Improvement of α-amylase to the metabolism adaptions of soil bacteria against PFOS exposure.

Toxicity of perfluoroalkyl substances (PFASs) in soils towards bacteria shows an impact on its ecosystem function. This study aims to obtain insight into the effect of hydrolase (e.g. α-amylase) in soil on metabolism adaptions of bacteria (e.g. Bacillus substilis) against PFOS exposure. Results show that exogenous α-amylase alleviates PFOS toxicity to bacteria growth, disturbance to membrane permeability and stimulation to reactive oxygen species (ROS) production. The mechanisms were owing to that α-amylase strongly influences the strategies of metabolism adaptions of bacteria against PFOS stress. In details, α-amylase prompts bacteria to regulate the secretion of extracellular polymeric substances (EPSs) and the production of metabolic signal (acetic acid), which leads to changes in the physicochemical properties (hydrophilicity, surface charge) of the bacterial surface and the inactivation of the interaction with PFOS, thereby reducing the PFOS toxicity. Molecular simulations show that PFOS combines with Srt A at Gly 53 and Trp 171, which may induce the increase of permeability and changes of surface characteristics. Meanwhile, α-amylase competes with Srt A to bind PFOS at Arg 125 and Lys 176. This competition changes the physicochemical characteristics of PFOS and its bioavailability, further improving the metabolism adaptions of bacteria against PFOS. Altogether, this work provides direct evidences about α-amylase buffering effect of PFOS and demonstrates that the presence of α-amylase affects the essential but complex metabolic response in bacteria triggered by PFOS.

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

BACKGROUND: Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. RESULTS: We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. CONCLUSIONS: Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

Biochemical, structural and functional diversity between two digestive α-amylases from Helicoverpa armigera.

BACKGROUND: Helicoverpa armigera (Lepidoptera) feeds on various plants using diverse digestive enzymes as one of the survival tool-kit. The aim of the present study was to understand biochemical properties of recombinant α-amylases of H. armigera viz., HaAmy1 and HaAmy2. METHODS: The open reading frames of HaAmy1 and HaAmy2 were cloned in Pichia pastoris and expressed heterologously. Purified recombinant enzymes were characterized for their biochemical and biophysical attributes using established methods. RESULTS: Sequence alignment and homology modeling showed that HaAmy1 and HaAmy2 were conserved in their amino acid sequences and structures. HaAmy1 and HaAmy2 showed optimum activity at 60°C; however, they differed in their optimum pH. Furthermore, HaAmy2 showed higher affinity for starch and amylopectin whereas HaAmy1 had higher catalytic efficiency. HaAmy1 and HaAmy2 were inhibited to the same magnitude by a synthetic amylase inhibitor (acarbose) while wheat amylase inhibitor showed about 2-fold higher inhibition of HaAmy1 than HaAmy2 at pH7 while 6-fold difference at pH11. Interactions of HaAmy1 and HaAmy2 with wheat amylase inhibitor revealed 2:1 stoichiometric ratio and much more complex interaction with HaAmy1. CONCLUSIONS: The diversity of amylases in perspective of their biochemical and biophysical properties, and their differential interactions with amylase inhibitors signify the potential role of these enzymes in adaptation of H. armigera on diverse plant diets. GENERAL SIGNIFICANCE: Characterization of digestive enzymes of H. armigera provides the molecular basis for the polyphagous nature and thus could assist in designing future strategies for the insect control.

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases.

α-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4-7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (ß/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (ß/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting ß-glucan-active hydrolases.

Carbohydrases in the digestive system of the spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae).

The spined soldier bug, Podisus maculiventris, is a generalist predator of insects and has been used in biological control. However, information on the digestion of food in this insect is lacking. Therefore, we have studied the digestive system in P. maculiventris, and further characterized carbohydrases in the digestive tract. The midgut of all developmental stages was composed of anterior, median, and posterior regions. The volumes of the anterior midgut decreased and the median midgut increased in older instars and adults, suggesting a more important role of the median midgut in food digestion. However, carbohydrase activities were predominant in the anterior midgut. In comparing the specific activity of carbohydrases, α-amylase activity was more in the salivary glands (with two distinct activity bands in zymograms), and glucosidase and galactosidase activities were more in the midgut. Salivary α-amylases were detected in the prey hemolymph, demonstrating the role of these enzymes in extra-oral digestion. However, the catalytic efficiency of midgut α-amylase activity was approximately twofold more than that of the salivary gland enzymes, and was more efficient in digesting soluble starch than glycogen. Midgut α-amylases were developmentally regulated, as one isoform was found in first instar compared to three isoforms in fifth instar nymphs. Starvation significantly affected carbohydrase activities in the midgut, and acarbose inhibited α-amylases from both the salivary glands and midgut in vitro and in vivo. The structural diversity and developmental regulation of carbohydrases in the digestive system of P. maculiventris demonstrate the importance of these enzymes in extra-oral and intra-tract digestion, and may explain the capability of the hemipteran to utilize diverse food sources.

