Study On The Production, Characterization, Application And Gene Clone Of The Protease From Serratia Sp. SYBC H

Proteases are a kind of enzymes which catalyze the hydrolysis of peptide bonds. They are widely used in food, feed, medicine, chemical industries and so on. They have theoretical and potential commercial values to study on proteases production, enzymatic properties and applications. The strain Serratia sp. SYBC H isolated from decayed Cyanobacteria in Taihu Lake （Jiangsu, China） was used as the protease producer. The contents in this thesis are as follows:Three statistical methods were used for shaken flask optimization for the protease production by Serratia sp. SYBC H with duckweed as nitrogen source. At first, a variety of variables influencing protease production were investigated with one-variable-at-a-time approach. Then, orthogonal design was applied to find the significant variables. Finally, response surface methodology （RSM） including Box-Behnken central composite experiments was used to determine the optimal concentrations and interaction of the three significant variables. The optimal level of the significant variables for the maximum protease production was duckweed 43.9 g/L, wheat flour 20 g/L, and sodium chloride 0.08 mol/L, respectively. The interaction between duckweed and wheat flour was significant.The operating conditions such as pH, aeration, stirring speed were studied in 5-liter fermentor, respectively. It is found that the yield of the protease was higher when pH value was controlled in fermentation process than that of control experiments with no pH control. The maximum protease activity （1450.4 U/mL） was observed from the ventilation rate at 6.0 vvm, and stirring speed at 800 r/min. The effect of Tween 80 supplemented in fermentation medium is to improve the permeability of Serratia sp. SYBC H cell membranes, and to promote the protease secretion into supernatant.Three statistical methods were used for shaken flask optimization for proteases production by Serratia sp. SYBC H with cyanobacteria as nitrogen source. At first, a variety of variables influencing protease production were investigated with one-variable-at-a-time approach. Orthogonal design was then applied to find the significant variables. Finally, response surface methodology （RSM） including Box-Behnken central composite experiments was used to determine the optimal concentrations and interaction of the significant variables. The optimal level of the significant variables for the maximum protease production was cyanobacteria 50.0 g/L, ZnSO4.7H2O 1.5 mmol/L and Urea 0.05 mol/L. The maximum protease activity reached 704.7 U/mL in the optimized medium. There was no interaction between the significant factors.The crude protease from fermentation supernatant was purified through three steps involving （NH4）2SO4 salt precipitation, DEAE-cellulose anion-exchange chromatography, and gel filtration. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH ranges between 5.0 and 11.0, with the maximum activity at about pH 8.0. It is also fairly active over the temperature ranges between 30°C and 80°C, with the maximum activity at about 40°C. The protease activity was substantially stimulated by Mn²⁺ and Na+ （5mmol/L）, respectively. Other metal ions inhibited the protease activity, especially, 5 mmol/L Co²⁺ can completely inhibit the protease activity. In addition, Mn²⁺ enhanced the thermostability of the protease significantly at 60°C. However, different from other serine proteases, the activity and stability of serine protease from Serratia sp. SYBC H were inhibited by Ca²⁺. The protease not only remained relatively high activity in 50% （v/v） hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH, but also showed high stability in hydrophobic solvents such as hexane, heptane and octane. The protease retained relatively high initial activity in the presence of nonionic surfactants 20% （v/v） Tween 80, 25% （v/v） glycerol and Triton X-100, respectively. But, there was obvious reduction in protease activity in the presence of the anionic surfactant SDS at 1% （w/v） concentration. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease.It is found that the protease from Serratia sp. SYBC H has significant capacity to remove blood. The protease efficiently catalyzed the degradation of the feather meal rich inβ-structure of proteins. In the hydrolytic supernatant, soluble polypeptides were 3-5 times that of control with the inactivated protease. However, the protease can not catalyzeα-rich proteins as hair and wool, even at its optimal temperature of 40°C for 24 h..The protease can catalyze sucrose esters syntheses in a variety of organic solvents such as pyridine, DMSO, DMF, methanol, acetone. As far as acyl donors were concerned, acetic anhydride and vinyl acetate can be catalyzed by the protease from Serratia sp. SYBC H. The protease can catalyze the formation of sucrose esters with varying degrees of substitution （DS）, with the performance of non-directed catalysis. It is also catalyze the synthesis of a single product, exhibiting directed catalysis. For example, in pyridine and hexane （1:1, v/v）, the protease catalyzed the acylation of sucrose with acetic anhydride to form sucrose octaacetate. The yield of sucrose octaacetatecan can reach 90% under 30°C, 200 r/min and 24 h.The serine protease from Serratia sp. SYBC H comprised of 1,288 bp encoding 429 amino acid residues. The proportion of the hydrophobic amino acids residues （43.62%） was higher than that of hydrophilic amino acids residues （28.64%） in the mature peptides of the serine protease from Serratia sp. SYBC H. Especially, the presence of high content of hydrophobic amino acid residues surrounding the catalytic residue Ser in C-terminal mature peptide may be related to the solvents stability of protease, which demonstrated the relationship between structure and function of the solvent-stable serine protease from Serratia sp. SYBC H.