Kinetic Inhibition of Human Salivary alpha-Amylase by a Novel Cellobiose-Containing Tetrasaccharide
Prakarn Rudeekulthamrong PhD*, Jarunee Kaulpiboon PhD**
Affiliation : * Department of Biochemistry, Phramongkutklao College of Medicine, Phramongkutklao Hospital, Bangkok, Thailand ** Department of Pre-Clinical Science (Biochemistry), Faculty of Medicine, Thammasat University, Pathumthani, Thailand
Objective : The aim of the study was to evaluate the inhibitory kinetics of a novel cellobiose-containing tetrasaccharide on
human salivary α-amylase (HSA).
Material and Method: Synthesis of cellobiose-containing tetrasaccharide was catalyzed by Paenibacillus sp. A11 CGTase
using β-CD as a donor and cellobiose as an acceptor under the optimal conditions. The reaction mixture was analyzed by
HPLC and a cellobiose-containing tetrasaccharide obtained was studied for its inhibitory kinetics.
Results : In vitro activity of human salivary α-amylase showed the optimum pH and temperature at 7.0 and 37°C, respectively.
The effects of metal ions, protective chemicals and saccharides on α-amylase activity, they were found that 10 mM concentration
of CaCl2 and NaCl enhanced the enzyme activity. In contrast, the enzyme activity was significantly inhibited by 10 mM of
HgCl2, α-cyclodextrin (α-CD) and synthetic cellobiose-containing tetrasaccharide. Chemicals often used as protective
substance for enzyme such as β-mercaptoethanol, EDTA or used as fungicide during enzyme purification (NaN3) had no effect
on the activity of this enzyme. As a cellobiose-containing tetrasaccharide was shown to have a pronounce inhibition on α-
amylase activity. Its inhibition kinetic was performed and found that cellobiose-containing tetrasaccharide was a competitive
inhibitor with a Ki value of 7.89 μM.
Conclusion : Inhibition kinetic of a cellobiose-containing tetrasaccharide on α-amylase activity was competitive type with Ki
value of 7.89 μM. In addition, these results will be a basic knowledge in controlling α-amylase actions that have influence on
blood glucose level of trial animal and human further.
Keywords : Beta-cyclodextrin (β-CD), Cellobiose, Enzyme inhibitor, Human salivary α-amylase (HSA), Tetrasaccharide