Salivary pellicles on titanium and their effect on metabolic activity in Streptococcus oralis.

BACKGROUND: Titanium implants in the oral cavity are covered with a saliva-derived pellicle to which early colonizing microorganisms such as Streptococcus oralis can bind. The protein profiles of salivary pellicles on titanium have not been well characterized and the proteins of importance for binding are thus unknown. Biofilm bacteria exhibit different phenotypes from their planktonic counterparts and contact with salivary proteins may be one factor contributing to the induction of changes in physiology. We have characterized salivary pellicles from titanium surfaces and investigated how contact with uncoated and saliva-coated titanium surfaces affects metabolic activity in adherent cells of S. oralis. METHODS: Salivary pellicles on smooth titanium surfaces were desorbed and these, as well as purified human saliva, were subjected to two-dimensional gel electrophoresis and mass spectroscopy. A parallel plate flow-cell model was used to study binding of a fresh isolate of S. oralis to uncoated and saliva-coated titanium surfaces. Metabolic activity was assessed using the BacLight CTC Vitality Kit and confocal scanning laser microscopy. Experiments were carried out in triplicate and the results analyzed using Student's t-test or ANOVA. RESULTS: Secretory IgA, α-amylase and cystatins were identified as dominant proteins in the salivary pellicles. Selective adsorption of proteins was demonstrated by the enrichment of prolactin-inducible protein and absence of zinc-α2-glycoprotein relative to saliva. Adherence of S. oralis to titanium led to an up-regulation of metabolic activity in the population after 2 hours. In the presence of a salivary pellicle, this effect was enhanced and sustained over the following 22 hour period. CONCLUSIONS: We have shown that adherence to smooth titanium surfaces under flow causes an up-regulation of metabolic activity in the early oral colonizer S. oralis, most likely as part of an adaptation to the biofilm mode of life. The effect was enhanced by a salivary pellicle containing sIgA, α-amylase, cystatins and prolactin-inducible protein which was, for the first time, identified as an abundant component of salivary pellicles on titanium. Further studies are needed to clarify the mechanisms underlying the effect of surface contact on metabolic activity as well as to identify the salivary proteins responsible for enhancing the effect.

Prolonged feed deprivation does not permanently compromise digestive function in migrating European glass eels Anguilla anguilla.

The effects of prolonged feed deprivation (40 days at 18° C) and re-feeding (30 days) on body mass, growth and the activity of selected pancreatic and intestinal enzymes were evaluated in migrating European glass eels Anguilla anguilla by comparison with a control group fed to satiation with hake Merluccius merluccius roe for the duration of the experiment. Feed deprivation resulted in mass loss and a reduction in digestive function, as revealed by a decrease in the total and specific activities of pancreatic (trypsin and α-amylase) and intestinal brush border (alkaline phosphatase and leucine aminopeptidase) enzymes. The total activity of intestinal brush border enzymes diminished after 5 days of feed deprivation, whereas that of pancreatic enzymes did not decrease until 10 days, indicating that the intestine is more sensitive to feed deprivation than the pancreas. Re-feeding A. anguilla that were starved for 40 days resulted in compensatory growth, with specific growth rates that were 2·6 times higher than the control group. This compensatory growth was associated with the recovery of trypsin and intestinal brush border enzyme activities, which were restored to control levels within 5 days of re-feeding. The ability to maintain pancreatic enzyme activity during 40 days of feed deprivation, and rapidly recover capacity for protein digestion upon re-feeding, would enable A. anguilla at this glass eel stage to withstand periods without food but rapidly provide amino acids for protein synthesis and growth when suitable food was available.

Digestive alpha-amylases from Tecia solanivora larvae (Lepidoptera: Gelechiidae): response to pH, temperature and plant amylase inhibitors.

