The Chemical Characterization Of Marine Low-temperature Alkaline Protease

Alkaline proteases constitute one of the most important groups of industrial enzymes, and have established roles in the detergent, food, pharmaceutical, leather and silk industries. And the low-temperature alkaline proteases producing by some kinds of marine bacterium are attracting more and more concerns all over the world for their special characters nowadays. We summarize the studies about the respects of the marine low-temperature alkaline protease all over the world and point out the developing direction for later research.Marine Low-temperature Alkaline Protease(MLAP) is a kind of extracellular enzyme from a kind of marine bacterium of Flavobacterium YS-80-122.The study mainly concerned about the study of chemical characters of MLAP. During our experiments, we studied the purification conditions of enzyme, the characters of purified enzyme and enzyme kinetic parameters, and structural model by computer analysis. Through these methods, we got the data of the enzyme's characters and many useful structural information about the area of protein-ligand interactions. These results can provide theoretical foundation for further industrial use of this kind of enzyme.The main results and conclusions are as follows:Isolation and purification of MLAP: (1)A preliminary study on industrial abstraction technology is carried out by centrifuging the fermentation medium with high speed (6000r7m) at low temperature(4 ) and ultra filtrating (capturing molecular weight 10000Dal). After cooling dryness we get the raw samples. (2)Enzyme were precipitated by, ammonium sulphate (30%-65%). The precipitate was dialyzed against 20mM Na2HPO4-KH2PO4 (pH=6.5 ) buffer. The dialyzate was applied to IEF producing system(12W) and column Sephacry I S-200. At last we get the electrophoreticly pure sample after cooling dryness and the purity is checked by SDS- PAGE and C8 HPLC.Chemical characterization of MLAP: (1)We get its molecular weight--49,300+1000Dal by SDS PAGE gel and HPLC gel, and its pI-8.5 by IEF analysis system. For its native character is different from all the alkaline protease people knewed, we can classify it as a new kind of protease. (2)We have studied the effects of temperature and pH on enzyme activity and stability. With the experiments results, we have made a model of enzyme activity- temperature-pH (r2>0.9). And through this model we knew its pH optima is at 9.5, temperature optima is at 30 . The enzyme was stable at low temperature (t 30 ) and keeping relatively low pH C pH 8 ) is helpful for its stability. (3)In addition we studied the effects of various metal ions and some other ingredients which often occurr in detergents such as some kinds of surfactants and oxidants on enzyme activity. In our experiments we knew that the MLAP is compatible with most of the metal ions and surfactants. Few metal ions such as Pb2+, Ag+ , Cu2+act as strong inhibitors of enzyme activity. Also strong inhibition was observed with EDTA, and the finding was different from former alkaline protease.Study of kinetics parameters of MLAP: (1)We studied the effects of pH and temperature on the kinetic parameters of the enzyme hydrolyzing casein reaction. We got the data of activation energy(Ea=33.2kJ/mol) and reaction temperature modulus( Q10=1017433[l/ (T1-T2) ]). Also we get the values of pKa=9.3 and pKb=10.7, and which offers many useful information concerning the groups involvedin enzyme funcion. (2)By studying the kinetics parameters changing by adding its inhibitor such as EDTA, we deduce that the inhibitor EDTA is a kind of competitive inhibitor to this enzyme. So we knew there must be some metal elements in the protein-ligand interactions area.Homoibgy modeling and structural prediction of MLAP: (1)By analyzing the sequencing result with DNASTAR software, we get the result as follows: The molecule weight of the enzyme is 49903.92Dal; the pi is 8.52; there are 459 amide acids and 148 ones of which are hydrophobic and 153 ones are polar. It has a high homology to pseudomonas(nearly 76%), so it should has the similar...