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| Gene Synthesis | Among the tested strains, one strain named RM-2 from RM showed promising protease production ability based on the preliminary screening and rescreening. Further identification was on the basis of the 16S rDNA sequence analysis. Genomic DNA was extracted and purified using a genome DNA extraction kit according to the manufacturer's instructions, then it was used as the template for polymerase chain reaction (PCR) (Sangon, Shanghai, China). The 16S rDNA was amplified with the upstream primer 27F 5′-AGA GTT TGA TCC TGG CTC AG-3′ and downstream primer 1541R 5′-AAG GAG GTG ATC CAG CCG CA-3′. PCR amplification was performed under the following conditions: denaturation at 95 °C for 5 min, 29 cycles at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1.5 min, with a final extension at 72 °C for 10 min. DNA sequencing was carried out by Genscript Biotechnology Corporation (Nanjing, Jiangsu, China). The resulting DNA sequence of 1458 bp obtained by 16S rDNA sequencing was analyzed with Blast through the NCBI server. A phylogenetic tree was constructed using MEGA 5.0 (Christensen & Martin, 2016). | Get A Quote |
To reduce the high cost caused by steam sterilization of fermentation substrates and equipments in mesophilic solid-state fermentation of rapeseed meal (RM), a thermophilic protease-producing strain RM-2 from RM was isolated and employed in thermophilic solid-state fermentation of unsterilized RM for polypeptide preparation. The isolate was identified as Geobacillus stearothermophilus by appearance of colonies and microscopic observation as well as 16S rDNA sequencing. The highest yield of polypeptide (9.67%) was obtained by RM-2 at 50% moisture content, 55 °C and 24 h fermentation time. Analysis of microbial community diversity in fermented RM revealed Geobacillus stearothermophilus as dominant strain... More
