Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by Clostridium histolyticum

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.


Introduction
Enzymes in general are of fundamental importance in food, cosmetic and pharmaceutical industry due to their activity and wide substrate specificity. Collagenases have the ability to break down the peptide bonds in collagen, which is present in all animal tissues and consists of three polypeptide chains wound in a triple helical structure. Microbial collagenases split each polypeptide chain of collagen at multiple sites [1] and are able to hydrolyze both water-insoluble native and water-soluble denaturated collagen [2]. 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]. Clostridium histolyticum is an anaerobic, pathogenic, gram-positive peritrichal bacterium. Straight sticks of bacteria ((0.5 -0.9) × (1.3 -9.2) µm) are forming couples or short strings. Variety of proteolytic enzymes as collagenases, elastase, clostripain and neutral proteases are produced by Clostridium. Collagenases, a zinc-dependent metalloproteases, are divided into the two classes: Class I, encoded by gene ColG, and class II, encoded by gene ColH. 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).
Currently, the collagenase digestion method is the only way for successful pancreatic islets isolation from the surrounding acinar tissue for experimental as well as clinical purposes [4]. The enzyme is injected into the pancreatic duct of the exocrine system with the aim to selectively dissociate the pancreatic tissue and obtain intact pancreatic islets which constitute only 1% of the whole pancreas. Collagen is distributed within the pancreatic parenchyma and represents a major part of connective tissue fibers in capsule surrounding pancreatic islets; therefore collagenase is highly essential for islet release from pancreatic tissue. Important parameters for successful isolation are specific collagenolytic activity, possible presence of other proteolytic enzymes, absence of ballast or toxic substances, and of course the isolation costs.
Successful pancreatic islets isolation depends on many important factors [5] including digestion phase with the use of collagenase. While for clinical islet isolation only few more or less approved collagenase products have been accepted, for experimental purposes it is possible to use less purified and less expensive blends for achieving satisfactory results. At the present time, the batch to batch variability of commonly used crude collagenases for pancreatic islet isolations is caused by irregular concentrations of several proteases (collagenases, clostripain and neutral proteases). Assays for collagenase activity cannot accurately reflect the digestion effectiveness in vivo and for each new batch it is necessary to optimize the digestion phase in terms of concentration and duration. As the isolation outcome strongly depends on the enzyme efficacy, constant composition

Clostridium histolyticum and Collagenase Preparation
Clostridium histolyticum MB 204 was retained deep-frozen at −80˚C or as lyofilizate at 4˚C. After thawing, bacterial strain was cultured in trypton growth medium: Trypton 60 g/l, pepton 1.5 g/l, glucose 1.25 g/l, Na 2 HPO 4 3.4 g/l, collagen 50 g/l with vitamin K 0.01‰ and L-cystein 0.53 g/l (pH 8.4) for 24 hours at 37˚C ± 1˚C in anaerobic conditions. After that Clostridium was cultured in fermentor at the same conditions and pH 7.8 ± 0.5 till the bacterial growth was visible.

Bacterial Genome Sequencing
The bacterial genome of C. histolyticum strains were sequenced using the Nextera XT DNA Sample Preparation kit and MiSeq Reagent Kit v3 in the MiSEQ system (Illumina, USA). Obtained reads were assembled into contigs by de novo assembler in CLC. Rast were used for automatic annotation of contigs.

Assessment of Collagenase Activity
Collagenase activity was determined using spectroscopic method that measures collagenase cleavage of synthetic substrate Pz-PLGPR The assay was performed in 100 mM Tris buffer (pH 7.1). After 15 minutes of enzymatic reaction, yellow tripeptide fragment of Pz substrate was extracted into the ethyl acetate and absorbance was measured at 320 nm. Collagenase activity was defined as amount of degraded substrate micromoles by one gram of enzyme in one minute at 25˚C and expressed as PZS/g.

