Molecular Characterization And Domain Engineering Of Chitinase Chi3

Chitin is a linear polysaccharide composed of ?(1?4)N-acetylglucosamine(GlcNAc),widely found in the shells of shrimps,crabs,insects,and fungi and the yeast cell walls.It is the most abundant polysaccharide after the biomass cellulose and is synthesized in various forms in excess of 1×1011 tons of chitin per year.However,due to its insolubility in water,acid,alkalih,and organic solvents,millions of tons of chitin are discarded every year,resulting in great pollution and waste.In recent years,chitin oligosaccharides obtained from chitin and its deacetyl derivative chitosan have attracted wide attention because of their solubility in water,anti-tumor,antibacterial activity,immune stimulation,the inducibility of plant defense response and a certain degree of sweetness.Chitinase is able to hydrolyze ?(1?4)glycosidic bond in chitin to produce low molecular oligosaccharides under mild conditions.It is widely distributed in various organisms,including bacteria,fungi,plants,insects and even mammals.Screening enzymes with natural properties and their biochemical and structural characterization are still important because these studies provide us with new valuable information on the structure and function of the enzyme and explore more effective catalysts.In previous studies,we isolated an amylase-producing bacillus B.pseudofirmus 703 from saline-alkali land and performed genome-wide sequencing.Through sequence alignment and search,we found a putative chitin degrading enzyme genes.In this study,we cloned Chi3 gene from B.pseudofirmus 703 and expressed it in E.coli.Sequence analysis showed that the enzyme belongs to the glycoside hydrolase 18 family and is only 52%similar to the reported and characterized glycosidase.The optimum pH and temperature of Chi3 for pNP-(GlcNAc)3 is 7 and 40?.In addition,the enzyme has a wide range of pH stability and retains more than 50%of its activity from 4 to 9.The metal ions Li+,Na+,K+,Ca2+,Mg2+had almost no effect on the activity of Chi3,but Mn2+could significantly increase the activity of Chi3 enzyme.The enzyme has a wide range of substrates and can not only hydrolyze pNP-(GlcNAc)3 but also hydrolyze pNP-(GlcNAc)2,pNP-(GlcNAc),colloidal chitin and chitosan.Chi3 degradation of collide?-chitin resulted in a range of different chitooligosaccharides(GlcNAc)3 and(GlcNAc)4(main product)with a small proption of GlcNAc and,(GlcNAc)2,indicating endo-activity.With the prolongation of the time,the ratio of(GlcNAc)2 and GlcNAc increased until the final product was GlcNAc after6 h hydrolysis,indicating exo-activity.In this study,we compared amino acid sequences in NCBI and found that the N-terminus of chitinase Chi3 is very poorly homologous to the N-terminus of reported chitinase.To explore the role of the N-terminus on the catalysis of Chi3 substrates,the N-terminal of Chi3 was truncated according to the homologous protein structure or the characterize enzyme.The truncation of N-termianl domain didn't affect the binding ability of Chi3 to the substrate.In addition,after truncating 144 amino acids,the activities towards several substrates increased from 24.2 to 50.5%with the hydrolysis patterns remaining unchanged,and they still showed endo-and exo-activity towards chitin.Meantime,compared to the wild type Chi3,the hydrolysis time was shorter from 6 h to 2 h under the the same amount of chitinase.