Expression Of Acid Protease Gene From Saccharomycopsis Fibuligera In Yarrowia Lipolytica And Application Of The Recombinant Acid Protease

The acid protease, also known as aspartic protease, is a sort of enzyme which can break down protein into polypeptide or amino acid. Recently, the acid protease is widely applied in many fields of industry,such as dairy, feed and fermentation.Milk coagulation is a basic step in cheese manufacturing. The calf rennet, the conventional milk-clotting enzyme obtained from the fourth stomach of suckling calves, has been the most widely used as the coagulant in cheese making to manufacture most cheese varieties. However, milk-clotting enzymes from alternative sources, such as bacteria and fungi, are used as the calf rennet substitutes and have received increasing attention due to the consumer constraints on the use of rennet, reduced supply and the increased price of calf rennet, in addition to the ever-increasing cheese production and consumption. It has been well documented that Yarrowia lipolytica appears as one of the most attractive microorganisms because it has naturally secreting high amount of proteins and a large range of genetic markers and molecular tools. The yeast has been used in several industrial processes and is non-pathogenic (GRAS, generally regarded as safe). The Y. lipolytica expression/secretion system has been validated by the efficient production of active proteins of various origins.It has been found that Saccharomycopsis fibuligera A11 strain can produce acid protease. As this acid protease is a cell-bound enzyme and can't be secreted into media, it is very difficult to purify and characterize it. So the gene encoding an acid protease natively produced by Saccharomycopsis fibuligera was cloned and overexpressed in Yarrowia lipolytica and the resultant recombinant acid protease was purified and characterized. The molecular mass of the purified enzyme was estimated as 99 kDa by gel filtration chromatography. The optimal pH and temperature of the purified acid protease were 3.5 and 33℃, respectively, and the enzyme was very stable over a pH range of 1.0-3.0. The recombinant acid protease was activated by Zn²⁺, but was inhibited by Hg⁺, Fe²⁺, Fe³⁺, and Mg²⁺, EDTA, EGTA, iodoacetic acid, and pepstatin. The purified recombinant acid protease from the positive transformant 71 had high milk clotting activity, suggesting that it may be used as a rennet substitute in the cheese industry.While the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in the cells of Yarrowia lipolytica, the cells displaying the acid protease could form clear zone on the plate-containing milk indicating that they had extracellular acid protease activity. The cells displaying the acid protease can be used to effectively clot skimmed milk. The highest clotting milk activity (1,142.9 U/ml) was observed under the conditions of pH 3.0, 40°C, 20 mM of CaCl₂, and 10% skimmed milk powder. We found that the acid protease displayed on the cells of Y. lipolytica which has generally regarded as safe status could be easily isolated and concentrated compared to the free acid protease. Therefore, the displayed acid protease may have many potential applications in food and cheese industries. This is the first report that the yeast cells displaying the acid protease were used to clot milk.In recent years, many yeast strains from different sources have been found to contain high content of protein and can be used as single protein. The single cell protein can be utilized as feed for animals and food for human. In order to use Y. lipolytica 22a-2 as both the potential single cell protein and producer of the recombinant acid protease for milk clotting, the acid protease gene from S. fibuligera A11 was expressed in the cells of Y. lipolytica 22a-2 and the produced acid protease was used to clot the skimmed milk. It was also found that Y. lipolytica 22a-2 producing acid protease still contained high content of protein. We observed that hydrolysis products fromκ-casein under catalysis of the recombinant acid protease were similar to those fromκ-casein under catalysis of the calf rennet. This may suggest that mechanisms of action of the recombinant acid protease are similar to those of action of the calf rennet.