Western Blot
Western blot analysis [7] was performed on samples of lyophilized collagenase

Pancreatic Islet Isolation and Quality Testing
All experimental protocols were approved by Experimental Animals Welfare Sigma-Aldrich), in a humidified incubator at 37˚C and a 5% CO 2 atmosphere.
The quality of isolated islets was assessed by differential staining of live/dead cells and by glucose stimulated insulin secretion, as previously described [8].
Briefly, islet vitality was evaluated after staining with propidium iodide (dead red

Statistical Analyses
Results are expressed as mean ± SD. Lilliefors test for normality was performed to test a normal distribution of our data. Based on normality the Mann-Whitney

Results
Although To optimize fermentation and to enhance collagenase production, different growth media were tested: Trypton medium with beef extract, brain heart infusion (BHI), pepton or concentrated trypton medium. The best conditions for bacterial growth and also the highest collagenase production was achieved by concentrated trypton medium with pepton as it was evaluated by collagenase activity assessment (see Table 1).
To eliminate the loss of collagenase during fermentation, bacteria optical density was measured along with collagenase activity. Loss of enzyme occurred during the stationary phase of bacterial growth; therefore shorter fermentation and fresh medium addition were tested. We found out that optimal bacteria fermentation should take approximately 28 -30 hours, while longer fermentation require fresh growth medium addition to improve culture condition and enhance or maintain collagenase production by bacteria. Supply of concentrated media during fermentation increased collagenase activity from 210 -350 PZS/g to 780 PZS/g.
During the process of collagenase extraction, samples were taken and collagenase activity was tested to reveal critical points of the purification method. We found out that after ammonium sulfate precipitation considerable amount of collagenase remained in supernatant. Skipping this step had fatal consequences, a significant decrease in collagenase activity ranged from 54 to 80 PZS/g. Conversely, as the most effective was centrifugation to discard bacteria before precipitation with collagenase activity enhancement to 702 PZS/g. The obtained results also revealed that dialysis time can significantly influence the final collagenase activity. We found out that optimal dialysis time was 80 -90 minutes, shortened dialysis time resulted in decreased collagenase activity and similarly also increased dialysis time considerably reduced collagenase activity.
Ultrafiltration was used to remove ballast proteins and 50 kDa, 30 kDa and 10 kDa cassettes alone or in combination with 300 kDa cassette were tested. However the results showed big diversity in collagenase activity in dependence on the cut off cassettes, optimal was combination of 300 kDa with 30 kDa cassette.
Although collagenase activity was measured in all tested batches, detection of correct molecular weight (126 kDa) was performed as well. Samples were analyzed by western blotting to determine the intact collagenase or potential fragmentation (see Figure 1). Our results revealed samples of collagenases with no intact whole enzyme and simultaneously with no degradation fragments ( Figure   1(a)). Moreover, samples with no collagenase but only fragmentation of the enzyme (Figure 1 Table 2). Significant differences of digestion time were seen in Group 1 and in Group 2 (P < 0.001). In both mentioned groups there was lower number of In vitro analysis of the islet quality in Group 1 was not performed due to small number of isolated islets or none. Vitality of the isolated islets in Group 2 and 3 was comparable in majority of measurements (see Table 3). Significant difference between novel and standard collagenase was found only in Group 2 after 24 hours of cultivation (P < 0.01). Furthermore, analyses of the glucose stimulated insulin secretion assessed as stimulation index (see Table 4) revealed significant difference only in Group 2 (P < 0.01).

Discussion
In this study we systematically studied the activity of a new collagenase product proteases. Clostridium collagenases C1 and C2 hydrolyze collagen chains sequentially from the ends into mixture of small peptides [9]. It was reported that C2 was more efficient in collagen degradation [10] whereas C1 played only supporting role in islet isolations [11]. Study by Brandhorst et al. [12] showed that neither C1 nor C2 alone were able to dissociate pancreatic tissue. Rapid and complete loss of peri-islet ECM depends on both collagenase isoform and is best achieved with assistance of other proteolytic enzymes [ In this study we found out that the loss of enzyme occurred in few critical points of the process of Clostridium fermentation and collagenase purification.
These points were solved by specific modifications during the fermentation and purification processes. However, we are aware that for possible use in human islet isolation, higher grade of collagenase purification and efficacy confirmation will be needed. Our experimental work thus represents a prerequisite for scaling up of the collagenase production and for development of further steps in purification and standardization.