Biochemical Characterization Of Chitinases From Paenibacillus Barengoltzii And Rhizomucor Miehei

Chitinase has been widely concerned because of its application in biological control,biomass conversion and functional oligosaccharide production and so on.Paenibacillus barengoltzii is a thermostable bacteria from ocean,it can secrete a variety of extracellular chitinases using chitin as the inducer.This paper primarily studied the screening and identification of producing chitinase microorganisms,optimization of fermentation conditions,purification and characterization of wild chitinase.Besides,the gene clonging,expression and characterization of two recombinant chitinases were also discusssed,respectively.The main results were as follows:（1）A chitinase producing marine bacterium CAU904 was isolated from the South China Sea.The strain was identified as Paenibacillus barengoltzii based on its morphological characters and 16S rDNA sequence.The fermentation conditions for chitinase production by strain CAU94 were optimized using the one-factor-at-a-time method,and the optimal fermentation conditions were obtained as follows:0.5%colloidal chitin,0.2%yeast extract,0.1%Tween 80（medium pH 7.0）45 oC for 72 h.Under the optimized fermentation conditions,the highest chitinase activity of 8.2 U mL-1 was achieved,which was 5.4 folds higher than that obtained before.The chitinase zymogram analysis showed that at least 11 chitinases were secreted into the fermentation broth.A novel chitinase PbChi67 from Paenibacillus barengoltzii was purified and biochemically characterized.The enzyme belongs to GH family 18 with a molecular massof 67.0 kDa。It exhibited optimal activity at pH 3.5 and 60℃.It was stable in the pH range 3.0-9.0 and thermostable up to 65℃.It could hydrolyze chitinous substrate to N-acetylglucosamine（GlcNAc）and N-acetyl chitobiose（GlcNAc）2.PbChi67 was considered to be an endo-chitinase by its hydrolysis characterization.（2）A novel chitinase gene（PbChi74）from Paenibacillus barengoltzii was cloned and heterologously expressedin Escherichia coli as an intracellular soluble protein.The gene has an open reading frame（ORF）of 2,163 bp encoding 720 amino acids.The recombinant chitinase（PbChi74）was purified to apparent homogeneity with a purification fold of 2.2 and a recovery yield of 57.9%.The molecular mass of the purified enzyme was estimated to be 74.6 kDa and 74.3 kDa by SDS-PAGE and gel filtration,respectively.PbChi74 displayed an acidic pH optimum of 4.5 and a temperature optimum of 65℃.The enzyme showed high activity toward colloidal chitin,glycol chitin,N-acetyl chitooligosaccharides,and p-nitrophenyl N-acetyl β-glucosaminide.PbChi74 hydrolyzed colloidal chitin to yield（GlcNAc）2 at the initial stage,which was further converted to its monomer N-acetylglucosamine GlcNAc,suggesting that it is an exochitinase withβ-N-acetylglucosaminidase activity.The purified PbChi74 coupled with RmNAG（β-N-acetylglucosaminidase from Rhizomucor miehei）was used to convert colloidal chitin to GlcNAc,and GlcNAc was the sole end product at a concentration of 27.8 mg mL-1 with a conversion yield of 92.6%.These results suggest that PbChi74 may have great potential in chitin conversion.（3）A novel chitinase gene was cloned from the thermophilic fungus R.miehei.It has an open reading frame（ORF）of 1,284 bp,encoding 427 amino acids.The deduced amino acid sequence of RmChi44 showed the highest identity of 50%with a glycoside hydrolase（GH）family 18 chitinase from Metarhizium acridum（M.a.XP₀07810721）.RmChi44 was cloned and successfully expressed in Escherichia coli as an active chitinase.The recombinant enzyme was purified to homogeneity with a purification fold of 3.3 and recovery yield of 40.9%by Ni-IDA chromatography.The molecular mass of the purified enzyme was estimated to be 44.6 kDa by SDS-PAGE and 44.7 kDa by gel filtration separately,suggesting the enzyme was a monomer.The optimal pH and temperature of the purified enzyme were pH 4.5 and 50 ℃,respectively.RmChi44 exhibited a broad range of substrate specificity,as it can catalyze the cleavage of colloidal chitin and glycol chitin.Besides,RmChi44 also exhibit great antifungal activity against tested pathogenic fungi,such as Colletotrichum sp.,Fusarium oxysporum,Fusarium solani and Trichoderma viride.The excellent properties may make this enzyme an appropriate candidate in biological control application.