ABSTRACT
Thiaminase degrade thiamine into separate thiazole and pyrimidine moieties, an enzyme that cleaves vitamin B1 and are sporadically distributed among prokaryotes and eukaryotes. The excess thiaminase in fish tissues causes thiamine deficiency syndrome like neurological and cardiac disorder, it also causes reproductive disorder in fishes and finally leads to death. Thiaminase in human causes thiamine deficiency which leads to beriberi and Wernicke Korsakov syndrome or Wernicke paralysis, it also prevent the conversion of carbohydrate to glucose. Fishes rich in thiaminase are not good for human consumption because it leads to chronic thiamine deficiency.
This present study was carried out to determine the distribution, level and the effect of thiaminase in different tissues of some fish species.
A total number of 21 tissues were extracted from 7 different fish species (Gills, gut and flesh i.e. 3 from each).
In the tissue, Tilapia Zilli gut had the highest thiaminase activity (27.05±2.08) while Parachanna Africana gill had the lowest activity (0.09±0.12). Tilapia Zilli gut also have the highest specific activity (0.0058±0.0004) Parachanna Africana gill has the lowest specific activity (0.0000±0.0000). Clarias Gariepinus flesh has the highest protein concentration (14387.44±179.13) Hepsetus Odoe gut has the lowest protein concentration (791.28±13.33). Saroterodon Galilaeus species has the highest level of thiaminase activity (10.136ª) and Parachanna Obscura species has the lowest level of thiaminase activity (0.463ᵇ). Tilapia Zilli species has the highest specific activity of (0.0023ª) while Parachanna Africanna species has the lowest specific activity of (0.0002ᶜ).
Gut has the highest thiaminase activity of (11.315ª) while flesh has the lowest thiaminase activity of (1.916ᵇ). Gut also has the highest specific activity of (0.00304ª) and flesh also has the lowest specific activity of (0.00022ᵇ).
Therefore, the significance of the study is that excess thiaminase degrades thiamin and causes thiamin deficiency in fishes which leads to various ailments in fish and finally leads to death.
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
Enzymes are proteins made by cells that act as catalysts to speed up chemical reactions that occur in cells. The human digestive system contains many good examples of how enzymes function in the body. An enzyme’s job is to change molecules. An enzyme may add atoms to a molecule, remove atoms from a molecule, split a large molecule into two smaller molecules, or join together smaller molecules to form a larger molecule. But the important point is that enzymes always change molecules.
Enzymes have unique shapes and reactive sites that allow them to bind with specific molecules (called substrate molecules).When bound to substrate the enzyme puts a slight strain on the substrate providing the “push” needed to get the reaction started. This “push” is called the activation energy. In many reactions the activation energy is very large and would require a large input of energy to overcome without an enzyme. This energy could be provided by increasing the temperature but many molecules in the cell would subsequently be destroyed by the heat. Enzymes provide a means of activating specific chemical reactions at temperatures that are safe for the cell.
Thiaminases (thiamine: base 2-methyl-4-aminopyrimidine-5-methenyl transferase, EC 2.5.1.2) degrade thiamine by replacing the thiazole moiety with a variety of nucleophiles. They were observed in varieties of foods more than six decades ago. The activity of thiaminase has been detected in a wide variety of sources, including bacteria, m d Chick, 1977; Murata, 1982). Two types of thiaminase activity have been identified, thiaminase I and thiaminase II; both types operate by a pingpong kinetic mechanism, but type I is thought to be solely responsible for the cited thiamine deficiencies and it is the better characterized enzyme (Lienhard, 1970; Agee et al., 1973; Abe et al., 1987). The enzyme is an extracellular, 42-kDa enzyme and was purified from Bacillus thiaminolyticus (Douthit and Airth, 1966). A homogeneous preparation of thiaminase was obtained for the first time, from carp liver, a nonbacterial source (Bos and Kozik, 2000).
Thiaminase I has been reported to have a wide tolerance to the nature of the nucleophile (Lienhard, 1970). Thiamine functions as a precursor of thiamine diphosphate which serves as a co-enzyme in a number of the major metabolic pathways (Friedrich, 1987). The ability of a wide range of aromatic amines, heterocyclic bases and sulphydryl compounds to participate in the hydrolysis of thiamine by thiaminase I has been recognised for many years. Fujita et al. (1954) showed that some bases were better co-substrates than others.
