
PPYL-3 | 2.1±0.0 |
PPYL-4 | 3.8±0.2 |
PPYL-5 | 3.3±0.2 |
PPYL-6 | 2.3±0.1 |
CCYL-1 | 1.0±0.2 |
CCYL-2 | 6.3±0.2 |
CCYL-3 | 2.5±0.1 |
CCYL-4 | 7.1±0.4 |
FVYL-1 | 4.7±0.1 |
CPGG-1 | 4.5±0.1 |
CPGG-2 | 3.6±0.0 |
CPGG-3 | 7.1±0.4 |
CPGG-4 | 2.3±0.5 |
CPGG-5 | 2.1±0.1 |
CPGG-6 | 4.1±0.2 |
CPGG-7 | 4.1±0.8 |
CPGG-8 | 1.9±0.0 |
AFGG-1 | 0.2±0.2 |
AFGG-2 | 3.9±0.5 |
AFGG-3 | 0.7±0.2 |
ACLJ-2 | 3.7±0.1 |
OSLJ-1 | 3.2±0.1 |
OSLJ-2 | 3.2±0.8 |
OSLJ-3 | 6.8±0.1 |
OSLJ-4 | 4.9±0.2 |
OSLJ-6 | 3.2±0.2 |
OSLJ-7 | 1.2±0.1 |
OSLJ-8 | 4.2±0.3 |
OSLJ-9 | 3.5±0.1 |
OSLJ-10 | 2.0±0.0 |
OSLJ-11 | 2.5±0.3 |
OSLJ-12 | 3.7±0.0 |
OSLJ-13 | 10.4±0.3 |
OSLJ-14 | 3.2±0.3 |
OSLJ-15 | 8.4±0.2 |
OSLJ-16 | 8.3±0.1 |
PALJ-1 | 6.1±0.0 |
PALJ-2 | 4.0±0.5 |
PALJ-3 | 2.7±0.3 |
PALJ-4 | 3.9±0.1 |
PALJ-5 | 2.8±0.3 |
PALJ-6 | 5.9±0.2 |
PALJ-7 | 9.6±0.4 |
BCPH-1 | 10.9±0.1 |
BCPH-2 | 8.4±0.4 |
BCPH-3 | 7.4±0.1 |
BCPH-4 | 3.2±0.3 |
BCPH-5 | 5.1±0.1 |
BCPH-6 | 4.8±0.0 |
BCPH-7 | 7.5±0.3 |
AMPH-1 | 2.5±0.1 |
AMPH-2 | 5.1±0.2 |
SOCY-1 | 2.7±0.1 |
SOCY-2 | 10.7±0.4 |
NSSC-1 | 11.8±0.3 |
NSSC-2 | 9.2±0.1 |
NSSC-3 | 9.9±0.1 |
NSBY-1 | 11.6±0.3 |
NSBY-2 | 18.4±0.1 |
NSBY-3 | 9.5±0.2 |
NSYT-1 | 29.9±1.8 |
NSYT-2 | 45.8±0.7 |
NSYT-3 | 49.9±0.2 |
NSCX-1 | 10.8±0.7 |
NSCX-2 | 4.3±0.3 |
NSCX-3 | 11.8±0.1 |
NSHM-1 | 6.2±0.2 |
NSHM-2 | 5.8±0.0 |
NSHM-3 | 6.5±0.2 |
以产蛋白酶细菌菌株的基因组DNA为模板,以27f和1492r为引物,PCR特异性扩增出一条长度为1 516 bp的DNA片段(
16S rRNA基因PCR扩增产物及其限制性内切酶酶切产物的琼脂糖凝胶电泳图
Gel-electrophoresis of PCR-amplified 16S rRNA gene fragment and the restriction pattern
对经PCR扩增获得的每一株产蛋白酶耐热菌株的16S rRNA基因片段,分别用限制性内切酶
产蛋白酶耐热菌株的RFLP分型
RFLP types of protease-producing pseudo-
Types of RFLP | Strains |
注:下划线菌株为16S rRNA基因测序菌株. | |
H1M1R3 | |
H1M3R2 | |
H1M2R2 | |
H2M2R1 | |
H2M3R2 | |
H1M2R1 | |
H2M1R2 | |
H2M3R1 | |
H1M3R3 | |
H1M2R3 | |
H2M1R1 | |
H2M2R2 |
对经核苷酸测序获得的产蛋白酶菌株16S rRNA基因序列与ATCC标准菌株进行比对和聚类分析。分析结果表明:本次筛选的菌株均为芽胞杆菌(
产蛋白酶菌株的16S rRNA基因序列的聚类分析
Phylogenetic analysis of protease-producing strains using 16S rRNA gene sequences
已有专利及文献表明,
本研究克隆的3种酶蛋白多肽链氨基酸序列的聚类分析
Phylogenetic analysis of amino acid sequences from subtilisin carlsberg, cephalosporin acetyl hydrolase and acetyl xylan esterase, respectively
分别以已结构解析的SCP (PDB数据库:1AF4)、AXE (PDB数据库:2XLB)和CAH (PDB数据库:1ODS)为模板,对从
从
3D structural model and hydrogen-bond network between the residues of the catalytic triad from
根际微生物与植物的互作可有效增强植物抗逆(及抗病)能力,提高植物吸收和利用各类营养物质,促进植物生长。如植物根际的
α/β水解酶折叠家族酶蛋白表现出来的过水解催化活性,属于其多功能催化活性(promiscuous activity),具有重要的开发价值。目前相关的专利及产品均为宝洁、杜邦等国际公司所垄断,因此,开发具有自主知识产权的过水解酶产品具有重要意义。本文从筛选到的芽胞杆菌菌株中克隆了不同类群具有过水解催化酶活的系列候选基因,候选基因后续的重组表达实验结果表明,从
本研究结果将为后续深入研究α/β水解酶过水解催化活性的催化机理、相关酶制剂的制备开发等奠定基础。
Dessaux Y, Grandclément C, Faure D. Engineering the rhizosphere[J]. Trends in Plant Science, 2016, 21(3): 266-278
Aloo BN, Makumba BA, Mbega ER. The potential of Bacilli rhizobacteria for sustainable crop production and environmental sustainability[J]. Microbiological Research, 2019, 219: 26-39
Ma XM, Zarebanadkouki M, Kuzyakov Y, et al. Spatial patterns of enzyme activities in the rhizosphere: effects of root hairs and root radius[J]. Soil Biology and Biochemistry, 2018, 118: 69-78
Achari GA, Ramesh R. Characterization of quorum quenching enzymes from endophytic and rhizosphere colonizing bacteria[J]. Biocatalysis and Agricultural Biotechnology, 2018, 13: 20-24
Wierzbicka-Woś A, Henneberger R, Batista-García RA, et al. Biochemical characterization of a novel monospecific endo-β-1, 4-glucanase belonging to GH family 5 from a rhizosphere metagenomic library[J]. Frontiers in Microbiology, 2019, 10: 1342
