Guleria Shiwani, Walia Abhishek, Chauhan Anjali, Shirkot C K
Department of Microbiology, DAV University, Jalandhar, Punjab144012, India.
Department of Basic Sciences (Microbiology Section), Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan 173230 (H.P.), India.
Int J Food Microbiol. 2016 Sep 2;232:134-43. doi: 10.1016/j.ijfoodmicro.2016.05.030. Epub 2016 Jun 1.
An alkaline protease gene was amplified from genomic DNA of Bacillus amyloliquefaciens SP1 which was involved in effective biocontrol of Fusarium oxysporum. We investigated the antagonistic capacity of protease of B. amyloliquifaciens SP1, under in vitro conditions. The 5.62 fold purified enzyme with specific activity of 607.69U/mg reported 24.14% growth inhibition of F. oxysporum. However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15mM) to purified enzyme. An 1149bp nucleotide sequence of protease gene encoded 382 amino acids of 43kDa and calculated isoelectric point of 9.29. Analysis of deduced amino acid sequence revealed high homology (86%) with subtilisin E of Bacillus subtilis. The B. amyloliquefaciens SP1 protease gene was expressed in Escherichiax coli BL21. The expressed protease was secreted into culture medium by E. coli and exhibited optimum activity at pH8.0 and 60°C. The most reliable three dimensional structure of alkaline protease was determined using Phyre 2 server which was validated on the basis of Ramachandran plot and ERRAT value. The expression and structure prediction of the enzyme offers potential value for commercial application in agriculture and industry.
从解淀粉芽孢杆菌SP1的基因组DNA中扩增出一个碱性蛋白酶基因，该菌株参与对尖孢镰刀菌的有效生物防治。我们在体外条件下研究了解淀粉芽孢杆菌SP1蛋白酶的拮抗能力。纯化了5.62倍、比活性为607.69U/mg的该酶对尖孢镰刀菌的生长抑制率为24.14%。然而，在向纯化酶中添加蛋白酶抑制剂即苯甲基磺酰氟（15mM）后未发现拮抗活性。蛋白酶基因的1149bp核苷酸序列编码43kDa的382个氨基酸，计算得到的等电点为9.29。推导的氨基酸序列分析显示与枯草芽孢杆菌的枯草杆菌蛋白酶E具有高度同源性（86%）。解淀粉芽孢杆菌SP1蛋白酶基因在大肠杆菌BL21中表达。表达的蛋白酶由大肠杆菌分泌到培养基中，在pH8.0和60°C时表现出最佳活性。使用Phyre 2服务器确定了碱性蛋白酶最可靠的三维结构，并根据拉氏图和ERRAT值进行了验证。该酶的表达和结构预测在农业和工业商业应用中具有潜在价值。
