Autoproteolytic stability of a trypsin from the marine crab Cancer pagurus

Hehemann, Jan‐Hendrik; Redecke, Lars; Murugaiyan, Jayaseelan; Bergen, Martin von; Betzel, Christian; Saborowski, Reinhard

doi:10.1016/j.bbrc.2008.03.128

Cited by 15 publications

(7 citation statements)

References 30 publications

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“…Other important difference between decapod trypsin and bovine trypsin is autoproteinolytic stability of trypsins in decapods (Hehemann et al . ). Chymotrypsins in P. vannamei preferentially hydrolyse peptide bonds involving Tyr, Phe and Trp.…”

Section: Discussionmentioning

confidence: 97%

Exogenous proteinases as feed supplement for shrimp:in vitroevaluation

2013

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To test the efficacy of adding enzyme supplements to feeds, an in vitro study was conducted by mixing bovine trypsin or proteinases from the gastric juice of the Cortez swimming crab Callinectes bellicosus with an enzyme extract from the digestive gland of the whiteleg shrimp Penaeus vannamei. Enzymes alone and mixtures were tested for hydrolysing proteinaceous natural substrates (bovine casein, bovine haemoglobin, and bovine serum albumin). All enzyme preparations hydrolysed casein. Shrimp enzymes hydrolysed haemoglobin but not serum albumin. Bovine trypsin and crab proteinases hydrolysed serum albumin but not haemoglobin. The mixture of shrimp and crab enzymes generated more hydrolytic products of serum albumin than shrimp enzymes alone. Shrimp enzymes mixed with bovine trypsin did not hydrolyse albumin because the bovine trypsin vanished; shrimp enzymes hydrolysed bovine trypsin. Results indicated that it is naive to assume that proteinolytic enzymes from different species will add their catalytic capabilities if mixed; here, we demonstrated that they may act as proteinases and will hydrolyse available protein regardless of its function. Our conclusion is that enzyme supplements should be tested in vitro before intending them as exogenous proteinases in feeds. This technique can be used to assess the compatibility and additivity of proteinases when mixed for biotechnological purposes. Besides, the technique can demonstrate who hydrolyses whom.

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Section: Discussionmentioning

confidence: 97%

Exogenous proteinases as feed supplement for shrimp:in vitroevaluation

2013

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“…Subsequently, the gel spots were digested overnight at 37 1C using trypsin (Sigma, Munich, Germany), according to Shevchenko et al (1996), with minor modifications described elsewhere (Hehemann et al, 2008). The resulting mixture of peptides was extracted two times with acetonitril/85% formic acid (50%/5.8%, v/v).…”

Section: Sample Preparation and Maldi-msmentioning

confidence: 99%

Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures

et al. 2008

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It is still a challenge to link specific metabolic activities to certain species in a microbial community because of methodological limitations. We developed a method to analyze the specific metabolic activity of a single bacterial species within a consortium making use of [ 13 C 7 ]-toluene for metabolic labelling of proteins. Labelled proteins were subsequently analyzed by 2D gel electrophoresis (2-DE) and mass spectrometry (MS) to characterize their identity as well as their 13 C content as an indicator for function and activity of the host organism. To establish this method, we analyzed the metabolic incorporation of 13 C carbon atoms into proteins of Aromatoleum aromaticum strain EbN1. This strain is capable of metabolizing toluene under nitrate-reducing conditions and was grown in either pure culture or in a mixed consortium with a gluconate-consuming enrichment culture. First, strain EbN1 was grown with non-labelled toluene or labelled [ 13 C 7 ]-toluene as carbon sources, respectively, and their proteins were subjected to 2-DE. In total, 60 unique proteins were identified by MALDI-MS/MS. From 38 proteins, the levels of 13 C incorporation were determined as 92.3 ± 0.8%. Subsequently, we mixed strain EbN1 and the enrichment culture UFZ-1, which does not grow on toluene but on gluconate, and added non-labelled toluene, [ 13 C 7 ]-toluene and/or non-labelled gluconate as carbon sources. The isotope labelling of proteins was analyzed after 2-DE by MS as a quantitative indicator for metabolic transformation of isotopic-labelled toluene by the active species of the consortium. Incorporation of 13 C was exclusively found in proteins from strain EbN1 at a content of 82.6 ± 2.3%, as an average calculated from 19 proteins, demonstrating the suitability of the method used to identify metabolic active species with specific properties within a mixed culture.

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“…The marked autolysis susceptibility of KT1 makes sense for a highly active trypsin to avoid self-digestion of the enzyme-producing tissues [68–71], but is not in agreement with reported cases of crustacean trypsins [72]. Ca 2+ binding-induced stabilization has been demonstrated to provide protection against autolysis in mesophilic trypsins [68,69].…”

Section: Resultsmentioning

confidence: 99%

Cold Adaptation, Ca2+ Dependency and Autolytic Stability Are Related Features in a Highly Active Cold-Adapted Trypsin Resistant to Autoproteolysis Engineered for Biotechnological Applications

et al. 2013

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Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca2+ dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10° C and at least 50% more active than pig trypsin up to 50° C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca2+ concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca2+ dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.

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Autoproteolytic stability of a trypsin from the marine crab Cancer pagurus

Cited by 15 publications

References 30 publications

Exogenous proteinases as feed supplement for shrimp:in vitroevaluation

Exogenous proteinases as feed supplement for shrimp:in vitroevaluation

Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures

Cold Adaptation, Ca2+ Dependency and Autolytic Stability Are Related Features in a Highly Active Cold-Adapted Trypsin Resistant to Autoproteolysis Engineered for Biotechnological Applications

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