Characteristics, protein engineering and applications of psychrophilic marine proteinases from Atlantic cod

Abstract

Hydrolytic enzymes, especially proteinases, have many uses and potential applications in industry, medicine and research. Among these are detergent production, leather processing, chemical modifications and food processing. Enzymes isolated from cold water marine organisms may prove to be especially useful for these purposes. The cold-active or psychrophilic enzymes are frequently more active at low temperatures than their mammalian or bacterial counterparts, a characteristic which could be beneficial in many industrial processes. The overall goal of this study is to investigate the structural mechanisms of temperature adaptation involved in cold-active enzymes. As a model system we will use cod trypsin-family serine proteinases which will be modified by site directed mutagenesis. Biochemical and biophysical methods will be used to study the native and modified forms of the enzyme. A mixture of proteinases, called Cryotin, was prepared by neutral extraction from Atlantic cod pyloric caeca. The preparation was shown to contain trypsin, chymotrypsin, elastase and collagenases. Trypsin was purified and resolved into three differently charged species termed cod trypsin I, II and III, with pI values of 6.6, 6.2, and 5.5 respectively, but a similar molecular mass of 24 kDa. The catalytic efficiency at 25°C expressed as k cat /K m was 17 times greater for cod trypsin I than bovine trypsin, when these enzymes were assayed as amidases. Atlantic cod trypsin demonstrated less resistance to thermal inactivation and treatment in mildly acidic solutions than bovine trypsin. The amino terminal sequence of cod trypsin enzyme I, the predominant species from Atlantic cod, showed similarities to other known trypsins, in particular the porcine and rat trypsins, with 30 identical residues out of 37, bovine trypsin, with 29 residues identical out of 37. Peptide mapping and partial amino acid sequencing of the three trypsin forms have shown that the cod trypsin isoenzymes are separate gene products. We have cloned and sequened the cDNA encoding cod trypsinogen III. Two chymotrypsins were purified with isoelectric points of 6.2 and 5.8, but a similar molecular mass of 26 kDa. It is not clear at this point whether they are separate gene products. The cod enzymes differed from bovine chymotrypsin in having more acidic isoelectric points and in being unstable in weakly acidic solutions. The cod enzymes were found to be more active than bovine chymotrypsin towards both ester and amide substrates. Cod chymotrypsin retained activity in 50% (v/v) of various organic solvent solutions. However, it was more thermolabile than bovine chymotrypsin. We have cloned and sequened the cDNA encoding one of the cod chymotrypsinogens. Elastase from cod has been purified extensively, and has recently been characterized. At least two distinct collagenases have been isolated from other known proteolytic activities in the Cryotin mixture. One of these has been highly purified and is precently being characterized. Cryotin, the mixture of proteinases from Atlantic cod, has many potential applications in industry and medicine, especially in food processing which require hydrolysis at low temperatures, inactivation under mild conditions or collagen digestion. It has proven promising in various fish processing applications such as skinning of fish, removal of membranes and ripening of herring. Cryotin also has potential as a digestive aid, both for humans and animals, and could be used as an adjunct in microdiets for fish larvae. Various food processing applications are also being considered, such as chill-proofing of beer, biscuit manufacture, tenderizing of meats, hydrolysis of various food proteins such as gelatin, vegetable proteins, and collagens.