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Microbial proline 4-hydroxylase screening and gene cloning.
1Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahimachi, Machida, Tokyo 194-8533, Japan.
Applied and Environmental Microbiology
|September 3, 1999
Summary
Researchers identified microbial proline 4-hydroxylases for L-proline biotransformation. A Dactylosporangium sp. strain RH1 gene was cloned and expressed in E. coli, yielding significantly enhanced trans-4-hydroxy-L-proline production.
Area of Science:
- Biotechnology
- Enzymology
- Microbial genetics
Background:
- Microbial proline 4-hydroxylases catalyze the conversion of L-proline to trans-4-hydroxy-L-proline.
- Establishing an industrial biotransformation system requires efficient enzyme sources.
Purpose of the Study:
- To screen for microbial proline 4-hydroxylases for industrial biotransformation.
- To clone and express the gene encoding proline 4-hydroxylase from Dactylosporangium sp. strain RH1.
Main Methods:
- Screening of microbial strains for enzyme activity.
- Partial purification of the enzyme from Dactylosporangium sp. strain RH1.
- Gene cloning using degenerate primers and a genomic library in E. coli.
- Recombinant gene expression in E. coli.
Main Results:
- Enzyme activity detected in eight strains, including Dactylosporangium and Amycolatopsis.
- Proline 4-hydroxylase gene isolated from Dactylosporangium sp. strain RH1.
- Recombinant E. coli expressing the gene showed a 13.6-fold increase in enzyme activity.
- Conserved histidine motif found, despite lack of homology to other dioxygenases.
Conclusions:
- Dactylosporangium sp. strain RH1 harbors a proline 4-hydroxylase gene suitable for biotechnological applications.
- Recombinant expression in E. coli significantly enhances trans-4-hydroxy-L-proline production.
- The identified enzyme represents a novel 2-oxoglutarate-dependent dioxygenase.