Studies On Lignocellulolytic Enzymes And Cloning Of Ligninolytic Enzyme Genes From Trametes Gallica

Lignocellulose is composed of cellulose, hemicellulose and lignin, and the most abundant and renewable bio-resource in nature. Cellulose and hemicellulose can be enzymatically hydrolyzed by cellulase and hemicellulase from microorganisms, respectively. However, the structure heterogeneity and recalcitrance of lignin greatly retard the efficient utilization of plant biomass. The white-rot fungi secrete ligninolytic enzymes to oxidatively decompose lignin and aromatic pollutants. These enzymes are greatly potential in bio-pulping, bio-bleaching of pulp and treatment of environmental pollutants, etc. Phanerochaete chrysosporium, a model microorganism in studying lignin bio-degradation, has been extensively studied, but it produces low ligninolytic enzymes and little laccase, which is the important member of ligninolytic enzyme system. Therefore, the screening of strains with high yield of ligninolytic enzymes, the cloning and the high efficient expression of ligninolytic enzyme genes are important research aspects in the lignin biodegradation. White-rot basidiomycete Trametes gallica is one of the main bio-degraders of poplar in China. Our previous study has proved its strong capacity to degrade lignocellulose. Using T. gallica as a donor strain, studies were carried out in the following 4 aspects: (1) production of lignocellulolytic enzymes by T. gallica; (2) purification and characterization of some ligninolytic enzymes; (3) comparison and analysis of the differentially expressed genes between T. gallica and the model strain P. chrysosporium by gene chip; (4) cloning of MnP cDNA and heat shock proteincDNA. The corresponding results obtained are as follows:Firstly, several procedures have been employed for growing T. gallica to produce lignocellulose-degrading enzymes. The results showed that nitrogen-limited nutrition (2.2 mmol/L) and shallow stationary cultivation favored production of extracellular MnP with activity of 36.9 U/L in glucose medium for 12d. However, laccase levels observed in high-nitrogen (22 mmol/L) agitated cultures were much greater than those found in low-nitrogen. Furthermore, only low laccase activity could be obtained under shallow stationary cultivation no matter whatever high or low nitrogen was used. MnP activity reached 118.1 U/L when T. gallica was grown in a low-nitrogen culture containing phenylalanine, hemicellulose and cellulose. The experiments showed that NH/ plays an important role in inducing production of MnP and laccase of the fungus. Results indicate that T. gallica produces a series of the lignocellulolytic enzymes (cellulase, hemicellulase, laccase, MnP and LiP), and needs sufficient nitrogen nutrient to produce all the enzymes during solid-state fermentation of wheat straw. In the presence of high concentration of nitrogen and Mn2+, the resulting 396.7 U/L of MnP in soaking fluid of the solid culture (with high concentration of nitrogen and Mn2+) was obtained, corresponding to 2.0 U per gram of straw powder.Secondly, one active component with cellulae, xylanase, laccase, MnP and LiP activity, was obtained by Sepharose-Q Fast Flow chromatography. By further fraction on Sephadex G-100 and recovery through native-polyacrylamid gel electrophoresis (PAGE), a purified MnP and a laccase were separately obtained. SDS-PAGE and isoelectric focusing (IEF) were used to evaluated molecular weight and isoelectric point (pi) of protein, respectively. The purified MnP had a molecular weight of 35.7 kDa and a pi of pH 2.8, and the laccase, 58.4 kDa, and pi 3.2, respectively. The result showed that MnP has a maximum absorbance at 407 run, optimal pH 5.25 and reaction temperature of 35°C. The laccase showed maximum activity at optimal pH 3.5 and 35°C respectively. A new tank for detection of protein pi without suing ampholytes was designed and constructed. The pi of bovine serum albumin was evaluated as pH 4.7 by the new mothod, and equal to that by IEF. Thispreliminarily verified the possibility of the new method for detection of pi of ampholytes.Thirdly, by cDNA microarray hybridization, the differentially expressed geneS of T. gallica cultivated in nitrogen-limited medium at different times were studied; Two total-RNA isolates of T. gallica cultured for 5d and 12d on the N-limited medium were extracted. Fluorescent dye- (cy-5- and cy-3-dCTP) labeled cDNA was produced through single primer amplification reaction. The labeled cDNA was hybridized to the cDNA microarray, which contained 2,596 cDNA fragments froman SSH (suppression subtractive hybridization) library of P. chrysosporium grown onishallow N-limited culture for 2 and 3 days. The results showed that there were 17^ positive clones detected on the microarray. There are 95.9% of the positive clones with a ratio (intensity of labeled 12d cDNA/intensity of labeled 5d cDNA) from 0.5 to 2.0. However, only 3 and 4 clones with the ratio over 2.0 and lower than 0.5 wer$ found, respectively, corresponding to 5d and 12d cultures of T. gallica. One hundred and twenty-two clones were randomly selected and sequenced, of which 118 clones could be perfectly located in the genome of P. chrysosporium. These clones can be grouped into 47 genes, among them, the function of 24 genes have been known, including 19 house-keeping genes. The result indicates that there is obvious sequence difference and far relative between these two fungi. Two interesting clones have been obtained from homoiogy comparison, one corresponding to a fragment of peroxidase ipoB gene of P. chrysosporium and the other encoding a sort of heat shock protein.Fourthly, a pair of specific primers corresponding to the conserved sequences of mature MnP cDNAs of T. versicolor and P. chrysosporium were designed and use<￡ to amplify MnP cDNA of T. gallica with PCR reaction, in which the total cDNA$ were used as templates. An MnP cDNA fragment with 580 bp was obtained and ha$ an open reading frame encoding a peptide with 193 amino acid residues and 20.5 kDa of molecular weight. There was an obvious codon usage preference and more than 90% of homoiogy with one MnP sequence of T. versicolor. Similarly, a pair of specific primers corresponding to the sequence of mature heat-shock protein (HSP) cDNA of T. versicolor was designed and used to amplify HSP cDNA from T. gallicausing RT-PCR reaction, in which the total RNAs were used as templates. A cDNA fragment with 848 bp was obtained and encodes a mature peptide with 256 amino acid residues and 28.4 kDa of molecular weight. The deduced amino acid sequence shares 90% of homology with that of HSP from T. versicolor fddl23b. The strongest codon usage preference has been found for codon CAA of Gin, AAG of Lys and TGT of Cys, whose usage frequency reached 100%.