Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50 °C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53 ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147 kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25 kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20 °C for 12 weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

Diverse activities and biochemical properties of amylase and proteases from six freshwater fish species.

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.

Recombinant Expression, Purification and Characterization of Pyridoxal 5'-phosphate Synthase from Geobacillus sp. H6a, Thermophilic Bacterium Producing Extracellular Vitamin B6.

Background: Pyridoxal 5' -phosphate synthase (PLPS) is present in deoxyxylose 5'-phosphate-independent of the de novo vitamin B6 biosynthesis pathway. This enzyme complex consists of PdxS and PdxT, which function as synthase and glutamine amidotranferase respectively to produce PLP. Objectives: This study aimed to clone, express, and purify PLPS of Geobacillus sp. H6a, followed by its characterization. Material and Methods: The PdxS and PdxT genes were amplified from Geobacillus (Gh) sp. H6a. Recombinant vectors pET28a-GhpdxS and pET28a-GhpdxT were constructed and the resulting His-tagged proteins were expressed in E. coli BL21(DE3). The soluble rGhpdxS and rGhpdxT were purified via nickel-affinity chromatography and cation-exchange chromatography. The mixture of rGhpdxS and rGhpdxT was further characterized. Results: The molecular weights of rGhpdxS and rGhpdxT were estimated to be 35 and 23 kDa by SDS-PAGE, respectively. The native form of rGhpdxS showed hexamer and dodecamer, whereas those of rGhpdxT were a monomer upon detection with non-denaturing gel electrophoresis and gel filtration. A molar ratio of 1:1 of rGhpdxS:rGhpdxT showed the highest PLP synthesis activity (4.16 U.mg-1) and was used for analyzing the biochemical properties. The kinetic values were obtained by using glyceraldehyde 3-phosphate, ribose 5-phosphate, and glutamine as the substrates. The rGhPLPS showed pentose phosphate isomerization without triose phosphate isomerase activity. The metal ions affected PLP synthesis activity. The optimum pH and optimum temperature of rGhPLPS were 9 and 70 °C, respectively. The rGhPLPS was active over a broad range of temperatures and pH values. Conclusions: These results support the potential of rGhPLPS as a candidate for industrial application.