Jochens H, Hesseler M, Stiba K, et al. Protein engineering of α/β-hydrolase fold enzymes[J]. Chembiochem, 2011, 12(10): 1508-1517
Jones BJ, Lim HY, Huang J, et al. Comparison of five protein engineering strategies for stabilizing an α/β-hydrolase[J]. Biochemistry, 2017, 56(50): 6521-6532
Rauwerdink A, Kazlauskas RJ. How the same core catalytic machinery catalyzes 17 different reactions: the serine-histidine-aspartate catalytic triad of α/β-hydrolase fold enzymes[J]. ACS Catalysis, 2015, 5(10): 6153-6176
Yin DT, Kazlauskas RJ. Revised molecular basis of the promiscuous carboxylic acid perhydrolase activity in serine hydrolases[J]. Chemistry, 2012, 18(26): 8130-8139
Carboni-Oerlemans C, Domínguez de María P, Tuin B, et al. Hydrolase-catalysed synthesis of peroxycarboxylic acids: biocatalytic promiscuity for practical applications[J]. Journal of Biotechnology, 2006, 126(2): 140-151
Tao WY, Xu Q, Huang H, et al. Efficient production of peracetic acid in aqueous solution with cephalosporin-deacetylating acetyl xylan esterase from
Despotovic D, Vojcic L, Blanusa M, et al. Redirecting catalysis from proteolysis to perhydrolysis in subtilisin carlsberg[J]. Journal of Biotechnology, 2013, 167(3): 279-286
Mathews I, Soltis M, Saldajeno M, et al. Structure of a novel enzyme that catalyzes acyl transfer to alcohols in aqueous conditions[J]. Biochemistry, 2007, 46(31): 8969-8979
Földes T, Bánhegyi I, Herpai Z, et al. Isolation of
Kasana RC, Salwan R, Yadav SK. Microbial proteases: detection, production, and genetic improvement[J]. Critical Reviews in Microbiology, 2011, 37(3): 262-276
Huang Q, Peng Y, Li X, et al. Purification and characterization of an extracellular alkaline serine protease with dehairing function from
Chen XL, Zhang YZ, Gao PJ, et al. Two different proteases produced by a deep-sea psychrotrophic bacterial strain,
Sun M, Zhu CG, Yu ZN. Cloning of parasporal body protein gene resembling to S-layer protein genes from
孙明, 朱晨光, 喻子牛.类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆[J].微生物学报, 2001, 41(2): 141-147
Ouattara HG, Reverchon S, Niamke SL, et al. Molecular identification and pectate lyase production by
Gorlach-Lira K, Coutinho HDM. Population dynamics and extracellular enzymes activity of mesophilic and thermophilic bacteria isolated from semi-arid soil of Northeastern Brazil[J]. Brazilian Journal of Microbiology, 2007, 38(1): 135-141
Santana MM, Portillo MC, Gonzalez JM, et al. Characterization of new soil thermophilic bacteria potentially involved in soil fertilization[J]. Journal of Plant Nutrition and Soil Science, 2013, 176(1): 47-56
Santana MM, Gonzalez JM. High temperature microbial activity in upper soil layers[J]. FEMS Microbiology Letters, 2015, 362(22): fnv182
Mhatre PH, Karthik C, Kadirvelu K, et al. Plant growth promoting rhizobacteria (PGPR): a potential alternative tool for nematodes bio-control[J]. Biocatalysis and Agricultural Biotechnology, 2019, 17: 119-128
de Mandal S, Singh SS, Kumar NS. Analyzing plant growth promoting
Gianfreda L. Enzymes of importance to rhizosphere processes[J]. Journal of Soil Science and Plant Nutrition, 2015, 15(2): 283-306
Nannipieri P, Giagnoni L, Renella G, et al. Soil enzymology: classical and molecular approaches[J]. Biology and Fertility of Soils, 2012, 48(7): 743-762
Shu ZY, Lin RF, Jiang H, et al. A rapid and efficient method for directed screening of lipase-producing
舒正玉, 林瑞凤, 江欢, 等.从植物根际定向批量筛选广谱有机溶剂耐受性脂肪酶产生菌[J].微生物学通报, 2009, 36(6): 809-814
Waghmare SR, Gurav AA, Mali SA, et al. Purification and characterization of novel organic solvent tolerant 98 kDa alkaline protease from isolated