| Chymosin, the major component of rennet, is an acid protease produced in the fourth stomach of ruminants in the form of an inactive precursor prochymosin. It is responsible for hydrolysis peptide bond between Phe 105 and Met 106 in theΚ-casein. Chymosin is an important industrial enzyme widely used in cheese manufacture. In cheese-making, it plays a important role of condensing milk and improving flavor.To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799 which is a promising host strain for expression of the chymosin gene, we amplified the prochymosin gene from the plasmid pUC18-preprochymosin by PCR, and then cloned the gene into the expression vector pKLAC1 resulting in pKLAC1-prochymosin. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain pc7-7 with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. which offered the precondition for our latter research. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.And then, following site-directed mutagenesis were carried out, this protocol was a modification of the QuikChange? site-directed mutagenesis kit method by using the high fidelity pyrobest DNA polymerase. the expression vector pKLAC1-prochymosin containing fusion gene of bovine chymosin was prepared. Four pairs of mutagenic primers were synthesized in vitro. After that DpnⅠwas used to digest the PCR products to remove the methylated, nonmutated parental DNA template. Optimal amount of the digested products were used to transform competent E. coli JM 109 cells and colonies were picked up randomly followed by being sequenced to directly identify the needed mutants.The two mutated strain which were proved by sequencing was selected by YCB and YEPDL medium, The new results were obtained as follows: All the mutants could produce chymosin. However, the activity of chymosin was low compared with the wild type. These results indicate that mutation had little effect on prochymosin refolding and great effect on catalytic activity of chymosins. The chymosin was B type if the 224th amino acid was Gly ,The chymosin was A type if the 224th amino acid was Asp,the tertiary structure predictiones showed that the mature chymosin in our research was B type chymosin.Mutation of amino acid residues that are important on the active site of chymosin B, such as mutant strain mpc3-7, Asn 333 mutated to leu, His 334 mutated to Ile, resulted in the decrease of optimal temperature from 55℃to 50℃and milk clotting activity. It means that the 333th and 334th amino acids of chymosin played a critical role in milk-clotting and the thermostability of the enzymes.Chymosin produced by recombinant and the mutant was purified by using ethanol precipitation, size-exclusion chromatography using Sephadex G-75 and ion-exchange chromatography using DEAE-52 cellulose, The result of SDS-PAGE showed a single protein band with 37kD. Like the wild-type chymosin, mutant chymosins could be exhibited a similar behavior on DEAE-52 column. The specific activity of chymosin wild type pc7-7 had reached 1609.66 U/mg, mutant mpc2-1a had reached 724.57 U/mg, and mutant mpc3-7 had reached 566.29 U/mg.Studies on Characterization of recombinant strain pc7-7 showed that the optimum pH was 4.5,the optimum temperature range for the reaction of milk-clotting was 30-55℃.Km and Vmax of strain pc7-7 were 0.02 mg/ml and 0.0214 mg/ml.min respectively.As for the enzymic properities, The milk-clotting activity kept stable in pH 4.5, the optimal reaction temperature was 55℃, under the condition, the enzyme produced by mutant strain mpc2-1a had the fastest reaction velocity, Km and Vmax of strain mpc2-1a were 0.0122 mg/ml and 0.002 mg/ml.min respectively.The characterization of mutant strain mpc3-7 showed that the optimum pH was 4.5, the optimum temperature was 50℃, compare to recombinant strain pc7-7 and mutant strain mpc2-1a, the temperature of this strain was decreased, Km and Vmax of strain mpc3-7 were 0.003 mg/ml and 0.0005 mg/ml.min respectively. |
PDF Full Text Request