Characterization of acetylcholinesterase from the gut of sea cucumber Stichopus japonicus

An acetylcholinesterase was purified from the gut of sea cucumber Stichopus japonicus by anion exchange chromatography followed by gel filtration chromatography. The enzyme was purified 35.49-fold with a total yield of 7.73 %. The molecular mass of purified acetylcholinesterase was 68 kDa as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme displayed maximum activity at pH 7.5 and 35 °C with acetylthiocholine iodide as substrate. The enzyme activity appeared to be stable over pH 6.0–8.0 and up to 40 °C. It displayed an apparent Michaelis–Menten behavior in the concentration range from 0.1 to 0.8 mM with K _m values of 0.62 mM for acetylthiocholine iodide and 2.53 mM for butyrylthiocholine iodide. More than 95 % of acetylcholinesterase activity was inhibited by 1 mM eserine or 1,5-bis(4-allyldimethylammonium phenyl)-pentan-3-one dibromide (BW284C51), but only 19.1 % of the activity was inhibited by tetraisopropylpyrophosphoramide (iso-OMPA) at the same concentration. On the basis of the substrate and inhibitor specificities, the purified enzyme appeared to be a true acetylcholinesterase. Nevertheless, the purified acetylcholinesterase exhibited insensitivity to substrate inhibition phenomenon. Its biochemical properties were compared with those reported for different species.     

Purification and characterization of chymotrypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991

Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30–70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg²⁺, Fe²⁺, Cu²⁺, Li¹⁺, Mg²⁺, K¹⁺, Mn²⁺, while Ca²⁺ had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K _m and V _max values were 0.72 ± 1.4 mM and 1.15 ± 0.06 μmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.     

Viscera of Labeo rohita: A Potential Source of Trypsin for Industrial Application

ABSTRACT

The serine protease trypsin was isolated and purified from the digestive system of carp Labeo rohita rohu by ammonium sulphate precipitation, ion exchange, and affinity chromatography. The purified enzyme showed high activity between pH 7.0 and 9.0. The activity was maximum at 40°C. Incubation of the purified enzyme with CaCl₂ (2 mM) stabilized the enzyme activity for 8 h. The enzyme showed stability at 30 and 40°C for 1 h, but above 40°C, enzyme activity was reduced. The kinetic constants were recorded as K_m (0.104 mM), k_cat (44.25 s^?1), and catalytic efficiency (427.54 s^?1 mM^?1). Monovalent, bivalent, and trivalent ions (Li⁺, K⁺, Hg²⁺, Al³⁺, Mg^2+, Cd^2+, Co^2+, Zn²⁺, and Al³⁺) influenced the enzyme activity. Phenylmethylsulfonylflouride, soybean trypsin inhibitor, and N-α-p-tosyl-_L-lysine chloromethyl ketone completely inhibited the enzyme activity, while ethylenediaminetetraacetate caused partial inhibition. Molecular mass of the purified enzyme was 22.46 kDa. The pH and temperature stability of enzyme may be useful for its industrial applications.     

Purification and properties of a histidine decarboxylase from Staphylococcus epidermidis TYH1 isolated from Japanese fish-miso

Histidine decarboxylase (HDC) from Staphylococcus epidermidis TYH1, a halotolerant histamine-producing bacterium isolated from Japanese fermented fish-miso, was purified to homogeneity for the first time. The enzyme was purified 182-fold from cell-free extracts by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The N-terminal amino acid sequences of two polypeptide chains of 27–30 and 7–9 kDa were highly homologous with those of α- and β-chains of other staphylococcal HDCs. The optimum pH and temperature for the enzyme were 6.0 and 60 °C, respectively. This enzyme did not decarboxylate lysine, arginine, tyrosine, tryptophan or ornithine. The enzyme activity decreased with the addition of NaCl. At pH 4.8, the V _max and K _m values were 45.5 μmol histamine min^?1 mg^?1 and 1.10 mmol/L, respectively. Moreover, this enzyme was resistant to heat treatment (80 °C for 15 min) and was stable upon freezing at ?30 °C for 7 days. The very similar physiological properties of this enzyme and the almost identical N-terminal amino acid sequence to that of the HDC from S. capitis indicated that this enzyme may be evolutionally highly conserved in the genus Staphylococcus. The biophysical properties of staphylococcal HDC were elucidated using native purified enzyme.     

