Construction Of A CDNA Library Of Rhizopus Stolonifer, And Study On The Cloning And Expression Of The Key Enzyme Genes Which Participating In The Degradation Of Lignocellulose

Rhizopus stolonifer TP-02is a strain of Ascomycetes, which has the advantages of high efficiency of lignocellulose degradation ability of zygomycotina Rhizopus stolonifer subspecies nod Rhizopus. The lignocellulose degrading enzyme gene was studied in this article, constructing the cDNA library, bioinformatic analysis of cloned gene, and expressed in E. coli.(1) This study successfully constructed Rhizopus stolonifer cDNA library. After identification, the original library storage capacity reached1.95×106cfu, library titer was7.84×106cfu/mL, with the high quality of the standard library. The library cDNA fragment size distribution in500-4000bp, library recombination rate was96%. Thus the library was highly representative and good integrity. (2) According to the substrate specificity, the screening medium was used to screen the key enzyme gene of lignocellulose degradation process in different plates, including5cellulase genes (EGIV, EGV, CBHI, CBHII, BGIII) and a hemicellulase cellulase gene(XynI). Among them, CBHI and CBHII was first discovered in Rhizopus. Each gene was sent to Sangon Biotech and was sequenced, reading frame being analyzed, gene sequences being uploaded to GenBank and got the gene accession numbers. Signal peptide was analyzed by the SignalP4.1, transmembrane region was predicted by the TMHMM server2, the family home and feature sequence was analyzed by the CDD database.(3) All cellulase recombinant strains were induced expression. EGIV and EGV were measured in20h and22h reached the maximum enzyme activity of0.73IU/mL and0.82IU/mL, the recombinant CBHI reached the maximum enzyme activity of0.46IU/mL at18h, the recombinant CBHII reached the maximum enzyme activity of0.41IU/mL at16h, the recombinant BGIII reached the maximum enzyme activity of1.54IU/mL at20h. Induced by IPTG, the fermentation products of the recombinant CBHI and CBHII was primary analyzed. Results showed that the optimum temperature of CBHI and CBHII were50℃and55℃, the optimum pH were5.0, CBHI had better tolerance to temperature. Protein Electrophoresis results showed the size of EGIV and EGV were35kDa and25kDa, CBHI and CBHII protein size were45kDa and56kDa, BGIII protein size was about50kDa, XynI protein size was about26kDa.(4) According to the fermentation experiment of the semi cellulose recombinant strain (XynI), the IPTG concentration, induction time, and incubation temperature was studied. The optimum culture conditions: IPTG induced concentration was0.3mmol/L,28℃after inoculation with8h induced expression. The medium composition was determined by orthogonal test and the result was:glycerol was10g/L, yeast powder was7.5g/L:, peptone was7.5g/L, Mg2+ion concentration was8mmol/L.In this condition, recombinant xylanase activity reached21.32U/mL,which was1.68times as basic culture medium and provided the basis for protein study on Purification and enzyme properties.