Analysis Of Functions And Properties Of Chitinase From Bacillus Clausii And Study On Its Application

Chitin is a kind of polymer mainly extracted from Marine industrial waste.It is insoluble in water,organic solvents,diluted acids and so on,which limits its application scope.Chitin can be converted into high value-added products such as chitin oligosaccharides（N-acetyl chitooligosaccharides;N-acetyl COSs）using chitinase.Chitobiose（Glc NAc）₂ and N-acetyl glucosamine（Glc NAc）as the main components of chitosaccharides have been used in a variety of fields,such as as food additives,arthritis treatment and so on.However,currently reported multifunctional chitinases that can efficiently produce both（Glc NAc）₂ and Glc NAc are still limited.Therefore,additional sources of novel chitinase genes remain to be discovered.In this study,the chitinase-producing strain was screened and identified by 16S r DNA.The strain was named Bacillus Clausii TCCC 11004.The CHI gene（chi）of the strain was cloned and the recombinant plasmid PET-Chi was constructed.The length of CHI gene fragment was 1797 bp,encoding 559 amino acids.A novel recombinant chitinase（rCHI）was heterogenically expressed and purified from Escherichia coli BL21（DE3）.The optimum reaction temperature and p H of rCHI were 60°C and 7.0,and the rCHI maintained good stability in a wide p H range（4.0-10.0）and below 60°C.A variety of metal ions,especially Ca²⁺,positively promoted the enzyme activity of rCHI.It can maintain good stability in high Na Cl environment.rCHI is a multifunctional chitinase.rCHI has exochitinase activity（product is（Glc NAc）₂）and N-acetylglucosinase activity（product is Glc NAc and has no hydrolytic activity to（Glc NAc）₂）when using colloidal chitinase as substrate.（Glc NAc）₂ and Glc NAc were simultaneously prepared by hydrolyzing chitin from crab shell waste with rCHI.The maximum yields of（Glc NAc）₂ and Glc NAc were 25.73 mg/m L and 3.25 mg/m L,respectively.Bioinformatics analysis of rCHI substrate binding domain（CBD）showed that it was a novel structure.The CBD structure of rCHI was deleted and replaced.The results showed that the CBD structure played a key role in the catalytic efficiency and salt tolerance of rCHI.In addition,the change of CBD did not affect the action mode of rCHI on chitin and did not change the type of hydrolysates.