Anionic Trypsin from the Pyloric Ceca of Pacific Saury (Cololabis saira): Purification and Biochemical Characteristics

Anionic trypsin from Pacific saury (Cololabis saira) pyloric ceca was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. It was purified to 53.7-fold with a yield of 6.1%. The apparent molecular weight of the enzyme was about 24 kDa, as determined by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On native-PAGE, trypsin showed a single band. The purified anionic trypsin displayed optimal activity at pH 8.5 and 55°C. The enzyme was stable at neutral and alkaline pH and in the temperature range of 20–50°C. The stability was affected by the calcium ion. The activity of purified anionic trypsin was completely inhibited by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and partially inhibited by ethylenediaminetetraacetic acid (EDTA). NaCl (0–30%) decreased the activity in a concentration-dependent manner. The kinetic trypsin constants K_m and K_cat were 0.19 mM and 210 s^?1, respectively, while the catalytic efficiency (K_cat/K_m) was 1105.26 s^?1 mM^?1. The N-terminal amino acid sequences of anionic trypsin, IVGGYECQAH, were found and were homologous to those of trypsin from other fish species.     

Isolation and Characteristics of Carboxypeptidase B from Zebra Blenny (Salaria basilisca) Viscera

Carboxypeptidase B (CPB) from zebra blenny (Salaria basilisca) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 28-fold increase in specific activity and 21.72% recovery. The molecular weight of the enzyme was estimated to be 34.5 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60°C, respectively, using Hippuryl-l-Arg as a substrate. The enzyme was unstable above 50°C and below pH 5.0. The enzyme was activated by Co²⁺ and Zn²⁺ and inhibited by ethylenediaminetetraacetic acid (EDTA). The N-terminal amino acid sequence of the enzyme was determined as S P S Y T K Y N T. The CPB kinetic constants, K_m and k_cat for Hippuryl-l-Arg, were 0.32 mM and 36.23 s^?1, respectively.     

Purification and characterization of phospholipase A2 isoforms from the hepatopancreas of red sea bream, Pagrus major1

Two phospholipase A₂ (PLA₂) isoforms, tentatively denoted as DE-1 and DE-2 PLA₂s, were purified from the hepatopancreas of red sea bream (Pagrus major) to near homogeneity by sequential column chromatography on S-Sepharose fast flow, DEAE-Sepharose fast flow and butyl-Cellulofine, and by ion-exchange, gel-filtration and reversed-phase HPLC. The purified DE-1 and DE-2 PLA₂s both showed a single band with the apparent molecular mass of approx. 13.5 kDa by SDS-polyacrylamide gel electrophoresis, and were found to be both related to group I PLA₂ based on the N-terminal amino acid sequences. DE-1 PLA₂ had a pH optimum in the alkaline region at around pH 10 and required approximately 10 mM of Ca²⁺ and 4-10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine and phosphatidylethanolamine as substrates. DE-2 PLA₂ also had a pH optimum in the alkaline region at around pH 8-9 and required >10 mM of Ca²⁺ and approximately 6 mM of sodium deoxycholate for its maximum activity with 2 mM of phosphatidylcholine as a substrate; its enzymatic activity towards phosphatidylethanolamine was greatly inhibited by the addition of sodium deoxycholate. The results demonstrate that red sea bream hepatopancreas contains two enzymatically distinct group I PLA₂ isoforms.     

Purification and characterization of phospholipase A2 from the pyloric caeca of red sea bream, Pagrus major

A phospholipase A₂ was purified 55,000-fold in a yield of 10% from the lipid-free extract of powder of the pyloric caeca of red sea bream to near homogeneity by sequential column chromatography on S-sepharose fast flow, butyl-cellulofine, Asahipak ES-502C cation-exchange HPLC, TSK gel G3000SW gel-filtration HPLC, and Asahipak ODP-50 reversed-phase HPLC. The final preparation showed a single band with the apparent molecular mass of 14 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an estimated specific activity was 717 µmol min^-1 mg^-1 protein. The purified enzyme had a pH optimum in the range of pH 8.0–9.0 and required the presence of both 8 mM of Ca²⁺ and from 2 to 10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine as a substrate. The purified enzyme preferentially hydrolyzed the 2-acyl ester bonds of both phosphatidylglycerol and phosphatidylcholine in the presence of sodium deoxycholate, followed in order by phosphatidylethanolamine and phosphatidyl-serine. In contrast to porcine pancreatic PLA₂, pyloric caeca PLA₂ hydrolyzed mixed-micellar phosphatidylcholine substrate effectively, regardless of the kinds of bile salts used. These results indicate that Ca²⁺-dependent low molecular mass PLA₂, so called secretory PLA₂, occurs in the pyloric caeca of red sea beam.     

