Atlantic Cod Trypsins: from Basic Research to Practical Applications Ágústa Gudmundsdóttir, Helga Margrét Pálsdóttir
Total Page：16
File Type：pdf, Size：1020Kb
Review ^OTCCmOLCX:Y Atlantic Cod Trypsins: From Basic Research to Practical Applications Ágústa Gudmundsdóttir, Helga Margrét Pálsdóttir Science Institute, University of Iceland, Læknagardi, Vatnsmÿrarvegi 16, Reykjavik, IS-101, Iceland Received: 27 May 2004 / Accepted: 24 June 2004/ Online publication: 15 March 2005 Abstract of exported goods from Iceland (Table 1). The decline of cod stocks in many areas has led to increased Atlantic cod trypsin I is an appropriate representa emphasis on cod farming. There is also heightened tive of the traditionally classified cold-adapted group awareness in many countries of the importance of I trypsins, and the recombinant form of cod trypsin Y increasing the value of seafood products by advanc is the only biochemically characterized member of ing the utilization of by-products from the fishing the novel group III trypsins. Trypsin Y is adapted to industries (Nafstad et aí., 2002; Rustad, 2003). The lower temperatures than all other presently known research presented here has been focused on that trypsins. This review describes the basic character issue from the beginning. istics of and practical uses for trypsins of Atlantic The pyloric cecum, serving the role of a digestive cod, as well as those of other organisms. Overex organ in the Atlantic cod, is a by-product of the pression of the recombinant forms of cod trypsins I fishing industry found in abundance in Iceland. As and Y in microorganisms is explained as well as the would be expected, it is rich in digestive enzymes advantages of using site-directed mutagenesis to in such as serine proteases (Asgeirsson et al., 1989). The crease their stability toward autolysis and thermal best known members of the serine protease family inactivation. Trypsins appear to play a key role in are trypsins, chymotrypsins, elastases, serine colla- the nutrition and development of marine fish. We genases, and brachyurins (Halfon and Craik, 1998). discuss the potential use of cod trypsins as biomar The serine proteases play important roles in a kers to evaluate the nutritional status of cod larvae number of biological functions. In digestion (Neu and describe the industrial applications of cod tryp rath, 1984) trypsin has a dual role in that it cleaves sin I and other trypsins. ingested proteins and activates the precursor forms of several other digestive proteases including chy- Key words: trypsins — fish — cold-adapted — motrypsin. Serine proteases from the Atlantic cod expression — practical applications such as trypsin (Asgeirsson et al., 1989), chymo- trypsin (Asgeirsson and Bjarnason, 1991), elastase (Asgeirsson and Bjarnason, 1993), and serine colla- Introduction genase (Kristjànsson et al., 1995) have typical char acteristics of cold-adapted enzymes. This review spans over two decades of research on The Atlantic cod is known to produce numer trypsins from the Atlantic cod (Gadus morhua). Its ous trypsin isozymes (Asgeirsson et al., 1989; main goal is to shed light on the basic properties of Gudmundsdóttir et al., 1993; Helgadóttir, 2002). these cold-adapted proteases as well as to view the Several of these have been isolated from their na numerous practical uses of trypsins from cod and tive source, with trypsin I being the most pre other organisms. The Atlantic cod is an economi dominant one. It also has the highest catalytic cally important fish species in Iceland and elsewhere efficiency and is by far the best characterized of in the Northern hemisphere. In 2002 cod products the trypsin isozymes (Asgeirsson et al., 1989; accounted for approximately 24% of the total value Jónsdóttir et al., 2004). The complementary DNAs of two trypsin isozymes (I and X) (Gudmundsdóttir et al., 1993), in addition to a novel trypsin termed Correspondence to: Ágústa Gudmundsdóttir,- E-mail: [email protected] trypsin Y (Spilliaert and Gudmundsdóttir, 1999), DOI: 10.1007/sl0126-004-0061-9 • Volume 7, 77-88 (2005) • © Springer Science+Business Media, Inc. 2005 77 78 Á g ú s t a G udmundsdóttir a n d H e lg a M a r g r é t P á l s d ó t t ir : A t l a n t ic C o d T r y psin s Table 1. Value of Exported Marine Products by Product Atlantic cod trypsin I has already proved useful Categories in 2002 Relative to All Exported Goods3 in industrial applications (Bjarnason et al., 1993; Product category Total (million ISK) (%) Value Bjarnason and Benediktsson, 2001) and medical All exported goods 204,000 100 applications (Bjarnason, 2000), as will be discussed Total marine products 129,000 63 later in this review. Other cold-adapted proteolytic Pelagic catch 25,000 12 enzymes have been applied as processing aids in the Demersal catch 78,000 38 food and feed industries, as was