科研之友
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摘要
Thermostability is considered a promising characteristic for industrial enzyme applications. Screening of the genomic library of thermophilic G. thermoleovorans YN (ac: AF385083) for esterase/lipase activity has led to the isolation of two positive clones. Sub-cloning and sequence analyses identified two potential genes estB (560 bps, partial sequence) and estC (2022 bps), encoding two different esterases. The complete ORF of estB (1500 bps) was amplified using degenerate primers based on multiple sequence alignment. EstB showed 95% identity to putative carboxyl esterases of G. kaustophilus HTA426 (ac: BAD77334) G. stearothermophilus (ac: AAN81910) and 84% similarity to G. thermodenitrificans NG80 (ac: ABO68347).
On the other hand, ORF of estC (2022 bps), encoded a protein with typical esterase/lipase motif GXSXG with 37% identity to acylpeptide hydrolase/esterase of hyperthermophilic Aeropyrum pernix K1 (ac: BAA80835). In addition, EstC showed high similarity to proteins with esterase and peptidase dual activity. Both, estB and estC were subcloned and efficiently expressed in E. coli DH5 alpha under control of temperature-inducible lambda p L-promoter using the vector pCYTEX-P1. SDS-PAGE of the recombinant E. coli crude cell lysates showed induction of 54.7 kDa and 75.4 kDa proteins corresponding to EstB and EstC respectively. The temperature optima of Est B and EstC were 65 degrees C and 55 degrees C respectively; however, they showed almost quite similar pH optima ranging from 8 to 9, The recombinant proteins (EstB or C) showed a complete stability at 65 degrees C after one hr and pH 10 after 24 hr exposure, moreover detergents addition to the EstB enhanced its activity.
- 出版日期2013-4
