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Purification and some properties of endo-1,4-beta-D-mannanase from a mud snail, Pomacea insularus (de Ordigny)
1Department of Applied Microbial Technology, Kumamoto Institute of Technology, Japan.
Abstract:
Endo-1,4-beta-D-mannanase (1,4-beta-D-mannanohydrolase, EC 3.2.1.78) was purified from viscera of a mud snail, Pomacea insularus (de Ordigny). The purified enzyme gave a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was estimated to be 44,000. The amino-terminal sequence was H.Gly-X-Leu-Arg-Arg-Gln- Gly-Thr-Asn-Ile-Val-Asp-Ser-His-Gly-His-Lys-Val-Phe-Leu-Ser-Gly-Ala-Asn- Thr-Ala-Trp-Val-Ala-Tyr-Gly-Tyr-Asp-. The enzyme was stable from pH about 5.0 to about 10.5 and had its maximum activity at pH about 5.5. The purified enzyme produced M2, M3, M4, and M5 from beta-1,4-mannan. Enzyme activity was greatly inhibited by Ag+, Hg2+, Cu2+, and dithiothreitol at 1 mM concentration. In addition, N-bromosuccinimide completely inhibited the enzyme activity.
Insights
Researchers purified endo-1,4-beta-D-mannanase from mud snail viscera. This enzyme efficiently breaks down beta-1,4-mannan into smaller oligosaccharides, showing stability across a wide pH range.
Area of Science:
- Biochemistry
- Enzymology
- Marine Biology
Background:
- Endo-1,4-beta-D-mannanase is a key enzyme in carbohydrate metabolism.
- Mud snails (Pomacea insularus) are a potential source of novel enzymes.
- Understanding mannanase properties is crucial for biotechnological applications.
Purpose of the Study:
- To purify and characterize endo-1,4-beta-D-mannanase from Pomacea insularus.
- To determine the enzyme's molecular weight, amino-terminal sequence, and optimal pH.
- To investigate the enzyme's substrate specificity and inhibition patterns.
Main Methods:
- Purification of endo-1,4-beta-D-mannanase from mud snail viscera.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for purity and molecular weight determination.
- Enzyme activity assays using beta-1,4-mannan as substrate.
- Analysis of enzyme stability across a pH range and inhibition by various agents.
Main Results:
- A single protein band of 44,000 molecular weight was obtained via SDS-PAGE.
- The enzyme exhibited stability between pH 5.0 and 10.5, with optimal activity at pH 5.5.
- Beta-1,4-mannan was hydrolyzed into mannooligosaccharides (M2-M5).
- Activity was significantly inhibited by Ag+, Hg2+, Cu2+, dithiothreitol, and N-bromosuccinimide.
Conclusions:
- Endo-1,4-beta-D-mannanase from Pomacea insularus is a stable enzyme with potential applications in mannan degradation.
- The enzyme's characterization provides valuable data for understanding its biochemical properties.
- Inhibition studies suggest the involvement of specific amino acid residues in the enzyme's active site.