The biochemical properties of the digestive alpha-amylase from Tecia solanivora larvae, an important and invasive insect pest of potato (Solanum tuberosum), were studied. This insect has three major digestive alpha-amylases with isoelectric points 5.30, 5.70 and 5.98, respectively, which were separated using native and isoelectric focusing gels. The alpha-amylase activity has an optimum pH between 7.0 and 10.0 with a peak at pH 9.0. The enzymes are stable when heated to 50 degrees C and were inhibited by proteinaceous inhibitors from Phaseolus coccineus (70% inhibition) and P. vulgaris cv. Radical (87% inhibition) at pH 6.0. The inhibitors present in an amaranth hybrid inhibited 80% of the activity at pH 9.0. The results show that the alpha-amylase inhibitor from amaranth seeds may be a better candidate to make genetically-modified potatoes resistant to this insect than inhibitors from common bean seeds.

Salivary alpha-amylase in biobehavioral research: recent developments and applications.

In the history of science, technical advances often precede periods of rapid accumulation of knowledge. Within the past three decades, discoveries that enabled the noninvasive measurement of the psychobiology of stress (in saliva) have added new dimensions to the study of health and human development. This widespread enthusiasm has led to somewhat of a renaissance in behavioral science. At the cutting edge, the focus is on testing innovative theoretical models of individual differences in behavior as a function of multilevel biosocial processes in the context of everyday life. Several new studies have generated renewed interest in salivary alpha-amylase (sAA) as a surrogate marker of the autonomic/sympathetic nervous system component of the psychobiology of stress. This article reviews sAA's properties and functions; presents illustrative findings relating sAA to stress and the physiology of stress, behavior, cognitive function, and health; and provides practical information regarding specimen collection and assay. The overarching intent is to accelerate the learning curve such that investigators avoid potential pitfalls associated with integrating this unique salivary analyte into the next generation of biobehavioral research.

[Saliva as a main component of oral cavity ecosystem. Part II. Defense mechanisms]. / Slina jako glówny skladnik ekosystemu jamy ustnej. Czesc II. Mechanizmy odpornosciowe.

Human saliva not only lubricates the oral cavity, making possible functions such as swallowing and speaking, but it also helps to maintain integrity of the hard tissues of the teeth. In addition to secretory immunoglobulins saliva contains several less specific antibacterial systems. This innate defense system includes: lysozyme, lactoferrin, peroxidase system, histatins, mucins, and other polypeptides with basic side chains. Some proteins of innate defense system have bactericidal or bacteriostatic effects; some can cause aggregation of oral bacteria resulting in their increased clearance from oral cavity.

Characterization of a partial alpha-amylase clone from red porgy (Pagrus pagrus): expression during larval development.

A partial alpha-amylase cDNA was isolated from red porgy (Pagrus pagrus, Teleostei: Sparidae) and its tissue specific expression during larval development was examined. The cDNA was 949 bp long and showed 90% identity with other fish amylases. A 545 bp fragment was used to study amylase expression using in situ hybridization and RT-PCR techniques. Both methods showed a similar pattern: high and relatively constant expression for the first 30 days after hatching (dah), subsequently decreasing until the end of the experiment at 60 dah. The goal of this work was to extend the existing knowledge of the functionality of larval fish digestive systems and to provide new information about alpha-amylase gene expression.

Isolation and identification of alpha-amylase producing Bacillus sp. from dhal industry waste.

A bacterial strain was isolated from dhal industry red gram waste and identified as Bacillus. A thermostable extracellular amylase was partially purified from the strain. Optimum temperature and pH for the enzyme were found to be 60 degrees C and 6.5, respectively. The maximum amylase production was achieved with maltose as carbon source. Among the nitrogen sources, peptone and yeast extract produced maximum amylase.

An increase in the transglycosylation activity of Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.

The 84th tryptophan residue in Saccharomycopsis alpha-amylase molecule was replaced by a leucine residue and the resulting site-directed mutant, W84L enzyme, showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. Analysis of the reaction products from p-nitrophenyl maltooligosaccharides indicated that these large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the tryptophan residue located at subsite 3 of the enzyme plays an important role not only to hold the substrate, but also to liberate the hydrolysis products from the substrate binding pocket.

Salivary amylase - The enzyme of unspecialized euryphagous animals.

