A carbohydrate binding module-5 is essential for oxidative cleavage of chitin by a multi-modular lytic polysaccharide monooxygenase from Bacillus thuringiensis serovar kurstaki

Manjeet K, Madhuprakash J, Mormann M, Moerschbacher BM, Podile AR

Abstract

Conversion of crystalline chitin to soluble sugar molecules, using lytic polysaccharide mono-oxygenases (LPMOs) has emerged as a new avenue for the production of biofuels. The present study describes the role of accessory domains in a multi-modular LPMO from Bacillus thuringiensis serovar kurstaki (BtLPMO10A). The full length BtLPMO10A (BtLPMO10A-FL) possesses an N-terminal LPMO of AA10 family (BtAA10) and a C-terminal CBM5 (BtCBM5) connected via two fibronectin (Fn) III domains (aligned as AA10-FnIII-FnIII-CBM5 from N- to C-terminus). To determine the role of individual domains, we generated truncation mutants of BtLPMO10A-FL. Substrate binding and kinetic studies revealed that BtCBM5 was involved in increasing binding efficiency of BtAA10 which otherwise has feeble binding towards β-chitin and could not bind to α-chitin. Furthermore, binding assays also indicated that the presence of CBM5 increases the binding efficiency of BtLPMO10A-FL under extreme pH conditions. FnIII domains neither bind nor assist BtLPMO10A-FL in chitin binding and serve as linkers in BtLPMO10A-FL. BtLPMO10A-FL and BtAA10 generated oxidized chito-oligosaccharides from the insoluble β-chitin substrate. It is concluded that BtCBM5 is responsible for increasing binding efficiency of BtLPMO10A-FL, whereas; BtAA10 domain is accountable for oxidative cleavage of recalcitrant chitin.