Characterization Of A Keratinase From A Novel Constructed Recombinant Pichia Pastoris Strain

Keratinase is a class of enzyme which can specifically degrade keratin. It has been widely used in the many fields especially in the feed industry. However, due to the level of enzyme production from the natural strains is relatively low, its application is limited in scope. Therefor, it is significantly important for the agriculture and industry to do the research on the recombinant strain.To obtain genetically engineered bacteria which can express keratinase(kerUS)efficiently, this study analyzed the coding sequences of keratinase gene kerUS derived from Brevibacillus brevis US575 and the gene was modified with codon optimization based on Pichia codon preference. The modified gene was inserted into the pPIC9 K vector to construct the recombinant plasmid and achieve a heterologous expression in Pichia pastoris. At the same time the properties of the recombinant keratinase was been systematically studied.The main results are as follows.KerUS gene removed the signal peptide sequence named the ori-KerUS and after codon optimization named opt-KerUS. After optimization the CAI value of KerUS sequence increased from 0.69 to 0.91, GC content fell from 45.27% to 42.22%; wherein the lower the values of the free energy of RNA secondary structure the more efficiently the protein expression. On base of small fluctuations in GC content several high secondary structure was modified in order to reduce the total free energy more effectively.The value of free energy decreased by optimizing from 209.8 kkal/mol to 157.9 kkal/ mol.Sequence analysis revealed that opt-KerUS contained 205 bp substitutions.The DNA sequence of KerUS gene obtained by total gene synthesis was inserted into the pPIC9 K vector of Pichia pastoris and was transformed into host strain Pichia pastoris GS115. Selected by the G418 antibiotic of different concentration gradient obteined the high expression pPIC9K-KerUS/GS115. In bottle fermentation level, keratinase activity was 340U/ml. In high cell density fermentation level, its activity was 2680U/ml, nearly eight times the level of bottle fermentation.Characterization analysis showed that the optimum temperature of the enzyme was60℃, the optimum pH was pH 9.5. Studies of the thermal stability have shown that theenzyme activity remained 60% after 8h water bath treatment at 60℃ and 40% at 80℃.pH stability experiments showed that after 12 h stored in the buffer of pH6-10 the remaining enzyme activity is still more than 40%, which has a wide range of pH stability.A low concentration of metal ions such as Mn2+(123%) and Ba2+(200%) promote the keratinase activity. In addition, the enzyme have a certain tolerance on organic solvents,but the tolerance on anionic surfactant and oxidant is not strong. The enzyme kinetic constants experiment show that the maximum reaction rate of the keratinase was0.019g/L/min,the Km value of the keratinase to feather meal was 4.64g/L,specific activity was 440U/mg.