Biomaterial Database

Improving the activity and stability of thermolysin by site-directed mutagenesis. 2007

Kiyoshi Yasukawa, and Kuniyo Inouye

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

In previous site-directed mutagenesis study on thermolysin, mutations which increase the catalytic activity or the thermal stability have been identified. In this study, we attempted to generate highly active and stable thermolysin by combining the mutations so far revealed to be effective. Three mutant enzymes, L144S (Leu144 in the central alpha-helix located at the bottom of the active site cleft is replaced with Ser), G8C/N60C/S65P (Gly8, Asn60, and Ser65 in the N-terminal region are replaced with Cys, Cys, and Pro, respectively, to introduce a disulfide bridge between the positions 8 and 60), and G8C/N60C/S65P/L144S, were constructed by site-directed mutagenesis. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM), the k(cat)/K(m) values of L144S and G8C/N60C/S65P/L144S were 5- to 10-fold higher than that of the wild-type enzyme. The rate constants for thermal inactivation at 70 degrees C and 80 degrees C of G8C/N60C/S65P and G8C/N60C/S65P/L144S decreased to 50% of that of the wild-type enzyme. These results indicate that G8C/N60C/S65P/L144S is more active and stable than the wild-type thermolysin. Thermodynamic analysis suggests that the single mutation of Leu144-->Ser and the triple mutation of Gly8-->Cys, Asn60-->Cys, and Ser65-->Pro are independent.

UI	MeSH Term	Description	Entries
D002364	Caseins	A mixture of related phosphoproteins occurring in milk and cheese. The group is characterized as one of the most nutritive milk proteins, containing all of the common amino acids and rich in the essential ones.	alpha-Casein,gamma-Casein,AD beta-Casein,Acetylated, Dephosphorylated beta-Casein,Casein,Casein A,K-Casein,Sodium Caseinate,alpha(S1)-Casein,alpha(S1)-Casein A,alpha(S1)-Casein B,alpha(S1)-Casein C,alpha(S2)-Casein,alpha-Caseins,beta-Casein,beta-Caseins,epsilon-Casein,gamma-Caseins,kappa-Casein,kappa-Caseins,AD beta Casein,Caseinate, Sodium,K Casein,alpha Casein,alpha Caseins,beta Casein,beta Caseins,beta-Casein Acetylated, Dephosphorylated,beta-Casein, AD,epsilon Casein,gamma Casein,gamma Caseins,kappa Casein,kappa Caseins
D002384	Catalysis	The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.	Catalyses
D004789	Enzyme Activation	Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.	Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D004795	Enzyme Stability	The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat.	Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006868	Hydrolysis	The process of cleaving a chemical compound by the addition of a molecule of water.
D001407	Bacillus	A genus of BACILLACEAE that are spore-forming, rod-shaped cells. Most species are saprophytic soil forms with only a few species being pathogenic.	Bacillus bacterium
D013379	Substrate Specificity	A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.	Specificities, Substrate,Specificity, Substrate,Substrate Specificities
D013820	Thermolysin	A thermostable extracellular metalloendopeptidase containing four calcium ions. (Enzyme Nomenclature, 1992) 3.4.24.27.	Thermolysin S
D016297	Mutagenesis, Site-Directed	Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.	Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses