Purification, Cloning, Expression And Surface Display Of Alkaline Protease From Marine Yeast Aureobosidium Pullulans HN2-3 And Application For Bioactive Peptide Production

Over 400 yeast strains were isolated from seawater, sediments, bowels of marine organisms and surface of thalassophyte, we found 5 strains( HN2-3, N13d, N13C, Mb5 and HN3-2) among them could form clear zone around the colonies on the double plates with 2.0 % casein. Strains HN2-3, N13d, N13C and HN3-2 were identified as Aureobasidium pullulans and strain Mb5 was identified as Yarrowia lipolytica using morphology, physiology and molecular methods. Strain HN2-3 had highest protease activity. Peptides in the hydrolysate produced by the proteases from strain HN2-3 had the highest angiotensin I-converting-enzyme (ACE)-inhibitory activity and the protease from HN2-3 was named as ALP2.The extracellular alkaline protease ALP2 in the supernatant of cell culture of the marine yeast A. pullulans HN2-3 was purified to homogeneity with a 2.34-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography(Sephadex G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamidegel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 33.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 52℃, respectively. The enzyme was activated by Mg²⁺,Na⁺and Mn²⁺ (at a concentration of 5.0 mM) and inhibited by Fe²⁺,Fe³⁺,Cu²⁺,Co²⁺,Ag⁺ and Hg²⁺. Zn²⁺,Ca²⁺,K⁺ and Li⁺ has no effect on the protease activity. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, and weakly inhibited by EDTA and iodoacetic acid. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strainsN3C (Y. lipolytica) and YA03a (Hansenia sporauvarum) was digested by the purified alkaline protease, the highest values of ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were 88.3% and 47.3 %, 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 pharmaceuticalindustries.The alkaline protease structural gene (ALP2 gene) was obtained from both the genomic DNA and cDNA of A. pullulans HN2-3 by degenerate PCR, inverse PCR and RT-PCR. DNA sequence of the ALP2 gene ( accession number: EU331441) was 1354bp. An open reading frame of 1248 bp (accession number: EU224431 ) encoding a 415 amino-acid protein with calculated molecular weight of 42.9 kDa was characterized. Eighteen amino acid at the beginning of the gene was signal peptide. The protein was found to have the conserved serine active site and histidine active site of serine proteases in the subtilisin family. The protease ALP2 gene was expressed by the plasmid pINA1317.The recombinant A. pullulans alkaline protease produced in Y. lipolytica formed clear zones on the double plates with 2.0 % milk and alkaline protease activity in the supernatant of the recombinant Y. lipolytica culture was detected, suggesting that the cloned ALP2 gene was protease gene which was expressed in Y. lipolytica and the expressed alkaline protease was secreted into the medium.When the cDNAALP2 gene was cloned into multiple cloning sites of the surface display vector pINA1317-YlCWP110 and expressed in cells of Y. lipolytica. the protease displaying cells could form the clear zone on the double plate containing milk protein. The alkaline protease displaying cells were also found to be able to produce bioactive peptides from different sources of proteins. The produced peptide from single cell protein of marine yeast strain G7a had the highest ACE inhibitory activity while the produced peptide from spirulina protein had the highest antioxidant activity. This is the first report that the yeast cells displaying alkaline protease were used to produce bioactive peptides. The properties of the surface displayed protease were examined. It was found that the optimal temperature of the displayed alkaline protease was lower than that of the free alkaline protease. However, the thermal stability of the displayed protease was enhanced compared to that of the free protease. The pH stability of the displayed protease was different from that of the free protease. The results reveal that the displayed alkaline protease exhibited a lower affinity to azocasein than the free one.