Thiamine deficiency causes disease in fish (Fisher et al., 1996), foxes, Vulpes spp.minks, Mustela vison (Petrova et al., 2003), cats, Felis silvestris catus (Anderson and Morrow, 1987), dogs, Canis lupus familiaris (Houston and Hulland, 1988), chickens, Gallus gallus (Ishihara et al., 1974), ruminants (Roberts and Boyd, 1974), and humans (disease termed beriberi: Fujita, 1954; Adamolekun, 1994; nushimune et al., 2000; Luxemburger et al., 2003). Adamolekun (1993) also reported the prevalence of seasonal ataxia in the western Nigeria as a result of acute thiamine deficiency among the poor community who were marginally deficient in thiamine because of a limited diet of high carbohydrate meals containing thiamine-binding cyanogenetic glycosides.
Tissues are groups of cells with a common structure (form) and function (job).Tissues provide the numerous functions of organs necessary to maintain biological life. This lab exercise seeks to introduce the various tissues found in the human body and to familiarize you with their composition and function. The study of tissues is called histology, and is important to the understanding of how the human body is able to function as a unit. There are four main tissues in the body – epithelium, muscle, connective tissue and nervous tissue.
Individual organisms can usually be recognized, but the larger units we use to describe the diversity of life, such as populations, subspecies, or species, are not so easilyidentifiable. Taxonomists further group species into genera, families, orders, and kingdoms, while ecologists group species into higher structures such as communities and ecosystems. The justification for these group terms is utility, rather than intrinsicnaturalness. However, there will be a virtually infinite number of different, albeit nested, ways of classifying the same organisms, given that life has evolved hierarchically. Darwin (1859) felt that species were similar in kind to groupings at lower and higher taxonomic levels; in contrast, most recent authors suggest that species aremore objectively identifiable, and thus more ‘‘real’’ than, say, populations or genera. Today, much of ecology and biodiversity appears to depend on the idea that the species is the fundamental taxon, and many have argued that these fields could be undermined if,say, genera, or subspecies, had the same logical status as species. Species conceptsoriginate in taxonomy, where the species is ‘‘the basic rank of classification’’ according to the International Commission of Zoological Nomenclature. The main use of species in taxonomy and derivative sciences is to order and retrieve information on individualspecimens in collections or data banks.
AIMS AND OBJECTIVES
The objective of this study is to determine the level of thiaminase in different tissues (Gills, gut, flesh) of seven fish species in osu reservoir, osun state south western Nigeria, under which seven species of fish would be studied.
Furthermore, the research will help us know which fish species has the highest thiaminase activity.
1.2 LlTERATURE REVIEW
1.2.1 HISTORY OF THIAMINASE
Thiaminase activity has been reported previously in aquatic animals. Green (1936) observed paralysis among silver foxes fed raw carp. Later, Green et al. (1937) related this Chastek paralysis to vitamin B, deficiency and, in 1942, found the vitamin B, inactivating factor in the abdominal viscera, skin and head of the fish. Evans et al. (1942) described the brain lesions of Chastek paralysis in foxes. Deutsch and Hasler (1943) studied the distribution of the vitamin destructive enzyme and found it to be common in fresh water fish but not in the 9 varieties of salt water fish tested. Neilands (1947) reported thiaminase in scallop muscle. (Smith and Proutt 1944) described lesions in vitamin B1 deficient pigeons similar to Wernicke’s paralysis in humans (1881).
Fig 1: Structure of Thiaminase
1.2.2 CLASSIFICATION
Thiaminases degrade thiamin into separate thiazole and pyrimidine moieties. Two classes of thiaminases have been identified: thiaminase I (Costello et al.,1996; Nicewonger et al.,1995; Kelleher et al., 1995; Kelleher et al., 1997; Wu et al., 2000) and thiamine II(Campobasso et al 1998). TenA was shown to be the thiaminase II in B. subtilis (Toms et al., 2005).
