参与芽孢镰刀菌生物防治的解淀粉芽孢杆菌SP1碱性蛋白酶的分子表征。,Internatio
参与芽孢镰刀菌生物防治的解淀粉芽孢杆菌SP1碱性蛋白酶的分子表征。
International Journal of Food Microbiology
(
IF
5.0
)
Pub Date : 2016-06-14
, DOI:
10.1016/j.ijfoodmicro.2016.05.030
Shiwani Guleria
1
,
Abhishek Walia
1
,
Anjali Chauhan
2
,
C K Shirkot
2
Affiliation
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.
从解淀粉芽孢杆菌SP1的基因组DNA中扩增了碱性蛋白酶基因，产后康复13825404095该基因参与了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。5.62倍纯化的酶的特异活性为607.69U / mg，据报道对尖孢镰刀菌的生长抑制率为24.14％。但是，向纯化的酶中添加蛋白酶抑制剂即PMSF（15mM）后，没有发现拮抗活性。蛋白酶基因的1149bp核苷酸序列编码了382个43kDa的氨基酸，计算的等电点为9.29。推导的氨基酸序列的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％）。淀粉芽孢杆菌SP1蛋白酶基因在大肠杆菌BL21中表达。表达的蛋白酶被大肠杆菌分泌到培养基中，并在pH8.0和60°C下表现出最佳活性。使用Phyre 2服务器确定了碱性蛋白酶最可靠的三维结构，该服务器根据Ramachandran图和ERRAT值进行了验证。该酶的表达和结构预测为农业和工业中的商业应用提供了潜在的价值。
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Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum.
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.