Screening And Identification Of A Proteinase-secreting Fungus, Cloning Of Proteinase Genes And Its Optimization For Protease Production

This research isolated strains producing protease from cattle rumen. The stainspecies was identificated through morphological observation and molecular. Inaddition, we cloned the protease genes and designed the optimization of enzymeproduction experiment for the strain to get the optimal coditions. This researchprovide theoretical basis for exploiting new sources of protease and the compositeproduction protease.In this experiment,16strains were selected from the34strains obtainedoriginally by the casein hydrolysis method. A strain, the highest proteinase activitystrain, whose enzyme activity is415.82u/ml, was selected from the strains applyingthe HE value and proteinase activity Comprehensive comparison method. Throughmorphological observation we found that the strain and the aspergillus oryzae inAspergillus genus had very similar growth characteristics of growth and morphology.Through the analysis of the sequence of a1800bp gene segment of18S rDNA, wefound that it had the highly sequence homologous with Aspergillus oryzae, whichreached99%. Combined with the morphological characteristics, the strain wasidentified as Aspergillus oryzae.We cloned the neutral protease gene （Alp） and alkaline protease gene （Npi） fromthe total RNA of Aspergillus oryzae. The gene Npi, containing1905bp nucleotides,encoding a634amino acid, molecular formula being C3057H4652N838O974S13, relativemolecular mass69.1443kD, isoelectric point5.07, half-life period greater than20h,unstable parameter21.94, was also proved to be a stable protein. The gene Alp,containing1212bp nucleotides, encoding a403amino acid, molecular formula beingC₁₈₇₈H₂₉₆₄N_520O60₃S₃, relative molecular mass42.5714kD, isoelectric point5.95,half-life period greater than20h, unstable parameter34.4, was proved to be a stableprotein. Similarly.The optimization of enzyme production experiment was designed to get theoptimal coditions. We got the best inoculation amount4%, the optimum temperature30℃, time60h through the single factor test. By the response surface analysis（RSA）methods, the optimal fermentation conditions were determined as follows:4%inoculation quantity, temperature29℃, time55h. Through repeated experiments,the stability of enzyme production was better. The highest enzyme activity reached527.57u/ml, which increased by22.6%than before under the optimal conditions thatinoculation quantity is4%, temperature29℃and time55h.