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Biodegradation Properties
Published in Chih-Chang Chu, J. Anthony von Fraunhofer, Howard P. Greisler, Wound Closure Biomaterials and Devices, 2018
These in vivo studies of the accelerated degradation of natural absorbable sutures, when exposed to alimentary tract fluids, are consistent with in vitro and in vivo studies. Jenkins and Hrdina observed that plain catgut sutures were digested in an average time of 11 h, and chromic catgut sutures in excess of 20 h, in HCl and pepsin solution.22 Okada also found that an accelerated loss of catgut suture mass and strength was found in a pH 1.6 aqueous solution (0.5% by weight NaCl) with a variety of concentrations of pepsin enzyme and the rate of weight loss depended on the concentration of pepsin.13 Mizuma et al. tested the loop-breaking strength of various sutures in saline, canine serum, bile, and activated and nonactivated pancreatic juice, and found that both plain and chromic catgut sutures disintegrated almost completely within 24 and 48 h, respectively, in enterokinase-activated pancreatic juice (i.e., trypsinogen was activated into trypsin).23 In an effort to explore the mechanism of degradation of the catgut sutures, the same authors used trypsin inhibitors, such as aprotinin and soybeans, to examine the rate of loss of loop strength of the sutures. It was found that these trypsin inhibitors did not protect the sutures from degradation.23 This suggests that trypsin may not be the major enzyme responsible for the degradation of catgut sutures.
Development of clone with novel TrpE fusion tag in E. coli for overexpression of trypsin in a bench-scale bioreactor
Published in Preparative Biochemistry & Biotechnology, 2021
Santhosh Nagaraj Nanjundaiah, Jayasri MA, Sunilkumar Sukumaran, Ganesh Sambasivam
Trypsin (EC 3.4.21.4) is a highly valuable serine protease of molecular weight 23.3 kDa, which targets basic amino acids such as lysine and arginine at the C-terminus. The zymogen form of the enzyme called trypsinogen gets converted to trypsin by the addition of either trypsin or enterokinase. Trypsin plays a major role in metabolism, digestion and coagulation in mammals.[1] Besides, the enzyme is useful in leather bating, food processing, pharmaceuticals and clinical diagnosis.[2] The application of trypsin in cell culture mainly lies in the removal of adherent cells from the culture surface and in the resuspension of cells.[3] The optimal pH for trypsin activity is 7–9, hence the formulation buffer should be of acidic pH to prevent self-activation.[4] To date, trypsin used in the laboratory as well as on the commercial scale is obtained from bovine and swine pancreas. Nonetheless, if extracted from these sources, the risk of microbial load being carried over even after the purification step is high.[5] The use of recombinant enzymes can help to overcome this complication. The bovine trypsin gene has been widely expressed in both prokaryotes and eukaryotes. Even though proteins are expressed without any fusion tag, the use of such tags are desirable as the enzymes are highly prone to degradation even when expressed in prokaryotic hosts. Fusion tags increase enzyme stability. However, the size of the tag is an important factor as it determines the final yield. The tag-protein ratio when using glutathione S transferase, maltose binding protein and thioredoxin fusion tag with trypsin is about 1:1–1:3. Although the expression levels are high, the total product percentage is only around 33–50% of the inclusion bodies produced. Besides, some amount of the protein is present in the soluble fraction, resulting in the reduction of yield after the purification and refolding steps. In order to convert the soluble fraction of protein into insoluble fraction, the use of an insoluble fusion partner with a hydrophobic core is desirable. In this context, the use of fusion tags that are less than 2 kDa in size and a ratio of up to 1:8–1:10 (tag: protein) might reduce the cost of production by increasing the protein yield. This approach could result in 80–90% of the protein being pushed into the inclusion bodies fraction.