Environmental Effects on Seafood Availability,Safety, and Quality (Chemical & Functional Properties of Food Components). Edited by E. Grażyna Daczkowska-Kozon and Bonnie Sun Pan

Trypsin, with molecular weight of 28 kDa from the intestine of genetically improved Nile tilapia (Oreochromis niloticus), was purified by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. Purified trypsin had maximal activity at pH 8.0 and 60°C for hydrolysis of N _α-p-tosyl-L-arginine methyl ester. The enzyme was stable at temperatures up to 50°C and pH range of 6.0–11.0. Its activity was strongly inhibited by metal ions such as Pb²⁺ and Fe³⁺ and protease inhibitors including soybean trypsin inhibitor and phenylmethylsulfonyl fluoride. Also, the ion Ca²⁺ slightly inhibited this activity. The Michaelis-Menten constant (K _m) and catalytic constant (K _cat) of purified trypsin were 0.036 mM and 152 s^?1, respectively. Furthermore, trypsin contained low amounts of hydrophobic and aromatic amino acids as well as β-sheet (20.2%) and β-turn (25.0%).     

Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro

Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2′,5′-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K _m values for G6-P and NADP⁺ were determined to be 0.026, 0.0068 mM, respectively. The V _max values for G6-P and NADP⁺ were 2.20 and 2.27 μM min^?1 mg protein^?1, respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10^?6 min^?1 mg protein^?1, respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC₅₀ values of Zn⁺², Mn⁺², Al⁺³, Cu⁺², and Cd⁺² were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn⁺², Al⁺³, Cu⁺², and Cd⁺² were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn⁺², Al⁺³, and Cd⁺² showed competitive inhibition, while Cu⁺² inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.     

Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn²⁺ and Cu²⁺ and inactivated by Fe³⁺. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.     

Exploration of the antibacterial proteins in pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> induced by bacterial inoculation

The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 10¹² colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.     

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.     

Isolation,Purification, and Biochemical Characterization of Trypsin from Indian Mackerel (Rastralliger kanagurta)

Trypsin from viscera of Indian mackerel (Rastralliger kanagurta) was purified by ammonium sulphate precipitation and chromatographic techniques such as size exclusion, ion exchange, and affinity chromatography, with a 14.4-fold increase in specific activity and 18.7% recovery. The molecular weight of the trypsin was estimated to be approximately 26 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified trypsin showed amidase-specific activity which was determined using benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for isolated trypsin activity were 9.0 and 50°C, respectively. The purified trypsin was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK). Purified trypsin showed almost 40% recovery at high NaCl concentration (30%). The N-terminal amino acid sequence of the first 10 amino acids of purified trypsin was IVGGYESQPH. The Michaelis-Menten constant (K_m) and catalytic constant (K_cat) of purified trypsin were 0.430 mM and 0.77 s^?1, respectively, determined using BAPNA as a substrate. Purified trypsin showed digestion of casein similar to bovine trypsin by the fluorometric method.     

Purification and characterization of three trypsin isoforms from viscera of sardinelle (<Emphasis Type="Italic">Sardinella aurita</Emphasis>)

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20–70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0–11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K _m and k _cat were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s⁻¹, respectively. The N-terminal sequences of the first 10 amino acids were “I V G G Y E C Q K Y” for trypsin A and “I V G G Y E A Q S Y” for trypsins B and C. These sequences showed highly homology to other fish trypsins.     

Trypsin Enzyme from Viscera of Common Kilka (Clupeonella cultriventris caspia): Purification,Characterization, and Its Compatibility with Oxidants and Surfactants

The purification of trypsin from the common kilka (Clupeonella cultriventris caspia) viscera (pyloric caeca) resulted in a 28.3-fold increase and 12% yield by ammonium sulfate precipitation (30–60%), Sephadex G-75, and DEAE-cellulose chromatography. Trypsin showed a molecular weight of 23.2 kDa and appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, and zymography. The trypsin had optimal activity at pH 8.0 and 60°C for the hydrolysis of α-N-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) substrate. Trypsin was stable up to 50°C and at pH range of 7.0–10.0. Activity was significantly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) inhibitors (p < 0.05). The enzyme was relatively stable toward oxidizing agents, retaining 59.7 and 98.0% of its initial activity after 1 h incubation in the presence of 15% H₂O₂ and 1% sodium perborate, respectively. Trypsin was significantly activated by surfactants and Ca²⁺, Mg²⁺, and Mn²⁺ and inactivated by Fe²⁺, Zn²⁺, Cu²⁺, Al³⁺, Ba²⁺, and Co²⁺ (p < 0.05). Nevertheless, Na⁺ and K⁺ had no significant effect on trypsin activity (p > 0.05). The purified trypsin showed significantly higher catalytic efficiency (k_cat/K_m) than porcine pancreatic trypsin against BAPNA and N-α-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) substrates (p < 0.05).     