Fig 2: Structure of Thiamine
Thiaminase I have been associated with early mortality syndrome in predatory fish of the Laurentian Great Lakes and the New York Finger Lakes (Honeyfield et al., 2005) as well as in Atlantic salmon in the Baltic Sea (Marquenski et al., 1997). Fish produce eggs with low levels of thiamin, which leads to a variety of illnesses and finally death between the time the fish are hatched and their first feeding. It is believed that the thiamin deficiency results from the egg-carrying female feeding on the nonnative forage fish alewife (Alosa pseudoharengus) that produces high levels of thiaminase I. The importance of thiaminase I is also indicated in the sidebar “Thiaminase I— The Culprit Behind a Disastrous Nineteenth Century Australian Expedition.” Catalytically, the difference between thiaminase I and II is seen in the substrates used to cleave the C-N bond connecting the two heterocyclic rings of thiamin. Thiaminase I can use aniline, cysteine, dithiothreitol, pyridine, quinoline, and veratrylamine as substrates (Costello et al., 1996,Lienhard .1970), and thiaminase II can only use water(Toms et al.,2005). Thiaminase I is also able to accept thiamin analogs, which vary extensively in the thiazole ring moiety but not in the pyrimidine moiety (Bos et al.,2000). The two classes of thiaminases have been shown to have dissimilar sequences and structures. It is also known that thiaminase II promotes the production of degradative enzymes—dubbed Deg proteins in B. subtilis. These proteins are expressed in bacteria during the transition between a growth phase and a stationary phase. The protein TenI has been shown to have the opposite effect on the production of Deg proteins.
Fig 3: Reactions of Thiaminase 1&11 in the cleavage of thiamin
1.2.3 DISTRIBUTION AND OCCURENCE
Thiaminases, enzymes that cleave vitamin B1, are sporadically distributed among prokaryotes and eukaryotes. Thiaminase I enzymes catalyze the elimination of the thiazole ring moiety from thiamin through substitution of the methylene group with a nitrogenous base or sulfhydryl compound. In eukaryotic organisms, these enzymes are reported to have much higher molecular weights than their bacterial counterparts. A thiaminase I of the singlecelled amoeboflagellate Naegleria gruberi is the only eukaryotic thiaminase I to have been cloned, sequenced, and expressed.
Fig 4: Stereoview ribbon diagram of the N. gruberi thiaminase I boundto 3-dzThi, a substrate analog (PDB ID code 4HCY). α-Helices are red and β-strands are blue. 3-dzThi, Glu241, Cys118, and Asp272 are green.
This structure was used to solve the structure of the enzyme in complex with 3 deazathiamin,a noncleavable thiamin analog and enzyme inhibitor (2.7 Å; R, 0.233; Rfree, 0.267). These structures define the mode of thiamin binding to this class of thiaminases and indicate the involvement of Asp272 as the catalytic base. This enzyme is able to use thiamin as a substrate and is active with amines such as aniline and veratrylamine as well as sulfhydryl compounds such as L-cysteine and β-mercaptoethanol as cosubstrates. Despite significant differences in polypeptide sequence and length, we have shown that the N. gruberi thiaminase I is homologous in structure and activity to a previously characterized bacterial thiaminase I.
Thiaminase I,an enzyme that degrades thiamin (vitamin B1) and thiamin diphosphate (TPP), the biologically active form of thiamin (Fulton et al., 2010). They showed that N. gruberi thiaminase I induces apoptosis via its enzymatic activity in that an enzymatically inactive mutant lost its ability to cause cell death (Fulton et al., 2010). Active thiaminase I depletes extracellular thiamin, which leads to subsequent depletion of intracellular TPP, an essential coenzyme for many enzymes involved in carbohydrate and energy metabolism, and in turn triggers apoptosis by an asyet- to-be-characterized mechanism. It has long been known that thiamin deficiency in animals can cause neurological and cardiac symptoms that ultimately lead to death of the animal. In humans, beriberi and Wernicke–Korsakoff syndrome are associated with chronic thiamin deficiency (Carpenter2000). Fulton and Lai have shown that the N. gruberi thiaminase I is also capable of killing cells resistant to commonly used chemotherapeutic agents and suggest that thiamin depletion-induced cell death may be a suitable candidate for use in tissue-targeted cancer therapies (Fulton et al., 2010)
1.2.4 THIAMINASE CATALYSIS
