Study On Synergistic Degradation Of Hemicellulase From Roots

With the rapid development of social economy, the oil crisis and the sharp increase in the world’s energy demands accelerate the development of clean renewable energy. Hemicellulose that was an important biomass resource was second only to cellulose in nature, it cross-linked with cellulose and lignin. Complete degradation of hemicellulose needed various combinations of enzymes, this paper focused on a class of hemicelluloses-xyloglucan degradation, in previous studies the importance of its degradation does not attract enough attention. Our study focused on overexpression of a key enzyme, that a-xylosidase, in the degradation of xyloglucan, its characterization analysis and the enzyme jointly degradation of xyloglucan in vitro. The recombinant xylanase degraded the purified xylan from birch, bagasse, corn cob and blueberries and analysed the degradation products by qualitative and quantitative methods.The xyloglucan gene cluster associated with xyloglucan degradation was predicted by analyzing the genome of Thermotoga maritima MSB. The gene of α-xylosidase Xy1G from gene cluster was cloned into the pHsh that was a high efficient heat shock expression vector, designated pHsh-xylG. High-level expression was achieved in Escherichia coli by site-directed mutagenesis. Firstly, we analyzed mRNA secondary structure of the recombinant plasmid TIR area by online predict and enable RBS and ATG sites released from the complementary pairing region of hairpin structure. Secondly, we modified the rare codons in N-terminus translation initiation region of target gene, designated pHsh-xylG Ⅱ and pHsh-xylGⅢ respectively. Above Two plasmids were transformate into E. coli DH10B for expression by heat shock-induced, and the expression lever of optimized recombinant plasmid increased15-fold. SDS-PAGE analysis showed the ultimate expression of the recombinant enzyme accounted for40%of the total soluble protein, which showed the modification of N-terminus affected the expression level significantly.The recombinant enzyme was purified by the heat treatment and DEAE Sepharose Fast Flow anion exchange chromatography. SDS-PAGE showed a single band that was88kDa. The specific activity of pure enzyme was55.7U/mg, enzyme purification factor was12.5times, and the yield was86.7%. Purification results showed that removal of host proteins by heat treatment, to achieve relatively pure protein, recovery rate of90%, the results indicate that in the future can be used in industrial applications such heat treatment of low-cost and easy way of preparation of the enzyme.Furthermore, we analyzed the properties of purified recombinant enzyme on substrate pNPS. The results showed that the optimum pH of5.0, and at the range of pH5.0-7.5enzyme activity could maintain at above60%, and within the optimum temperature85℃. The thermal stability of the recombinant enzyme showed that after incubation at85℃for1h, activity was maintained at80%, the recombinant enzyme showed high thermal stability.Impact of metal ions and inhibitors on the activity showed that Co2+, Ni2+, Li1+, EDTA had no significant effect on the enzyme activity; Zn2+, Cu2+, Fe2+on the enzyme activity was significantly inhibited. Mn2+, Sr2+, Mg2+, Triton-X100on the activity has a significant role in promoting. Graphical analysis of the recombinant enzyme kinetic parameters on pNPS substrates by Eadie-Hofstee plot method, According to the linear regression equation it is showed the Km of0.04mmol, Vmax was78U/mg.The genes of β-galactosidase GalC and a-fucose AfuD were cloned into the pHsh expression vector and achieved over-expression in E. coli., associated with previous cloned endoglucanase CelB, endoglucanase Cel74, β-glucosidase Bg1A and α-arabinosidase AraB to degrade the xyloglucan (tamarind). TLC analysis showed that XylG was important on degradation of xyloglucan. The efficiency of Cel74degrading xyloglucan was higher than CelB. The GalC and AraB also have certain effect.The gene aguA from T. maritima was cloned into pHsh, designated pHsh-aguA and overexpressed in E. coli. We used the AguA together with endo-xylanase XynB, β-xylosidase XylC, a-arabinosidase AraB to degrading the purified xylan from birch, bagasse, corn cob and blueberries, TLC analysis showed that for degrading different plant sources appropriate xylanase enzyme-series were needed for joint degradation. Through reasonable enzyme jointly reactions, xylan from corn cob and blueberries can be complete degraded into monosaccharides. At the processing of bagasse, we found that two oligosaccharide were not able to completely degraded, it may be judged by the color of the oligosaccharide composed of glucose, when the β-glucosidase was added, the two oligosaccharide chains was completely degraded. This result has not been reported before, we hypothesized that the bagasse contains a unique dextran.