Expression And Characterization Of Chitinases From Pig Fecal Metagenomic DNA,Paecilomyces Thermophila And Rhizomucor Miehei

Chitinases have been widely concerned because of its application in biological control,biomass conversion and functional oligosaccharide production and so on.The present study investigated gene cloning,expression and characterization of chitinases from Pig fecal environment DNA,Paecilomyces thermophila J18 and Rhizomucor mieheiCAU432,the main results were as follows:(1)A novel exochitinase gene(Echi47)was directly cloned from the pig fecal environment DNA using the genomic walking PCR technique and expressed in Escherichia coli BL21(DE3).Echi47 has an open reading frame(ORF)of 1,161 bp encoding 386 amino acids.The amino acid sequence of Echi47 showed 36%identity with that of chitinase from Coprinellus cangregatus.The recombinant exochitinase was purified with specific activity toward colloidal chitin of 6.84 U/mg.Echi47 was optimally active at pH 5.0 and 40 ?,respectively.When colloidal chitin was used as substrate,N-acetylchitobiose[(GlcNAc)2]was mostly produced at the initial stage,suggesting that it is an exochitinase.Echi47 exhibited excellent resistance to pepsin,trypsin,proteinase K,and flavor protease.Under simulated alimentary tract conditions,Echi47 was stable and active,releasing 21.1 mg of N-acetylchitooligosaccharides.from 80 mg of colloidal chitin.These properties make Echi47 a potential additive in the food and feed industries.(2)The second chitinase gene(Ptchi45)was cloned from the thermophilic fungus Paecilomyces thermophila J18 and expressed in Pichiapastoris.The gene belonging to glycoside hydrolase(GH)family 18 has an open reading frame(ORF)of 1275 bp encoding 424 amino acids with six introns.The recombinant chitinase(Ptchi45)was secreted at high levels of 126.4 U/ml in high cell density fermentor,which is the highest yield obtained for a chitinase.The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 45 kDa in SDS-PAGE.The enzyme exhibited a high specific activity of 16.1 U/mg.The optimum temperature and pH of Ptchi45 were determined to be 50? and pH 3.5,respectively.It was stable witihin pH 3.5-8.5.Ptchi45 hydrolyzed colloidal chitin to yield N-acetyl chitobiose[(GlcNAc)2]at the initial stage,suggesting that it is an exochitinase.These properties make Ptchi45 useful in the food industries.(3)The chitinase gene(Rmchi48)was cloned from the Rhizomucor miehei CAU432 and expressed in E.coli BL21.The gene belonging to GH 18 has an open reading frame(ORF)of 1849 bp encoding 445amino acids with seven introns.The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 48 kDa in SDS-PAGE.Based on crystal structure of Rmchi48,an unusual long loop,which is linked between C117 and C274,form a bridge streching across the substrate-binding cleft.To understand the loop,the mutants Rchi48-loop and Rchi48-C192/C349 were constructed by site-directed mutagenesis,expressed in E.coli,purified,and characterized.Mutants Rchi48-loop and Rchi48-C192/C349 exhibited a shift in the optimal pH from 5.0 to 6.0 and 5.5,respectively.Rmchi48,Rchi48-loop and Rchi48-C192/C349 were optimally active at 40 ?.The optimum temperature and pH of Ptchi45 were determined to be 50?.Rmchi48,Rchi48-loop and Rchi48-C192/C349 showed half-life of 462.2 min,211.7 min and 214.2 min,recepectly,at 40?.Rmchi48,Rchi48-loop and Rchi48-C192/C349 showed half-life of 167.6 min,34.8 min and 32.4 min,recepectly,at 50?.Rmchi48,Rchi48-loop and Rchi48-C192/C349 showed half-life of 48.0 min,26.2 min and 28.7 min,recepectly,at 60?.Compared with mutants Rchi48-loop and Rchi48-C192/C349,wild-type Rmchi48 showed increased thermal and thermal inactivation.When colloidal chitin was used as substrate,(GlcNAc)2 was mostly produced(74%),small amounts of GlcNAc(6.4%)and(GlcNAc)3(19.6%)were detected.When chitosan was used as substrate,(GlcN)2 was mostly produced(72.2%),small amounts of GlcN(15.3%)and(GlcN)3(12.5%)were detected.