Characterization of a Recombinant Glutaminase-Free L-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis

被引：19
|
作者：
Sudhir, Ankit P. [1 ]
Dave, Bhaumik R. [1 ]
Prajapati, Anil S. [1 ]
Panchal, Ketankumar [1 ]
Patel, Darshan [2 ]
Subramanian, R. B. [1 ]
机构：
[1] Sardar Patel Univ, BRD Sch Biosci, Vallabh Vidyanagar 388120, Gujarat, India
[2] Charotar Univ Sci & Technol CHARUSAT Changa, PD Patel Inst Appl Sci, Anand, Gujarat, India
来源：
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY | 2014年 / 174卷 / 07期
关键词：
Bacillus licheniformis; ansA1; ansA3; Cloning; Overexpression; Purification; Characterization; ESCHERICHIA-COLI-ASPARAGINASE; CAROTOVORA L-ASPARAGINASE; ERWINIA-CAROTOVORA; SUBSTRATE-SPECIFICITY; PURIFICATION; ACRYLAMIDE; EXPRESSION; AROIDEAE; LEUKEMIA; CLONING;
D O I：
10.1007/s12010-014-1200-z
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of similar to 37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 degrees C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K-m, V-max, k(cat), and k(cat)/K-m were calculated for recombinant ansA3.
引用
收藏
页码：2504 / 2515
页数：12
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