Study On Mechanism Of Chitosan Depolymerization By Commercial Lipase

Commercial lipases exhibit non-specific activity on chitosan depolymerization, however, no research on mechanicsm of chitosan deploymerization by lipase has been reported at home and abroad. In this study, characteristics and mechanism of chitosan hydrolysis by a commercial lipase were investigated, which is of important theoretical and academic value in elucidating mechanism of chitosan hydrolysis by non-specific enzymes and of practical significance in instructing industrial enzymatic production of low molecular chitosan and chitooligosaccharides (COS).Characteristics of chitosan hydrolysis by a commercial lipase from Aspergillus oryzae were investigated and hydrolysis products were analyzed. The lipase showed obvious hydrolytic activity on chitosans with different degrees of deacetylation (DD). The optimum pH values of the lipase on chitosans with DD of 64%, 73%, 82%, and 90% were 4.2, 4.4, 4.6, and 5.0, respectively, and optimum temperatures were all 60℃. The hydrolysis reactions of the lipase on different chitosans obeyed Michaelis-Meten equation, and kinetic parameters indicate that chitosans with DD of 73% and 82% were susceptible to be hydrolyzed. Products of chitosan hydrolysis by lipase included chitosan-oligomers with degree of polymerization (DP) 2~6, but the final product was glucosamine (GlcN), indicating some hydrolase with exo-mode action existing in the lipase. Products of hydrolysis of chitosans with different DD catalyzed by the lipase were identical.A hydrolase with chitosanolytic activity but no lipolytic activity was purified from lipase by using a combination of ultrafiltration, DEAE-Sepharose CL-6B ion exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic interaction chromatography, and Sephacryl S-200 gel filtration chromatography. Results of HPLC and SDS-PAGE show that the hydrolase had been purified to homogeneity. Because no other components of the lipase showed chitosanolytic activity during purification process, chitosanolytic activity the lipase exhibited was caused by the purified enzyme. Molecular mass of the purified enzyme estimated by SDS-PAGE and non-reducing SDS-PAGE was about 74 kDa and 130 kDa, respectively, indicating that the enzyme was composed of two identical subunits bound together with disulfide bonds.Amino acids composition of the purified enzyme was analyzed by auto-amino acids analyzer. The results show that concentrations of Asp and Glu were high, those of His, Arg, and Lys were low, while those of sulfur-containing amino acids were extremely low. The N-terminal sequence containing 12 amino acids of the purified enzyme determined by using the Edman degradation technique was Ala– Leu– Arg– Leu– Asn– Ser– Pro– Asn– Asn– Ile– Ala– Val. The similarity of the N-terminal sequence was blasted in the Non-redundant Protein Sequences database using the NCBI Blastp2 program, and it was found that this sequence was similar to that of a protein from Aspergillus oryzae, and the identity of the 12 amino acids was 100%.The purified enzyme was modified by DEPC, NBS, Ch-T, EDAC, PMSF, CHD, NAI, DTNB, and DTT. Modification of the purified enzyme by DEPC shows that 1 mole of histidine residue existed in the active site of the enzyme. Modification by NBS shows that tryptophan residues existed in the active site and there is at least 1 mole of tryptophan residue in the substrate binding site. Results of modification by Ch-T and EDAC indicate that methionine residues and carbosyl groups were essential groups of the purified enzyme. In addition, hydroxyl groups, arginine residues, tyrosine residues, sulfhydryl groups, and disulfide bonds were not essential groups of the purified enzyme.The enzyme showed the optimum action pH value and temperature were 4.6 and 60?C, respectively, and it was stable in pH range of 4.5~9.5 and at temperatures lower than 60?C. Metal ions such as Ni2+, Co2+, and Mn2+ had obvious activation effects on the enzymatic activity, while Fe3+, Sn2+, Pb2+, and Hg2+ inactivated the enzyme, and Na+, K+, Mg2+, Zn2+, and Cd2+ had no obvious effect on the purified enzyme. The enzyme exhibited higher chitosanolytic activity toward chitosans which were 73%, 81% and 82% deacetylated and lower activity to chitosans with DD of 64% and 90%.The action mode of the purified enzyme was studied. TLC method and HPLC method were used to analyze the hydrolysis products of standard compounds (chitosan-oligomers and chitin-oligomers) catalyzed by the purified enzyme. The results show that the purified enzyme acted in an exo-mode and released GlcN residues successively from chitosan-oligomers, which is the characteristic of exo-β-D-glucosaminidase (GlcNase). In addition, the enzyme acted in an exo-mode on and released (GlcNAc)2 successively from chitin– oligomers, which is the characteristic of chitobiosidase. Therefore, the purified enzyme exhibited both GlcNase activity and chitobiosidase activity, and split glycoside bonds between GlcN-GlcN and GlcNAc-GlcNAc.In this study, a hydrolase with chitosanolytic activity but no lipolytic activity was purified from a commercial lipase to homogeneity by using a combination of ion exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The purified enzyme exhibited both GlcNase activity and chitobiosidase activity, therefore, the chitosanolytic activity the lipase exhibited was caused by the purified enzyme.