Production, Purification, Characterization And Gene Cloning Of Alkaline Protease From Marine Yeast Aureobosidium Pullulans

Alkaline protease is one of the most important commercial enzymes. Microorganisms represent an attractive source of proteases as they can be cultured in large quantities in a relatively short time by established fermentation methods, and they produce an abundant, regular supply of the desired product. Furthermore, proteases from microorganism have a longer shelf life and can be stored under less than ideal conditions for weeks without significant loss of activity. In general, microbial proteases are extracellular in nature and are directly secreted into the fermentation broth by the producer, thus simplifying downstream processing of the enzyme as compared to proteases obtained from plants and animals.Production of alkaline protease employing marine yeast Aureobosidium pullulans 10 under solid state fermentation was optimized. The optimum medium is soybean meal with 5% rice hull and 50% NaNO₃ (2%) solution. The optimum inoculation size is 1.0×10⁷ yeast cells per gram dry medium. The optimum quantity of dry medium in 250-ml flask is 10 gram. The optimum temperature and initial pH are 28°C and 10.0, respectively. Maximum yields of 196.9U/g dry medium were achieved after 72 h under the optimum medium and optimum conditions.The extracellular protease secreted by Aureobosidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography (SephadexTM G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacryl- amide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0kDa. The optimal pH and temperature of the purified enzyme were 9.5 and 45°C, respectively. The enzyme was activated by Cu²⁺ (at a concentration of 1.0 mM) and Mn²⁺ and inhibited by Hg²⁺,Fe²⁺, Fe³⁺, Zn²⁺, and Co²⁺. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride. The Km and Vmax values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 mmol/min/mg of protein, respectively.After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.A genomic DNA fragment for the full length of gene encoding the alkaline protease(ALP1) from marine yeast Aureobosidium pullulans 10 has been cloned and sequenced by degenerate PCR and inverse PCR. ORF frame in the genomic DNA fragment consisting of 1349 bp is presumed by BLASTx in NCBI. The cDNA fragment is amplified by RT-PCR.After sequencing and blasting with ALP1 genomic ORF, the results show that ALP1 genome DNA fragment consists of three extrons of 311bp, 346bp, 588bp and two introns of 54bp and 50bp, respectively. The cDNA fragment of ALP1 encoded 415 amino acids. The molecular weight of alkaline protease precursor is 42887 Da.A putative promoter at–111 from the start codon is observed. A TATA box and two CAAT are observed at -101, -82, -25, respectively. Terminator at the 3′-noncoding region is observed with clip structure. A consensus sequence of a signal peptide consisting of 18 amino acids is found at the N-terminus and three N-glycosylation sites are found. The primary structure of the mature region shares extensive homology with that of subtilisin families and peptidase S8, and the two active sites (Histidine active site and serine active site) are preserved. Multiple alignment and phylogeny analysis of deduced amino acid sequence of pre-protease with other alkaline protease from representative species are done by using the software of Clustal X and PHYLIP-NP respectively.