Abstract
Clostridium histolyticum is used for production of several proteolytic enzymes such as elastase, neutral proteases, clostripain and in particular collagenase. Besides industrial applications, collagenase has been indispensable for medical purposes including isolation of pancreatic islets for diabetes treatment. The aim of this study was to optimize the method for production and partial purification of a new collagenase blend and to test its suitability for successful pancreatic islets isolation in a rat model. Bacterial strain of C. histolyticum was sequenced for presence of the collagenase genes. Different fermentation conditions were tested and the process of collagenase extraction was modified and optimized. Samples of collagenases were taken for western blot detection, activity assessment, and ability for dissociation of pancreatic tissue. Findings indicate that concentrated trypton growth medium with pepton was the most suitable for Clostridium growth and collagenase production. Whole genome sequencing revealed two genes for collagenase and also gene for clostripain. Western blot specific detection helped to select useful production modifications. Following these modifications was also improved the yield, morphology and in vitro function of intact pancreatic islets which were finally comparable or better than those achieved using standard blends of collagenase. The results support the use of the new collagenase blend for islet isolation giving thus the opportunity to choose an alternative product. Our next steps would lead to further enzyme purification through scaling up of the production method for a wider use.
Highlights
Enzymes in general are of fundamental importance in food, cosmetic and pharmaceutical industry due to their activity and wide substrate specificity
PCR confirmed the presence of collagenase gene, whole genome sequencing could better characterize Clostridium histolyticum strains
Collagenase from the selected strain was used for pancreatic islet isolation and the parallel results were used as a feed-back for modification of the culture and purification method in order to achieve stable results in terms of the islet isolation outcome
Summary
Enzymes in general are of fundamental importance in food, cosmetic and pharmaceutical industry due to their activity and wide substrate specificity. The research in the last decades has been focused on collagenase characterization and isolation from different tissues of variety of animals as well as from diverse species of microorganisms. One of the most important and studied producers of collagenase is Clostridium [1] [2] [3]. Variety of proteolytic enzymes as collagenases, elastase, clostripain and neutral proteases are produced by Clostridium. Molecular weight of different collagenase isoforms ranged from 68 to 130 kDa. In this study was used Clostridium histolyticum MB 204 (owner MB Pharma Ltd., Prague, Czech Republic), a bacterial strain selected by long-term passaging of different bacterial strains during adaptive laboratory evolution (DSM 627, DSM 1126 and DSM 2158 obtained from German microorganism collection)
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