INTRODUCTION: Alpha-amylase (EC 3.2.1.1) is the most abundant enzyme in the saliva of man and of several vertebrates. In humans, salivary amylase is mainly formed in the parotid gland; its activity is of high inter-individual and intra-individual variability. The physiological functions of α-amylase have not yet been explored completely. It is well known that the enzyme cleaves the α-(1,4)-glycosidic bonds of polysaccharides. Furthermore it plays an important role in initial bioadhesion in man, facilitating carbohydrate metabolism and bacterial adherence at the tooth surface and therewith caries initiation. Nevertheless, it is still a matter of interest why humans have such high amounts of salivary amylase. OBJECTIVE: The review presents an evolutionary approach by considering salivary amylase in the animal kingdom with special focus on mammalians divided into the three main nutritional types carnivores, herbivores, and omnivores; it was postulated that for most mammalian animals salivary α-amylase is essential. RESULTS: The enzyme has been detected in saliva of some herbivores and many omnivorous animals, but not in pure carnivores. Focusing on ruminants, negligible levels or an absence of α-amylase was determined. Presence and activity probably differ depending on the species-specific diet. Animals feeding on unripe fruits, seeds, roots and bulbs exhibit higher activity of salivary α-amylase than species consuming ripe fruits, insects, and vertebrates. CONCLUSION: In contrast to carnivores and most herbivores, omnivores have considerable amounts of amylase in their saliva. Though, the starch-digesting enzyme has been investigated well, the physiological function of amylase in saliva has not yet been explored completely. It can be hypothesized that nutritional habits affect expression of enzymes in the saliva of animals. It has to be verified, whether α-amylase is genetically or epigenetically determined. As a consequence of the development of agriculture, and following dietary changes, amylase can be recognized as a nutritional and evolutionary marker. Interdisciplinary evolutionary research might offer new perspectives for preventive dentistry.

Alpha-Amylase Activity in Blood Increases after Pharmacological, But Not Psychological, Activation of the Adrenergic System.

BACKGROUND & AIM: Alpha-amylase in both blood and saliva has been used as a diagnostic parameter. While studies examining alpha-amylase activity in saliva have shown that it is sensitive to physiological and psychological challenge of the adrenergic system, no challenge studies have attempted to elucidate the role of the adrenergic system in alpha-amylase activity in blood. We set out to examine the impact of psychological and pharmacological challenge on alpha-amylase in blood in two separate studies. METHODS: In study 1, healthy subjects were examined in a placebo-controlled, double-blind paradigm using yohimbine, an alpha2-adrenergic antagonist. In study 2, subjects were examined in a standardized rest-controlled psychosocial stress protocol. Alpha-amylase activity in blood was repeatedly measured in both studies. RESULTS: Results of study 1 showed that alpha-amylase in blood is subject to stronger increases after injection of yohimbine compared to placebo. In study 2, results showed that there was no significant effect of psychological stress compared to rest. CONCLUSIONS: Alpha-amylase in blood increases after pharmacological activation of the adrenergic pathways suggesting that sympathetic receptors are responsible for these changes. Psychological stress, however, does not seem to have an impact on alpha-amylase in blood. Our findings provide insight into the mechanisms underlying activity changes in alpha-amylase in blood in healthy individuals.

Cytochemical and immunocytochemical characterization of a fibrillar network (GP2) in pancreatic juice: possible role as a sieve in the pancreatic ductal system.

The secretory product of the exocrine pancreas contains sedimentable and non-sedimentable materials. Electron microscopy of the pellet obtained after ultracentrifugation reveals two major components: microvesicles (pancreasomes) and a fibrillar network of small mesh size. Negative staining of an unfixed pellet demonstrated that these structures are not fixation artifacts. Cytochemical analysis showed that pancreasomes are reactive to osmication and uranyl acetate staining, whereas the fibrillar network was unreactive thereby indicating that the latter does not contain lipids; however, lead citrate staining reveals the network. Alcian blue, known to bind sulfate groups of mucosubstances, reacted strongly with the fibrillar network. The pellet was also characterized by immunocytochemistry with specific antibodies to amylase and glycoprotein 2 (GP2). Both antibodies were located only on the fibrillar network. Washing of the pellet with 100 mM KCl-250 mM NaBr had little effect on GP2 content, but reduced considerably alpha-amylase associated with the reticular matrix. It appeared that GP2 was the major component of the scaffolding that gives rise to the fibrillar network and that other proteins such as alpha-amylase could reversibly bind to it. When double-labeling immunocytochemistry was carried out on the unwashed pellet, labeling of the first antigen reduced the labeling of the second. Removal of amylase by washing the pellet increased the GP2 signal. These results indicate that amylase is bound on the GP2 network. Although the function of the GP2 network is still not clearly defined several possibilities could be envisaged at the level of the pancreatic duct system: 1) The network could drain off any aggregates or precipitates forming in small ducts. 2) The small mesh of the network would present a physical barrier to infecting bacteria that could enter into the duct system from the intestine, especially in conditions of low flow rates. 3) The network may exert a mechanical pressure on the membranes bordering the acinar lumen and small ducts thereby preventing their collapse in basal conditions.

