Purification Of Trypsins From Euphausia Superba And Study On Cutaneous Wound Healing

Euphausia superba which belongs to Arthropoda Crustacea,is a psychrophilicspecies living in Antarctic area. It has become an increasing attention because of itshuge biomass and potential applications. Antarctic krill contains a variety of proteases,its protease degradation system is relatively unique and has high degradationefficiency, and40%of protease activity comes from trypsins. Trypsins from Antarctickrill are cold-adapted enzymes with high catalytic activity at low temperature andhave a lower optimal catalytic temperature.Because protease system in Antarctic krillhas both exonuclease and endonuclease activity, usually used to clear the necrotictissues, fibers and blood crust and can significantly accelerate woundhealing.Therefore, the Antarctic krill enzyme has a wide use in medical purposes.In this study, trypsin extracted from Antarctic krill.The separation andpurification of trypsin was conducted by saturated-precipitation methods using20-70%ammonium sulfate, DEAE Sepharose Fast Flow ion exchangechromatography and Sephacryl S100gel chromatography.After SDS-PAGE,onecomponent was obtained,which used as pure trypsin in animal experiments and itsmolecular weight was about32.0Kda.The animal model of full-thickness skin defect was established with mechanicaltrauma. Each rabbit was cut four wounds which were treated with crude tryspins,puretrypsins,papin and saline. Then pathological morphology change of the wounds wereobserved, the scars were rated with the Vancouver Scar Scale (VSS),the wounds areawas measured,the histological change were observed with tissue sections.The resultshowed that, compared with papain, the crude tryspins and pure trypsins extractedfrom Antarctic krill can significantly promote the cutaceous wound healing, theeffects of the crude trypsins were the best. The results indicated that crude trypsinscan significantly promote wound healing, shorten wound healing time, and alsosignificantly increase hydroxyproline contents in the trauma. Preliminary research indicated the Antarctic krill trypsin can increase collagen deposition, increase proteinsynthesis,accelerate epithelial tissue regeneration,and promote the formation ofgranulation tissue.EGF and bFGF are two crucial growth factors in wound healing. The EGF andbFGF contents of wound skin tissue were essayed. At the tenth and fiftieth day aftermodeling, the crude and pure trypsins promoted the expression of EGF and bFGF,andthe crude showed the fastest speed in the expression of EGF and bFGF. The resultsindicated that the crude trypsins may promote wound healing by regulating theexpression of EGFR and bFGF. There may have two aspects about wound healingmechanism: The first is the crude enzyme solution containing a variety of other activesubstances such as polysaccharides,proteins,chitin,which has been proved to promotewound healing,there may be a synergistic effect in the crude enzyme trypsin andchitin and other polysaccharides to commonpromote the repair of the wound,so thecrude showed the best effect. The second is the trypsin may be induced to releasegrowth factor in the process of wound repair,then speed up the repair process,improve repair quality.