Cloning, Expression And Site-directed Mutagenesis Of Chitosanase From Aspergillus Sp. CJ22-326

Chitosanases hydrolyze chitosan into chitooligosaccharides, which have various applications in many areas. However, the activity of microorganism chitosanase was low according to the reports. Our lab got a strain Aspergillus sp. CJ22-326 from the sea mud, which could produce endo-chitosanase (CSN) and exo-β-D-glucosaminidase (CSX). In order to improve the activity of CJ22-326 chitosanase, we cloned and expressed the gene. Then we studied the site-directed mutagenesis of the recombinant enzyme.At first, we identified CJ22-326 with 18s rDNA sequence and the shape. With the universal primers NS1 and NS8, 18s rDNA sequence was amplified by PCR using genome DNA as template. After identification and sequencing of PCR product, we found the 18s rDNA sequence of CJ22-326 was 96% identical to that of Aspergillus niger, Aspergillus proliferans, Penicillium expansum and Penicillium chrysogenum KCTC6052. According to the 18s rDNA sequence and shape of spore, we identified CJ22-326 was a new species of Aspergillus.The cDNA of CJ22-326 endo-chitosanase was amplified by 3'-RACE (rapid amplification of cDNA ends) and 5'-RACE. With a pair of specific primers, the structure gene (717 bp) of CJ22-326 endo-chitosanase was amplified and registered in the GenBank (EU302818). The coding gene of CJ22-326 exo-β-D-glucosaminidase was amplified by RT-PCR and the coding gene sequence (2652 bp) was registered in the GenBank (FJ449570).The structural cDNA encoding endo-chitosanase and exo-β-D-glucosaminidase was inserted into the vectors pET-28a(+) respectively. The recombinant pET28a-CSN was introduced into host Escherichia coli BL21(DE3)pLysS. After induced by IPTG, endo-chitosanase gene was successfully expressed. The recombinant protein was checked by SDS-PAGE and western-blot method. The result showed that about 29 kDa protein has been expressed in the host. The optimization of expression of recombinant endo-chitosanase showed that the best condition for expression was 37°C and 1mM IPTG. And induction of exo-β-D-glucosaminidase (100 kDa) was detected by SDS-PAGE and western-blot after pET28a-CSX was introduced into E. coli BL21(DE3)pLysS. After purifying by Ni-NTA affinity chromatography respectively, there were 2.3 mg/L of endo-chitosanse and 1.2 mg/L of exo-β-D-glucosaminidase. The activity of endo-chitosanse and exo-β-D-glucosaminidase measured by DNS method was 1.18 U/mL and 6.96 U/mL respectively.Study on the viscosity changes of reaction solution and analysis of enzymatic hydrolysis products by TLC showed that CSN was an endo-type cleavage enzyme. It decreased viscosity of reaction solution through hydrolyzingβ-(1, 4) glucosidic bond of chitosan. And CSX was an exo-type amino-glucosidase which mainly cleaved glucosamine residue from non-reducing end of chitosan. Studies on characterization showed optimum pH and temperature of endo-chitosanse are 6 and 65°C, while 4.1 and 50°C for exo-β-D-glucosaminidase. Km and Vmax of endo-chitosanse were 0.826 mg/ml and 0.094 mg/ml·min respectively. Km and Vmax of exo-β-D-glucosaminidase were 0.146 mg/ml·min and 7.758 mg/ml respectively. Conserved Glu and Asp are the candidates of potential essential amino acid of catalytic activity for site-directed mutation. After mutation, Asp160 and Glu169 lost all the activity. Circular dichroism assay showed that the secondary structure of mutant protein (E169Q) was identical with wild-type endo-chitosanase and mutant protein (D160E) that had some activity.