Inspection of active sites of human salivary alpha-amylase isozymes by means of non-reducing-end substituted maltooligosaccharides with 2-pyridylamino residue.

The modes of action of four alpha-amylase isozymes, which were purified from human saliva, on p-nitrophenyl alpha-maltopentaoside (G5P), maltohexaitol (G6R), and their 2-pyridylamino derivatives, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG5P) and O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O- alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D- glucitol (FG6R) were examined at various pH values. No differences in their modes of action on the substrates was found. Irrespective of which enzyme was used, the molar ratio of the hydrolysis products of G5P or G6R was almost constant at any pH examined. On the other hand, those of FG5P and FG6R varied with pH such that predominantly O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG3) was formed at high pH ranges, while the formation of O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-gl ucose (FG4) increased at lower pH. The result indicates that the binding mode of FG5P or FG6R to the active sites of the enzymes changed with pH; namely, interactions between the 2-pyridylamino residue of the substrates and some amino acid residue(s) located in the active sites were influenced by pH.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of alpha-amylase in the perception of oral texture and flavour in custards.

The role of salivary alpha-amylase in odour, flavour, and oral texture sensations was investigated in two studies in which the activity of salivary amylase present in the mouth of human subjects was either increased by presenting custards with added alpha-amylase or decreased by presenting custards with added acarbose, an amylase inhibitor. For starch-based vanilla custard desserts, amylase resulted in increased melting and decreased thickness sensations, whereas acarbose had the opposite effect, i.e., decreased melting and increased thickness. Other affected attributes included creamy mouth feel, creamy after feel, and fatty after feel. Creaminess, which is considered to be a highly desirable food quality, decreased by as much as 25% with added amylase and increased by as much as 59% with added acarbose. Neither additional amylase nor acarbose affected sensations for a nonstarch-based carboxy methylcellulose (CMC) vanilla custard dessert. This indicates that the effects of amylase on viscosity-related sensations of starch-based custards, such as perceived melting and thickness, are caused by amylase-induced breakdown of starch. Partial Least Square (PLS) analysis indicated that the effects of amylase and acarbose on perceived creaminess are not only driven by their effects on perceived melting and thickness, but also by their effects on perceived flavour.

Alpha-amylase circadian rhythm of young rat parotid gland: an endogenous rhythm with maternal coordination.

The circadian rhythm of alpha-amylase, E.C. 3.2.1.1. alpha-1,4-glucan-4-glucanohydrolase) in the parotid glands of 25-day-old rats were studied under different experimental designs (fasting, reversed photoperiod, constant lighting conditions and treatment with reserpine and alpha-methyl-p-tyrosine). The rhythm of fasted rats did not change. There were modifications in the rhythm of rats submitted to a reversed photoperiod or treated with reserpine or alpha-methyl-p-tyrosine. The rhythm was present, with changes in the acrophase, in parotids of rats kept during their gestation and postnatal life in constant light or dark. Results suggest that the circadian rhythm of alpha-amylase in parotid gland of young rats is endogenous, synchronized by the photoperiod, and with maternal coordination.

Starch digestion in tropical fishes: isolation, structural studies and inhibition kinetics of alpha-amylases from two tilapias Oreochromis niloticus and Sarotherodon melanotheron.

alpha-Amylases from the intestinal cavity of two tilapia species, Oreochromis niloticus (ONI-AMY) and Sarotherodon melanotheron (SME-AMY), were purified using ammonium sulfate precipitation, affinity chromatography and chromatofocusing procedures. The purification was approximately 100-fold. The amylolytic activity, specific activity, product distribution, pH and temperature profile of ONI-AMY and SME-AMY are quite similar. The molecular mass differs slightly: 56600 (ONI-AMY) vs. 55500 (SME-AMY). As shown by isoelectric focusing analysis, both amylases contain two isoforms A and B with distinct pI: 7.2 (A) and 7.8 (B), vs. 8.3 (A) and 8.8 (B), respectively. It was not possible to isolate B, since B converts into A with time. The kinetics of the inhibition of ONI-AMY and SME-AMY activity by alpha-, beta- and gamma-cyclodextrin (alpha-, beta- and gamma-CD) were investigated using amylose as the substrate. Statistical analysis of the kinetic data expressed using a general velocity equation and assuming rapid equilibrium showed that the inhibition is of the mixed noncompetitive type. Similar results were obtained with ONI-AMY and SME-AMY. beta- and gamma-CD are stronger inhibitors than alpha-CD. ONI-AMY and SME-AMY are then closely related and show the general features common to the members of the alpha-amylase class (family 13). They enable ONI and SME tilapias to digest starch in food.