Study On The Purification Of Fraction With The Chitosanastic Activity From Papain

Papain with non-specific action on chitosan depolymerization have been well characterized, but the mechanism of this non-specific catalysis and the action pattern of the non- specific chitosanolysis is greatly limited. We study on the purification of fraction with the chitosanastic activity from papain in this paper, it is important to illustrate the characterization and mechanism of non-specific enzyme hydrolysis on chitosan and to give the instruction to produce chitooligosaccharides by enzyme.Firstly we chose one kind of papainⅡwith good chitosanastic activity by comparing the activity of five kinds of commercial papain. Effects of different deacetylated degree of chitosans on the actions showed that this enzyme's best substrate was chitosanⅠ,which of D.D. was 85.7%; The enzyme showed maximum activity towards chitosan at the constration of substrate 0.5%,pH4.0,40℃and E/S 10%; under these optimum conditions, the viscosity decreased 70.77% in 30 min and it tended to be stable after 1h. Sepharose CL-6B gel column was utilized to determine the relative molecular weight distribution of the chitosan after 6h degradation by papainⅡ, the results showed that the relative molecular weight was from 2899 to 11943.PapainⅡwith the chitosanase activity was separated by ultra-filtration, The results showed that when papain was separated in the room temperature, 0.5% of concentration, 0.1MPa of pressure and eight times concentrated liquid, the specific activity of protease and chitosanase on papain increased to 531.39 U/mg and 2.65 U/mg from 435.10 U/mg and 2.17 U/mg respectively after ultra-filtration, and the degree of purification was 1.22-fold. Protein content of papainⅡincreased to 79.96% from 29.56% and reducing sugar content of papain decreased to 11.27% from 57.46% after ultra-filtration. SDS-PAGE diagram showed the low molecular weight protein was cleaned by ultra-filtration. The ingredient analysis indicated the reducing sugar content of papainⅡdecreased to 6.27% from 57.46% after ultra-filtration.Papain after ultra-filtration was purified by preparation electrophoresis, which of the experiment conditions were 4 % stacking gel and 10 % separating gel. The preparation electrophoresis spectrum showed five obvious bands, one of which had chitosanastic activity by analyzing activity. Single enzyme fraction with better chitosanastic activity from the band 2 was consentrated. Its homogeneity was demonstrated by SDS-PAGE and HPLC. The result indicated that there is the enzyme fraction with chitosanastic activity in the papainⅡ. The amino acid sequence of the enzyme fraction was analyzed by LC-MS/MS,the result was MRITISGSPG SGTTTLGRSI AEKYSYRYVS AGEVFRGLAK ERNMDLAAFG KIAETDPAID LEIADARQKEI GESSDDIILE GRLAGWMVEN ADLKILLCAS PECRSTRIAA REGLTEKQAF EMTIEREACE AGRYMEYYEI DILDFSPYDL ILNSETFSAN ELFAIVDAAV SSLLKRE。...