Thiaminases catalyze the cleavage of biologically active thiamin into its pyrimidine and thiazole ring moieties (Evans 1975), (Murata 1982)). These enzymes can be grouped into two classes defined by the nucleophile used in the mechanism by which the cleavage is accomplished. The thiaminase II (EC 3.5.99.2) class of enzymes exclusively uses water to accelerate the hydrolysis of thiamin into 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) and 4-methyl-5-(2-hydroxyethyl)thiazole and is found only in bacteria, fungi, and yeast (Wittliff etal.,1970). The thiaminase I (EC 2.5.1.2) class of enzymes uses a variety of aromatic and heterocyclic amines and sulfhydryl compounds as substituting bases in a nucleophilic displacement reaction on the methylene group of the pyrimidine moiety (Murata, 1982;Lienhard, 1970; Costello et al.,1996; Fujita, 1955; Wittliff, 1968). Thiaminase I is found in specific species of microorganisms such as Bacillus and Clostridium as well as multicellular organisms including certain ferns, insects, shellfish, and freshwater and ocean fish (Murata,1982; Fujita, 1955;Agee et al.,1973; Bos et al 2000; Fujita, 1954; Fujita,1972; Hayashi, et al.,1964; Nishimune et al., 2008; Nishimune et al., 2000; McCleary et al., 1977; Wittliff et al., 1970; Okonji et al., 2012; KuKu et al, 2012). Although distributed throughout the kingdoms, the phylogenetic distribution of thiaminase I, unlike most other enzymes, appears to follow no easily discernable evolutionary pattern. A physiological role has yet to be assigned for thiaminase I (Jurgenson et al., 2009), and several studies report that thiaminase I enzymes from eukaryotic organisms are produced as larger holoenzymes, 55–200 kDa, some of which are active as smaller fragments (Bos et al2000, Nishimune et al., 2008; Nishimune et al., 2000; McCleary et al., 1977; Okonji et al., 2012; KuKu et al., 2012). The presence of this enzyme in the diet of animals can have several deleterious effects and can even be fatal. It is responsible for thiamin deficiency and early mortality syndrome in Great Lakes salmonines and polioencephalomalacia in sheep.
Fig 5: Thiaminase degrading thiamin into its thiazole and pyrimidine moiety.
1.2.5 Thiaminase I
The structure of thiaminase I from Bacillus Thiaminolyticus shows an overall fold similar to that of group II periplasmicbinding proteins, such as the maltose-binding protein and the spermidine putrescine binding protein potD. The periplasmic binding proteins consist of two domains, each with an α/βfold. The two domains form a deep cleft and are connected by three crossover segments. This led to the proposal that thiaminase I may have evolved from an ancient periplasmic binding protein responsible for the uptake of thiamine (Campobasso et al., 1998). The physiological function of thiaminase I is not yet understood. The active site of thiaminase I is located in the cleft between the two domains. Six tyrosine residues and four acidic residues line the cleft. The active site was identified using the mechanism-based inhibitor 4-amino-6-chloro-2,5-dimethylpyrimidine, which bonds irreversibly to the active-site cysteine residue (Cys113)Thiamin is positioned in the active site by two hydrogen bonds between the pyrimidine moiety and Asp272, one of which occurs through an intervening water molecule. Glu241 then activates Cys113 for attack at C6 of the pyrimidine to form a zwitterionic intermediate. Nucleophilic attack and protonation by Glu241 result in cleavage of the bond between the thiazole and pyrimidine and release of products.
1.2.6 Thiaminase II
Ten A was shown to be thiaminase II, which uses water exclusively as the nucleophile to cleave the C-N bond connecting the pyrimidine and thiazole moieties (Toms et al., 2005). The crystal structure of Thiaminase II shows a bundle of 11 helices that surround a deep acidic pocket. The walls of the pocket are made of helices α4, α5, α9, and α10 with a total volume of approximately 700 ˚A3. The biological unit is a tetramer, with 222-point symmetry. Each monomer in the quaternary structure interacts with two other monomers. One interaction involves helices α7 and α11, and the other involves helices α4 and α5.
1.3 FISH TISSUES
1.3.1 FISH GILL
The location and basic structures of the gills of the agnathan hagfishes and lampreys, elasmobranchs, and teleosts are illustrated in Fig. below. In all fishes, the gills are bilaterally situated on either side of the pharynx and are composed of a series of pouch-like or arch-like structures that provide the physical support for the delicate gill filaments also term primary lamellae. The gills of the lampreys and higher fishes share a similar arch-like arrangement with intervening branchial slits for the water to pass laterally from the buccopharyngeal cavity through the gills and out. The hagfish gill does not have recognizable arch-like structures, and so its pouch gills will be dealt with separately.
Lining the sides of the gill arches are rows of regularly spaced filaments projecting posteriolaterally. Each row or stack of filaments constitutes a hemibranch, while a set of hemibranchs, one on each side of the arch, constitutes a holobranch. The gills of teleost fishes are composed of four such holobranchs spaced between five branchial slits(chambers) while the elasmobranchs have an extra hemibranch on the anterior side of the first branchial slit. The posterior side of the final branchial chamber does not have a hemibranch. Not included in the above count of hemibranchs is the pseudobranch, which is a reduced hemibranch present in most teleosts and elasmobranchs in association with the spiracles.
