No abstract

This study investigated the biochemical properties, enzyme activities, isoenzyme pattern, and molecular weight of three types of digestive enzyme from six freshwater fish species: Puntius gonionotus (common silver barb), Puntioplites proctozysron (Smith’s barb), Oreochromis niloticus (Nile tilapia), Hemibagrus spilopterus (yellow mystus), Ompok bimaculatus (butter catfish), and Kryptopterus geminus (sheatfish). The optimum pHs for amylase and alkaline protease activities were 7.0–8.0 and 8.0–10.0, and the optimum temperatures were 45–60 °C and 50–55 °C, respectively. A pepsin-like enzyme was detected in all three carnivorous fishes (Ompok bimaculatus, Kryptopterus geminus, and Hemibagrus spilopterus) with optimum reaction pH of 2.0 for each and optimum reaction temperatures 50–55 °C. In optimum reaction conditions, the amylase and alkaline protease from Puntioplites proctozyron showed the highest activities. Lower activities of all enzymes were observed at temperature (29 °C) of Lam Nam Choen swamp than at the optimum reaction temperatures. The fish species contained one to three and five to eight isoforms of amylase and alkaline protease, respectively, with molecular weights from 19.5 to 175 kDa. Both the alkaline proteases and amylases were stable in wide pH and temperature ranges.

Cellulase plays an important role in cellulose degradation. The enzyme catalyzes the cleavage of b-1,4 glycosidic bond between glucose residues. The Macrotermes gilvus cellulase was purified by using ammonium sulfate precipitation and anion exchange column with 1.38% recovery and 22-fold purification. The SDS-PAGE coupled with zymogram analysis revealed the molecular weights approximately of 54 kDa. The biochemical properties of the enzyme exhibited the optimum temperature and optimum pH of 45°C and 5.2. Interestingly, the enzyme was active over a wide range of temperatures (7-70°C) and a broad range of pH values (4.5-8). At the indicated temperatures and pH values, the enzyme exhibited more than 84 and 50% of its activity. The thermal stability and pH stability of the enzyme were also investigated. The result showed that the enzyme retained nearly 40% of its original activity after incubation in mild acidic (pH 5.2), neutral (pH 7.0) and basic (pH 10.0) conditions for 5 h. The enzyme retained its activity more than 70% of initial activity at both 37 and 45°C after incubation for 3 h. Moreover, the activity of the enzyme was strongly inhibited by Cu slightly increased the enzyme activity. Due to the wide temperature and pH range of enzyme activity, the Macrotermes gilvus cellulase might be potential enzyme for industrial or agricultural application.

Pyridoxal kinase encoded by pdxK gene, is the important key enzyme in the salvage pathway of vitamin B6 biosynthesis. The enzyme catalyzes the phosphorylation of the 5′ alcohol groups of free form vitamin B6 into their 5′-phosphate forms that requires metal ion and ATP. Pyridoxal kinase have been reported in many organisms except in the thermophilic bacterium. Therefore, this study aimed to clone, express and characterize pyridoxal kinase of Geobacillus sp. H6a isolated from the hot spring in the North of Thailand. The GhpdxK gene (810 base pairs) was inserted into pET28a(+) plasmids at restriction site of NdeI and BamHI and transformed into E.coli BL21(DE3). The expressed pyridoxal kinase of this bacterium exhibits a homodimer, in which each subunit had a molecular mass of about 32 kDa when examined by SDS-PAGE and gel filtration. The enzyme showed maximal activity at 70°C and at pH 8.0. The expressed enzyme obtained in this study was found to be more active (>50%) in the broad pH range (6.0 – 9.0) than those previously reported. This enzyme prefers Mg2+ and also accepts other cations to the less extent. Under optimal conditions, the expressed enzyme has higher affinity toward PN (20 ± 1.35 µM), while it showed the same affinity to pyridoxal (100 ± 0.76 µM) and pyridoxamine (100 ± 1.21 µM). The Km value for ATP and 4-amino-5-hydroxymethyl-2-methylpyridine were 8.99 ± 1.76 µM and 19 ± 0.85 µM, respectively. With high activity at high temperature and active in the broad pH range, it could be considered as a potential candidate for future application particularly bioconversion of vitamin B6.