Overexpression, immobilization and biotechnological application of Pseudomonas lipases

被引:68
作者
Reetz, MT
Jaeger, KE [1 ]
机构
[1] Max Planck Inst Kohlenforsch, D-45470 Mulheim, Germany
[2] Ruhr Univ Bochum, Lehrstuhl Biol Mokroorganismen, D-44780 Bochum, Germany
关键词
Pseudomonas lipase; overexpression; lipase-specific foldase; immobilization; in vitro evolution; enantioselectivity;
D O I
10.1016/S0009-3084(98)00033-4
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
Pseudomonas lipases play an important role in biotechnology both as hydrolases for detergent additives and as synthases catalyzing the kinetic resolution of racemic compounds. Large-scale production of Pseudomonas lipases requires correct folding and secretion through the bacterial membranes. Controllable expression of the gene lipH encoding a lipase-specific foldase proves to be important for overexpression in the homologous host Escherichia coli. Construction of appropriate His-tagged fusion proteins permitted overexpression, secretion and one-step purification of lipase from culture supernatants of the homologous host Pseudomonas aeruginosa. The immobilization of lipases in hydrophobic sol-gel materials derived from alkylsilane precursors of the type RSi(OCH3)(3) or mixtures of RSi(OCH3)(3) and Si(OCH3)(4) provides highly active chemically and thermally stable heterogeneous biocatalysts. The entrapped lipases are excellent catalysts in a variety of synthetic organic transformations. Using directed evolution based on error prone PCR, the enantioselectivity of the hydrolysis of a chiral ester, catalyzed by the lipase from P. aeruginosa, can be increased from ee 2 to ee 81% in just four mutagenesis cycles. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.
引用
收藏
页码:3 / 14
页数:12
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