Structure and Function of Xanthomonas campestris alpha-amylase

1996 Fiscal Year Final Research Report Summary

• Project/Area Number: 07650964
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 生物・生体工学
• Research Institution: Kagoshima University
• Principal Investigator: ABE Jun-ichi Department of Biochemical Science and Technology Faculty of Agriculture, Kagoshima University Associate professor, 農学部, 助教授 (80128404)
• Project Period (FY): 1995 – 1996
• Keywords: Xanthomonas campestris / alpha-amylase / loop structure / crystallization / inactivation by oxidation / mutagenesis / substrate specificity / Resistance against oxidation

Research Abstract

This study aimed to get further structure / function relationship of Xanthomonas campestris alpha-amylase and three results as below were obtained.
1. Mutant enzymes of which the 6th loop in (alpha / beta) 8 barrel was shortened by 4,8, or 12 amino acid residues were created. The selectivity of enzymes toward glycogen increased fivefold and twentyfold in the case of 4 and 8 residues-deleted mutant enzyme, respectively. This suggests that the long 6th loop of X.campestris alpha-amylase caused the steric hindrance between enzyme and glycogen. It is possible to say that a new enzyme can be created by partial deletion of loop in alpha-amylase,
2. X.campestris alpha-amylase was crystallized by the hanging drop method. Conditions were as follows ; enzyme concentration, 10 mg / ml ; pH 4.5 ; temperature, 20ﾟC ; precipitant in reservoir and drop, 15% and 10%, respectively, saturated ammonium sulfate in 10 mM sodium acetate (pH 4.5).
3. Auto-oxidation of methionine 82 was suspected to be a reason for the inactivation of X.campestris alpha-amylase, thus, the residue was replaced by alanine. The mutant enzyme was found to be quite stable in the presence of oxidizer of high concentration.