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
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