thoroughly described Cod products 49,000 24 in a review by Shahidi and Janak Kamil (2001). Other marine products 26,000 13 Sunde et al. (2001) demonstrated a significant “The value is given in million ISK (Icelandic kronas), fob. Also correlation between trypsin activity and the specific shown is the percentage value relative to all exported goods. Source: Statistics Iceland (External Trade, available at http:// growth rate of Atlantic salmon (Salmon salai). The www.hagstofan.is ). secretion rate of trypsin and chymotrypsin in marine fish has also been shown to be related to feed intake and filling of the stomach (Einarsson et al., 1996). have been isolated from a cod pyloric ceca cDNA Moreover, fish development appears to be highly library. Characterization of the recombinant tryp dependent on serine protease activity, and trypsin, in sin Y polypeptide demonstrated that it is very particular, has been suggested to play a key role in different from the classical trypsins, such as tryp larval survival (Porter and Theilacker, 1999). This is sin I (Pálsdóttir and Gudmundsdóttir, 2004), and due to the fact that trypsin activity, which is already may be the digestive enzyme produced under cold- present in fish larvae at first feeding or prior to the shock conditions (Roach 2002). development of the digestive system, is mainly In general trypsins from the Atlantic cod and responsible for the digestion of ingested food (Ko- other fish adapted to cold environments differ lovski, 2001). Trypsin is also a suitable short-term somewhat from their mammalian analogues in that indicator of nutritional quality in both farm-raised they have higher catalytic efficiencies, especially at and field-caught marine fish larvae (Nolting et al., low temperatures (Asgeirsson et al., 1989; Gerday 1999). Currently we are using knowledge about the et al., 2000; Schroder Leiros et al., 2000). These en Atlantic cod trypsins accumulated in our laboratory zymes are aiso more sensitive to inactivation by in the past in a research project aimed at increasing heat, low pH, and autolysis than their mesophilic the survival rate of farm-raised Atlantic cod larvae. analogues (Simpson et al., 1989; Asgeirsson et al., 1989). In addition, native proteins are easily hydro Trypsins I and Y lyzed by the cold-adapted fish proteases. These traits and the fact that the cold-adapted enzymes function Trypsin I. Most trypsins can be classified into 3 properly at low temperatures have stimulated inter basic groups, termed I, II, and III, based on their est in their commercial use, as they are generally amino acid sequence identities. The largest of these, better suited for enzymatic processes than their group I includes members like Atlantic cod trypsin I mesophilic counterparts (Bjarnason et al., 1993; and salmon trypsin I. All 3 trypsin groups, share the Bjarnason, 2000; Bjarnason and Benediktsson, 2001; catalytic triad residues His57, Aspl02, and Serl95 in Shahidi and Janak Kamil, 2001). addition to the obligatory Asp 189 localized at the The recent data presented on the expression of bottom of their substrate-binding pocket (Figure 1). Atlantic cod trypsin I (Jónsdóttir et al., 2004) and They also contain the Gly216 and Gly226 residues trypsin Y (Pálsdóttir and Gudmundsdóttir, 2004) are, lining the sides of the substrate-binding pocket and to our knowledge, the first reports on the expression Tyri 72, considered to be a key residue in trypsin of psychrophilic or cold-adapted proteolytic enzymes substrate specificity (Hedstrom et al., 1994). from fish in an active form. Difficulties related to the Three native trypsin isozymes, termed trypsins I, sensitivity of cold-adapted proteases to autolytic II, and III, were previously isolated from the pyloric degradation, thermal inactivation, as well as ceca of Atlantic cod and purified (Asgeirsson et al., molecular aggregation may account for the limited 1989). Trypsin I, the most abundant and best char number of publications in this area (Asgeirsson acterized form, also shows the highest catalytic et al., 1989; Asgeirsson and Bjarnason, 1991; Kris- efficiency (kcat/Km), which is approximately 20 times tjànsson et al., 1995; Helgadóttir, 2002). Site-directed higher than that of its mesophilic bovine analogue. mutagenesis of their cDNAs may be used to produce The high catalytic efficiency and low thermal sta new and improved recombinant enzyme variants bility of Atlantic cod trypsin I makes it an interest (Narinx et al., 1997; Benjamin et al., 2001; Gerike ing enzyme to study with respect to its structure- et al., 2001). function relationship. Atlantic cod trypsin I and Á g ú s t a G udmundsdóttir a n d H e l g a M a r g r é t P á l s d ó t t ir : A t l a n t ic C o d T r y p sin s 79 Group III -a 10 ia 30 40 30 57 60 70 w[ qJn p y A Q 1 A V V G D H H 1 WM Y E P olivaceus 3 M 1 G L i v [ l]