Trypsin from the viscera of Bogue (<Emphasis Type="Italic">Boops boops</Emphasis>): isolation and characterisation

Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS–PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-l-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants K _m and k _cat on BAPNA were 0.13 mM and 1.56 s⁻¹, respectively, while the catalytic efficiency k _cat /K _m was 12 s⁻¹ mM⁻¹. Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.     

Physicochemical and kinetic characteristics of rhodanese from the liver of African catfish <Emphasis Type="Italic">Clarias gariepinus</Emphasis> Burchell in Asejire lake

Two forms of rhodanese were purified from the liver of Clarias gariepinus Burchell, designated catfish rhodanese I (cRHD I) and rhodanese II (cRHD II), by ion-exchange chromatography on a CM-Sepharose CL-6B column and gel filtration through a Sephadex G-75 column. The apparent molecular weight obtained for cRHD I and cRHD II was 34,500 ± 707 and 36,800 ± 283 Da, respectively. The subunit molecular weight determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis was 33,200 ± 283 and 35,100 ± 141 Da for cRHD I and cRHD II, respectively. Atomic absorption spectrophotometric analysis revealed that cRHD II contained a high level of iron (Fe), which presumably was responsible for the brownish colour of the preparation. In contrast, no Fe was identified in cRHD I, and its preparation was colourless. Further characterization of cRHD II gave true Michaelis–Menten constant (K _m) values of 25.40 ± 1.70 and 18.60 ± 1.68 mM for KCN and Na₂S₂O₃, respectively, an optimum pH of 6.5 and an optimum temperature of 40°C. The Arrhenius plot of the effects of temperature on the reaction rate consisted of two linear segments with a break occurring at 40°C. The apparent activation energy values from these slopes were 7.3 and 72.9 kcal/mol. Inhibition studies on the cRHD II enzyme showed that the activity of the enzyme was not affected by Mn²⁺, Co²⁺, Sn²⁺, Ni²⁺ and NH₄ ⁺, but Zn²⁺ inhibited the enzyme considerably.     

Analysis of the α-amylase gene sequence and the enzyme activity of Indian rock oyster Saccostrea forskali

Indian rock oyster Saccostrea forskali is an important commercial species in Thailand. In this study, its full-length α-amylase (SfAmy) cDNA nucleotide sequence was investigated. The SfAmy cDNA was 1,689 bp long and contained a 1,563-bp open reading frame encoding 520 amino acid residues, including a 17-amino acid signal peptide. The molecular mass and the estimated isoelectric point (pI) of the deduced mature S. forskali α-amylase (SfAMY) were 55.948 kDa and 6.45, respectively. The deduced protein sequence showed 45–88 % identity to other mollusk AMYs. The molecular weight was confirmed by the weight of the purified native enzyme. The specific activities of crude and purified native enzymes toward 1 % starch were 29.53 and 187.42 U/mg. In addition, the obtained recombinant SfAMY also showed activity in digesting 1 % starch. The specific activities of the crude and purified recombinant proteins were 11.8 and 46 U/mg. Both enzymes showed optimal activity temperature at 40 °C but their optimum pH values were different, 6.0 for the native and 5.0 for the recombinant. The expression of SfAmy examined by RT-PCR showed the highest levels in the digestive gland but none was observed in the adductor muscle.     

Influence of feeding habits of fish on the characteristics of their visceral alkaline proteases with special reference to detergent compatibility

Technical characteristics and detergent compatibility of visceral alkaline proteases of three freshwater fish, namely Labeo rohita, Pangasianodon hypophthalmus and Cyprinus carpio of different feeding habits, were studied. Crude enzyme extract was partially purified by (NH₄)₂SO₄ precipitation and dialysis. The molecular weight was in the range of 20–63 kDa. The enzyme purification folds post‐dialysis were found to be 1.55, 1.81 and 2.17 in case of Rohu, Pangas and Common carp respectively. The alkaline protease from Rohu, Pangas and Common carp exhibited maximum activity at pH 10.0, 9.0 and 11.0 respectively. The enzyme temperature optima observed were 60°C (Rohu and Pangas) and 70°C (Common carp). SBTI and EDTA inhibited more than 90% of the activity at conc. of 50 mM. Exposure of the proteases to non‐ionic surfactants like Tween 20–80 retained about 92%–100% and 76%–100% of their activity at conc. (v/v) of 1% and 5% respectively. Proteases were found less stable in the presence of SDS. There was moderate to lesser influence of H₂O₂ and sodium perborate on the proteolytic activity. The alkaline protease from omnivorous fish was found superior compared to the herbivore and carnivore in respect of pH and temperature optima and stability with detergents and oxidizing agents.