A highly active and oxidation‐resistant subtilisin‐like enzyme produced by a combination of site‐directed mutagenesis and chemical modification

Abstract

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1 -10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222.It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H 2 0 z revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cy~~~~lsubtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cy~~~~]subtilisin, exhibited a kca,/Km value of 56% ofthat obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-N02 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cy~~~~]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kca,lKm for the hydrolysis of Suc-Ala-AlaPro-Phe-NH-Ph-NO,. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.The subtilisin family is currently being studied in several laboratories by in vitro mutagenesis. Single amino acid substitutions have resulted in enzyme derivatives with altered specificity [l], increased activity [2], altered pH dependence [3], improved alkaline stability [4] and altered stability towards oxidation [5].Sensitivity of subtilisins to oxidation has been reported and chemical analysis as well as X-ray crystallography has revealed that the apparent inactivation can be ascribed to the conversion of the methionyl residue, neighboring the cataCorrespondence to K. Breddam,

Commercial proteases: Present and future

“…Interestingly, under oxidizing conditions of 0.1 to 1.0 M H 2 O 2 , replacement of Met 222 to non-oxidizable amino acids under these conditions, Ala, Ser or Leu, improved the oxidative tolerance of subtilisin. Based on the fact that the high specific activity of subtilisin is linked to the presence of a sulfur atom at position 222, Breddam and co-workers chemically modified an M222C mutant by thiomethylating the cysteine with the thioalkylating reagent, methyl methane thiosulfonate (Me-MTS) [19]. The engineered thiomethylated subtilisin derivative exhibited an extraordinarily enhanced stability and catalysis under oxidative conditions compared to both wild-type and the unmodified M222C variant during treatment with hydrogen peroxide.…”

Synthesis of copper ion incorporated horseradish peroxidase-based hybrid nanoflowers for enhanced catalytic activity and stability

“…Furthermore, the time-consuming and labor-intensive synthetic procedures have limited application of the chemical modification method. [6][7][8] In contrast to chemical modification, immobilization of enzymes in/on solid supports has offered a simple, alternative, generally requiring one-step reaction. In general, immobilized enzymes show improved stability, making them efficient, reusable and economical.…”

Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

“…Previous mutagenesis studies on subtilisin Bacillus amyloliquefaciens (SBA) showed that the methionine sulfur atom next to the catalytic serine (M222 in SBA) was prone to oxidize. The formation of a sulfoxide at this position resulted in enzyme inactivation, probably as a result of destabilization of the transition‐state intermediate . This might be the reason why an inert atmosphere (addition of argon) is required to obtain conversion for the model reaction by using SC.…”

“…The formation of as ulfoxide at this position resulted in enzymei nactivation, probably as ar esult of destabilizationo ft he transition-state intermediate. [44][45][46] This might be the reason why an inert atmosphere (addition of argon) is required to obtain conversion for the model reactionb yu sing SC. Estell et al created 19 variants on position M222 in SBA and evaluated the oxygen resistance.…”