耐有机溶剂蛋白酶产生菌MSP05的筛选、鉴定及酶学性质研究
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摘要
目的:从燕尾港受污染海域筛选耐有机溶剂蛋白酶产生菌,确定该菌的分类地位,并对其所产溶剂稳定性蛋白酶的酶学性质进行研究。方法:通过在培养基中添加20%(V/V)甲苯作为筛选压力,筛选耐有机溶剂极端微生物,通过产蛋白酶筛选培养基筛选产蛋白酶的菌株,并利用摇瓶发酵进行复筛,最后对产酶菌株进行鉴定并对粗酶的性质进行测定。结果:筛选获得耐有机溶剂蛋白酶产生菌MSP05,结合16S rDNA序列分析、形态学观察和生理生化性质将其鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。该酶的最适反应温度和pH分别为50℃和10.0,Cu~(2+)、Mn~(2+)对酶有激活作用,Mg~(2+)、Fe~(3+)和Ba~(2+)对酶具有抑制作用。在50%乙醇中,25℃、140 r/min振荡72 h后仍能保持50%以上酶活力。结论:分离获得耐有机溶剂蛋白酶产生菌解淀粉芽孢杆菌菌株MSP05,对有机溶剂具有较好的耐受性,具有潜在的工业应用价值。
Objective:Organic-solvent tolerant protease-producing strains were isolated from polluted area in Yanwei Port,the taxonomic status were clarified,and the enzymatic characteristics of the protease were studied.Methods:Organic-solventtolerant strains were isolated from organic-pollution water by adding 20%(V/V)methylbenzene into medium as selective pressure.Protease-producing strains was obtained through the first screening with selection medium plate.The high-producing strains were re-screened by shake flask fermentation.Then the strains were identified and the enzymatic characterization was measured.Results:MSP05 was obtained and identified as Bacillus amyloliquefaciens by its morphological,physiological,biochemical characteristics and 16S rDNA sequence homology.The optimum pH and temperature values of this protease were pH10 and 50℃,respectively.The enzyme was activated by Cu~(2+),Mn~(2+)and inhibited by Mg~(2+),Fe~(3+)and Ba~(2+).This protease has good organic-solvent tolerant.It remained more than 50%activity after 72 h incnbated with 50%enthol at 25℃and140 r/min.Conclusion:One high-producing organic-solvent tolerant protease producer Bacillus amyloliquefaciens MSP05 was obtained,which would have potential industrial value.
Objective:Organic-solvent tolerant protease-producing strains were isolated from polluted area in Yanwei Port,the taxonomic status were clarified,and the enzymatic characteristics of the protease were studied.Methods:Organic-solventtolerant strains were isolated from organic-pollution water by adding 20%(V/V)methylbenzene into medium as selective pressure.Protease-producing strains was obtained through the first screening with selection medium plate.The high-producing strains were re-screened by shake flask fermentation.Then the strains were identified and the enzymatic characterization was measured.Results:MSP05 was obtained and identified as Bacillus amyloliquefaciens by its morphological,physiological,biochemical characteristics and 16S rDNA sequence homology.The optimum pH and temperature values of this protease were pH10 and 50℃,respectively.The enzyme was activated by Cu~(2+),Mn~(2+)and inhibited by Mg~(2+),Fe~(3+)and Ba~(2+).This protease has good organic-solvent tolerant.It remained more than 50%activity after 72 h incnbated with 50%enthol at 25℃and140 r/min.Conclusion:One high-producing organic-solvent tolerant protease producer Bacillus amyloliquefaciens MSP05 was obtained,which would have potential industrial value.
引文
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[18]Sanatan P T,Lomate P R,Giri A P,et al.Characterization of a chemostable serine alkaline protease from Periplaneta americana[J].Bmc Biochemistry,2013,14(1):1-9.
[2]胡学智,王俊.蛋白酶生产和应用的进展[J].工业微生物,2008,38(4):49-61.
[3]Zahir Z,Seed K D,Dennis J J.Isolation and characterization of novel organic solvent-tolerant bacteria[J].Extremophiles,2006,10(2):129-138.
[4]Priya J D,Divakar K,Prabha M S,et al.Isolation,purification and characterisation of an organic solvent-tolerant Ca2+-dependent protease from Bacillus megaterium AU02[J].Applied Biochemistry and Biotechnology,2014,172(2):910-932.
[5]Bai Y,Huang H,Meng K,et al.Identification of an acidicα-amylase from Alicyclobacillus sp.A4 and assessment of its application in the starch industry[J].Food Chemistry,2012,131(4):1473-1478.
[6]Geok L P,Che N a R,Rahman R N Z A,et al.Isolation and screening of an extracellular organic solvent-tolerant protease producer[J].Biochemical Engineering Journal,2003,13(1):73-77.
[7]Xu J,Jiang M,Sun H,et al.An organic solvent-stable protease from organic solvent-tolerant Bacillus cereus WQ9-2:purification,biochemical properties,and potential application in peptide synthesis[J].Bioresource Technology,2010,101(20):7991-7994.
[8]尹静.沙雷氏菌产耐有机溶剂蛋白酶的研究[D].无锡:江南大学,2011.
[9]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[10]Holt J G,Krieg N R,Pha S,et al.Bergey’s manual o determinative bacteriology 9th Ed[M].Williams and Wilkins company,1994:520-521.
[11]Fang Y W,Shu L,Wang S J,et al.Isolation and screening o a novel extracellular organic solvent-stable protease producer[J].Biochemical Engineering Journal,2009,43(2):212-215.
[12]Ogino H,Watanabe F,Yamada M,et al.Purification and characterization of organic solvent-stable protease from organic solvent-tolerant Pseudomonas aeruginosa PST-01[J].Journal o Bioscience and Bioengineering,1999,87(1):61-68.
[13]Sareen R,Mishra P.Purification and characterization o organic solvent stable protease from Bacillus licheniformis RSP-09-37[J].Applied Microbiology and Biotechnology,2008,79(3):399.
[14]Karan R,Singh S P,Kapoor S,et al.A novel organic solven tolerant protease from a newly isolated Geomicrobium sp.EMB2(MTCC 10310):Production optimization by response surface methodology[J].New Biotechnology,2011,28(2):136-145.
[15]Uttatree S,Charoenpanich J.Isolation and characterization of a broad p H-and temperature-active,solvent and surfactant stable protease from a new strain of Bacillus subtilis[J].Biocatalysis and Agricultural Biotechnology,2016,8:32-38.
[16]Maruthiah T,Immanuel G,Palavesam A.Purification and characterization of halophilic organic solvent tolerant protease from marine Bacillus sp.APCMST-RS7 and its antioxidant potentials[J].Proceedings of the National Academy of Sciences,India Section B:Biological Sciences,2015,87(1):207-216.
[17]Maruthiah T,Somanath B,Immanuel G,et al.Investigation on production and purification of haloalkalophilicorganic solvent tolerant protease from marine shell waste and its bioconversion to chitin by aquatic Bacillus sp.APCMST-CS4[J].Waste and Biomass Valorization,2016,8(3):811-827.
[18]Sanatan P T,Lomate P R,Giri A P,et al.Characterization of a chemostable serine alkaline protease from Periplaneta americana[J].Bmc Biochemistry,2013,14(1):1-9.