Protein engineering of bacterial chitinase for N-acetylchitooligosaccharide production

Abstract

In this study, genetic engineering and protein engineering techniques were used to modify bacterial chitinase for the production of N-acetylglucosamine and/or N-acetylchitooligosaccharide. Beta-chitin and colloidal chitin were used as substrate for sugar production by the chitinase derivatives. Bacillus licheniformis and Serrtia sp. chitinases were selected for studying of the oligosaccharide production. Chitinase A1 (ChiA1), chitinase 60 (Chi60) and chitinase B (ChiB) were cloned, expressed, partially purified and studied. ChiA1, Chi60 and ChiB have both of exo- and endo-type modes for hydrolysis depending on the type of the substrate. The different hydrolysis mode of enzymatic hydrolysis can be used to prepare NAG or N-acetylchitooligosaccharide. Exo-chitinase can be use for the production of N-acetylchitobiose while endo-chitinase can be used for the production N-acetylchitooligosaccharide. The engineered chitinase with deletion and insertion of the accessory domains in ChiA1 and Chi60 was constructed and characterized. The chitinase derivatives with high endo-type activity should be a good candidate for oligosaccharide production. The homologous recombination derivatives between chitinase 60 and chitinase B was constructed and characterized. Interestingly, the chimeric chitinase (RecSK-1) can hydrolyze pNP-NAG [subscript 2] and NAG [subscript 2] to produce monomer of NAG. In addition, the chimeric enzyme can be useful for the preparation of N-acetyl-D-glucosamine from chitin. Chi60 mutant, Chi60W33F/W245F, was used to prepare N- N'-diacetylchitobiose from beta-chitin and the product was separated by dialysis. The amounts of chitobiose from dialysis method yielded 57% (w/w) purity. After 6 days of incubation, the HPLC yields of NAG and NAG [subscript 2] from the hydrolysis of g beta-chitin were 85 mg and 565 mg, respectively, representing 9% and 57% (w/w). In addition, the ChiA1 ChBD was used to prepare oligosaccharide from colloidal chitin. In the condition used, chitotetraose can be prepared.