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Cloning, sequence, expression and characterization of human beta-mannosidase.
Zahoor Qadir Samra1, Muhammad Amin Athar
1Institute of Biochemistry and Biotechnology, Quaid-i-Azam Campus, University of the Punjab, Lahore, Pakistan. samra201@hotmail.com
Acta Biochimica Polonica
|September 19, 2008
Summary
This study reports the successful expression and characterization of recombinant human beta-mannosidase (MANB) enzyme. The findings provide insights into MANB enzyme activity and its potential role in glycoprotein metabolism.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Beta-mannosidase (MANB) is crucial for glycoprotein processing in eukaryotic cells.
- Understanding human MANB is essential for studying related genetic disorders.
Purpose of the Study:
- To clone, express, and characterize the recombinant human beta-mannosidase (MANB) enzyme.
- To investigate the enzyme's activity, optimal conditions, and inhibition profiles.
Main Methods:
- Human MANB gene amplification via RT-PCR, cloning, and sequencing.
- Recombinant protein expression in E. coli using pET vectors.
- Enzyme purification using ion exchange and size exclusion chromatography.
- Characterization of enzyme activity, kinetics, and inhibition.
Main Results:
- Sequenced human MANB showed nucleotide differences but retained enzyme activity.
- Recombinant MANB enzymes (MAN22 and MAN28) were successfully expressed and purified.
- The enzyme exhibited optimal activity at 37°C and pH 5.0, with a K(m) of 2.53 mM.
- Specific inhibitors and metal ions affected enzyme activity, while others did not.
Conclusions:
- This study presents the first report on the expression and characterization of recombinant human MANB.
- The characterized enzyme provides a valuable tool for further research into beta-mannosidases and associated diseases.