ArticleApplied and environmental microbiology1998
Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system.
Article in Applied and environmental microbiology, 1998. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed.
- Looking Back: A Short History of the Discovery of Enzymes and How They Became Powerful Chemical Tools.ChemCatChem · 2020Review
- Increment in protease activity ofIranian journal of microbiology · 2020Article
- Thirty-degree shift in optimum temperature of a thermophilic lipase by a single-point mutation: effect of serine to threonine mutation on structural flexibility.Molecular and cellular biochemistry · 2017Article
- Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.Frontiers in microbiology · 2016Review
- Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart.Applied and environmental microbiology · 2015Article
- Microbial enzymes: tools for biotechnological processes.Biomolecules · 2014Review
- Cold-adapted proteases as an emerging class of therapeutics.Infectious diseases and therapy · 2013Article
- Biotechnology of cold-active proteases.Biology · 2013Article
- Contributions of microorganisms to industrial biology.Molecular biotechnology · 2008Review
- Protein engineering of a cold-active beta-galactosidase from Arthrobacter sp. SB to increase lactose hydrolysis reveals new sites affecting low temperature activity.Extremophiles : life under extreme conditions · 2006Article
- Cold-active winter rye glucanases with ice-binding capacity.Plant physiology · 2006Article
- Molecular basis of cold adaptation.Philosophical transactions of the Royal Society of London. Series B, Biological sciences · 2002Review
- Streptomyces serine protease (DHP-A) as a new biocatalyst capable of forming chiral intermediates of 1,4-dihydropyridine calcium antagonists.Applied and environmental microbiology · 2002Article
- Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase.Applied and environmental microbiology · 2002Article
- The complete amino acid substitutions at position 131 that are positively involved in cold adaptation of subtilisin BPN'.Applied and environmental microbiology · 2000Article
- Simultaneous enhancement of thermostability and catalytic activity of phospholipase A(1) by evolutionary molecular engineering.Applied and environmental microbiology · 2000Article
- A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85.Applied and environmental microbiology · 1999Article
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46-51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the kcat/Km value) 100% higher than that of the wild type at 10 degrees C when N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased Km value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60 degrees C and circular dichroism spectra at 10 degrees C.
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