| China is rich in the resource of rice straw, but characteristics of hard degradation limited to utilize the rice straw. In recent years, improving the utilization of rice straw by microbial resources have increasingly become the focus of reasearchers. Endophytes Pantoea ananatis Sd-1, isolated from Gumei 4 rice seed after strict surface sterilization in our lab, exhibits efficient ability of degradating rice straw. In order to further explore the degradation conditions of rice straw and degradation mechanisms by Sd-1, the research contents in this paper mainly include following three parts.To investigate the optimal degradation conditions of P. ananatis Sd-1. Using rice straw as sole carbon source, we got its optimal degradation conditions i.e. inoculum size 1.5%; pH 10 and the rice straw content 2.5% by singal-factor experiments and orthogonal experiments. The degradation of rice straw reached to 46% in the optimal conditions. When using pretreated rice straw as sole carbon source, maximum degradation reached to 61.8% at 3th day, and some low molecule benzene compounds such as phenol, phenyl acetic acid which were considered as the composition of lignin was detected by GC-MS in fermentation broth. Moreover, acid compounds which could not only transformate to biofuel but also access in TCA cycle to provide ATP for Sd-1 were also detected in the fermentation broth.(2) We confirmed that Sd-1 had the ability to secrete several ligno-cellulolytic enzymes such as peroxidase, laccase and cellulase and hemicellulase. We further checked the activies of lingo-cellulolytic enzymes produced by Sd-1 during cultured in pretreated rice straw for 6 days. The maxium activities of lignin perosidase, laccase and cellulase were 5.9 U/m L, 600 U/L and 295 U/mL respectively at 3rd day 3, while the maximum activity of hemicellulase was 1345 U/mL at 1st day. The degradation of lignin, cellulose and hemicellulose were reached to 35.62%, 75.2% and 78.8% respectively.(3) Genomic analysis of Sd-1 identified four putative laccases named from Lac1 to Lac4. However, only Lac4 contains the complete signature sequences of laccase, and it shares at most 64% of sequence identities with those of other characterized bacterial multicopper oxidases. The recombinant Lac4 showed high activity towards non-phenolic and phenolic compounds under the condition of low pH and temperature from 30 ℃ to 50 ℃: Km values of Lac4 for ABTS at pH 2.5 and for guaiacol at pH 4.5 were 1.03±0.154 mM and 6.05±1.658 mM, respectively. The enzyme activity of Lac4 was increased efficiently by 0.8 mM Cu2+ and 5 mM Fe2+. In addition, Lac4 could decolorize different types of synthetic dyes, especially showed the decolorization rate of 89% for the azo dye Congo red in 4 h. Lac4 also exhibited the degradation rate of 38% for lignin and GC-MS confirmed that Lac4 could degrade the lignin to low molecular weight aromatic compounds. Q-PCR detection showed that the expression of lac4 gene was induced by pretreated rice straw. Taken together, these data show a promising biotechnological potential for laccase Lac4 and Lac4 may participate in degradation of rice straw. |
PDF Full Text Request