Purify The Collagenase From Bacillus Cereus R75E And Its Recombinant Expression In Pichia Pastoris

collagenase, a class of proteases highly specific for collagen, has been the focus of much practical interest with respect to its potential medical, cosmetic and food-production applications. Proteases from microorganisms have been extensively utilized in various fields because large amounts of these enzymes can be produced quickly at low cost and have higeh range of substrates.At present, a lot of collagenase products are crude, they have low purity, complex composition.Meanwhile the application of microbial collagenase in our country mainly relys on imports, which limits the application of collagenase.So this research has two parts about collagenase, one is to acquire the pure collagenase in Bacillus cereus R75 E fermentation, the other is to find a safe and stable method to produce collagenase in vitro.Investigate the optimal collagenase-producing conditions of Bacillus cereus R75 E and acquire the collagenase in high purity. We measured the effects of the culture temperature, culture time, inoculation amount, carbon source and nitrogen source et al, to the production of collagenase by Bacillus cereus R75 E using single factor experiment and orthogonal test. Then, ammonium sulfate precipitation, Butyl FF hydrophobic chromatography and Superdex 200 gel filtration chromatography were used to purify the collagenase from Bacillus cereus R75 E. The optimal ferment conditions for collagenases production were as follows: ferment temperature was 41°C, inoculation volume was 6%, ferment time was 36 h, 10 g /L glucose was used as carbon source, 5 g /L peptone was used as nitrogen source and initial pH was 7.0. The total activities of collagenase after optimization were raised to 2.9-fold higher than non-optimization. The collagenases were purified up to 90% purity in SDS-PAGE gel with an expected molecular weight of nearly 110 kD after sequential purification procedures. The final purified fold and enzymatic activity recovery reached to 18.4 and 1.1%, respectively. We acquired the optimal condition for collagenase production by Bacillus cereus R75 E, and constructed the procedure of collagenase purification, which facilitates the potential industrial exploitation.In order to find a safe and stable method to produce collagenase in vitro, we expressed the Bacillus cereus collagenase colR75 E in Pichia pastoris. With the Bacillus cereus genomic DNA as template, we successfully amplified the collagenase colR75 E DNA fragment by PCR and cloned it into pPICZαA plasmid. The pPICZαA/colR75 E recombinant plasmid was lineared with SacⅠ, which was then transformed into Pichia pastoris X-33 competent cell in order to integrate the colR75 E fragment controlled by inducible AOX1 promoter into Pichia pastoris X-33 genome. The successfully Pichia pastoris X-33 integrant was cultured and induced by methanol supplementation. The induced recombinant collagenase ColR75 E by methanol was precipitated and concentrated from Pichia pastoris X-33 culture’s supernatant with ammonium sulfate at the concentration of 80%. The activity of recombinant collagenase ColR75 E was then subsequently analyzed by collagenase activity assay, SDS-PAGE, zymography, and typeⅠcollagen degradation analysis. The highest level of collagenase ColR75 E induction was observed by 1 % methanol addition after 72 hours induction. The molecular weight of the recombinant collagenase is close to 110 kDa, which was consistent with the expected molecular weight of ColR75 E. The results of zymography and typeⅠcollagen degradation analysis indicated that the recombinant collagenase ColR75 E had an excellent collagen proteolysis activity. Its specific activity reached to nearly 1.454 U/mg under standard conditions. Pichia eukaryotic expression system is suitable for the expression of Bacillus cereus collagenase ColR75 E in a high collagen proteolysis activity, which laid a solid foundation for future investigation and industrial application.To sum up, this study acquire the from the purification of collagenase from Bacillus cereus R75 E fermentation, and purification of the collagenase has high activity than collagenase in markets. This shows that the purification method is feasible; At the same time, we lay a foundation for application of collagenase in the industry by collagenase gene cloning and eukaryotic expression expresses.