Improving the catalytic activity of Candida antarctica lipase B by circular permutation

被引:104
作者
Qian, Z [1 ]
Lutz, S [1 ]
机构
[1] Emory Univ, Dept Chem, Atlanta, GA 30322 USA
关键词
D O I
10.1021/ja053932h
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Lipases (EC 3.1.1.3) play an important role in asymmetric biocatalysis. Tailoring these enzymes to novel, unnatural substrates is one of the primary challenges of protein engineering. We have used circular permutation, the intramolecular relocation of a protein's N- and C-termini, to explore the effects of altered active site accessibility and protein backbone flexibility on the catalytic performance of lipase B from Candida antarctica (CALB). Our combinatorial approach identified 63 unique functional protein permutants of CALB, and kinetic analysis of selected candidates indicated that a majority of enzyme variants either retained or surpassed wild-type CALB activity on a series of standard substrates. Beyond the potential benefits of these tailor-made lipases as new catalysts for unnatural substrates, our study validates circular permutation as a promising general method for lipase engineering. Copyright © 2005 American Chemical Society.
引用
收藏
页码:13466 / 13467
页数:2
相关论文
共 22 条
[1]   Circular permutation and receptor insertion within green fluorescent proteins [J].
Baird, GS ;
Zacharias, DA ;
Tsien, RY .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (20) :11241-11246
[2]   Random circular permutation leading to chain disruption within and near α helices in the catalytic chains of aspartate transcarbamoylase:: Effects on assembly, stability, and function [J].
Beernink, PT ;
Yang, YR ;
Graf, R ;
King, DS ;
Shah, SS ;
Schachman, HK .
PROTEIN SCIENCE, 2001, 10 (03) :528-537
[3]   Controlling lipase enantioselectivity for organic synthesis [J].
Berglund, P .
BIOMOLECULAR ENGINEERING, 2001, 18 (01) :13-22
[4]  
BORNSCHEUER B, 1999, HYDROLASES ORGANIC S
[5]   Optimizing lipases and related enzymes for efficient application [J].
Bornscheuer, UT ;
Bessler, C ;
Srinivas, R ;
Krishna, SH .
TRENDS IN BIOTECHNOLOGY, 2002, 20 (10) :433-437
[6]   Enzymatic resolution of (RS)-p-hydroxy selenides in organic media [J].
Costa, CE ;
Clososki, GC ;
Barchesi, HB ;
Zanotto, SP ;
Nascimento, MG ;
Comasseto, JV .
TETRAHEDRON-ASYMMETRY, 2004, 15 (24) :3945-3954
[7]  
Freemantle M, 2004, CHEM ENG NEWS, V82, P25, DOI 10.1021/cen-v082n006.p025
[8]   Random circular permutation of genes and expressed polypeptide chains: Application of the method to the catalytic chains of aspartate transcarbamoylase [J].
Graf, R ;
Schachman, HK .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (21) :11591-11596
[9]  
Henke E, 2002, ANGEW CHEM INT EDIT, V41, P3211, DOI 10.1002/1521-3773(20020902)41:17<3211::AID-ANIE3211>3.0.CO
[10]  
2-U