Fig 6: Structure of Fish gill
1.3.2 FISH FLESH
Fish fillet, as the main edible portion of fish, hold the main economic and nutritional interest of fish production. Increasing fillet yield, without any negative effect on flesh quality, is a major challenge for fish farmers. Even small differences in yields have a considerable economic impact for fish processing companies as the fillet constitutes the most valuable part of the product. For example, for the French trout production the price of the fillet is treble the price of the whole fish (unpublished data from an economic survey of the French “Comité Interprofessionnel des Produits del’Aquaculture”), so an increase of 5 units of fillet yield corresponds to an increase of 15 % in terms of added value. During fish processing for fillet production the main wastes are viscera (and associated perivisceral fat), head, skeleton, skin, fins and subcutaneous adipose tissue. Fish fillet is organized in myomeres, consisting mainly of muscle fibres, connective and adipose tissues. Fish with high fillet yield present higher muscle mass compared to fish with low fillet yield. This higher muscle mass is achieved all along the fish growth by the combined development of all muscle tissues.
Fig 7: Structure of Fish flesh/fillet
1.3.3 FISH GUT
Researchers found microplastics in the guts of all species, with no difference in amounts between pelagic and demersal species. Across all species, the average number of pieces of microplastic per fish was just less than two, although there were examples of fish ingesting up to 15 pieces. The quantities per individual fish were, on average, very low, and probably do not present a hazard to the fish. Neither do the results suggest a risk linked to human consumption, as the plastic was found in the gut – which we do not normally eat. However, the research does demonstrate that the problem of microplastics is widespread, not only in natural habitats but also in marine organisms.
Fig 8: Structure of Fish gut
1.4 FISHES
Fishes are highly important in the development of Nigeria both economically and health wise as source of protein with low cholesterol level in the diets of many populace as well as an intermediate host to some parasites. In sub-Saharan Africa, fish accounts for 10% of the animal protein consumed, and 98% of this is finfish (Delgado and McKenna, 1997). The average per capital consumption of fish in Africa in 1992 was about 8 Kg having increased from an average of 7 Kg per annum from 1969 to 1974 (Ahmed, 1997). Approximately 40% of fish consumed in Africa, south of the Sahara is freshwater fish as compared to the global average of 25% (Bonga, 1999). Fish is given different definitions depending on the purpose and the persons involved. Fish according to the zoologist is a streamlined cold-blooded aquatic vertebrate that moves by means of fins and breaths by means of gills while an economist considers fish to be any living aquatic resource exploitable by man for food, income, recreation and other economically useful purposes (Moses, 1983, 2002). However, Onuoha (2009) defined fish as jawed (Gnathostomata), aquatic (freshwater and marine), poikilothermic (cold-blooded), oviparous or oviviparous (producing eggs that develop and hatch outside or develop within and hatch within or immediately after extrusion from the parent), streamlined vertebrate with gill for respiration and fins for movement. Depending on usage, the term ‘fish’ may cover not only finfish, but also other aquatic vertebrates such as reptiles (crocodiles and turtles), mammals (whales and porpoises) and aquatic invertebrates often referred to shellfish e.g. crustaceans (shrimps, crabs and lobsters) and mollusks (oysters, clams, periwinkles and cephalopods). Historically, living aquatic resources have provided livelihoods and revenue for Nigerians, particularly, those living along the riverine and coastal areas (Moses, 1990). Federal Department of Fisheries (FDF), reported that in the agricultural sector of the Nigerian economy which employs about 70% of the active labour force, fish occupies a unique position in that it is the cheapest source of animal protein consumed by the average Nigerian, accounting for up to 50% of the total animal protein intake.
1.5 FISH SPECIES
1.5.1 TILAPIA ZILLI
Tilapia zilli considered a potential competitor with native fish for food and spawning areas, is a voracious herbivore which constitutes a serious threat to native aquatic plants, and the organisms that depend on these. Osinmo reservoir is located in the South-Western region of Nigeria. The reservoir is home to diverse species of fishes with Tilapia species ranking tops. The reservoir serves to provide part of the region with inland water and fishing activities is the major occupation of the locals. Proteins, essentially the amino acids are required for foetal development and growth. In dietary protein, the amino acids are needed principally for growth, metabolism and maintenance, especially in the young ones.