Enzymatic Reaction Kinetics of Endo-&#946--1,4 Xylanase Recombinant Wild Type and N63D Mutant from Bacillus sp. Termite Abdominal in Beechwood Hydrolysis to Xylooligosaccharides
Anak Agung Istri Ratnadewi, Zuhrotul Mahfudhoh,Wuryanti Handayani1Sudarko1

Department of Chemistry, Faculty of Mathematics and Natural Science, University of
Jember, 68121, Jember, Indonesia

Abstract

Endo-&#946--1,4-D xylanase has been isolated from Bacillus sp. It plays a role in the hydrolysis of xylan substrates into xylooligosaccharides (XOS) and a small portion of xylose with the ability to hydrolyze for 15-20 hours. Based on the hydrolysis time, further research is needed to increase the effectiveness of the enzyme through modeling with the docking method and continued experimentally through the Site-directed Mutagenesis method to produce the N63D mutant. The interaction between N63D and the substrate has a lower Gibbs free energy value (&#916-G) compared to the interaction between the unmutated enzyme (wild type). The purpose of this study was to determine the characterization of the N63D mutant compared to its wild type by measuring the kinetics parameters and enzyme activity. Measurement of kinetic parameters and enzyme activity was carried out after incubation at 40 &#730-C for 1 h. The activity of the N63D mutant increased by 3.0 times compared to the wild type, respectively 0.3558 (U/mL) and 0.1183 (U/mL). Kinetic measurements showed that the N63D mutant had a 3.28-fold greater catalytic efficiency than the wild type, respectively 6.65442x10-7 (mL/sec.mg) and 2.02834x10-7 (mL/sec.mg). the kinetic efficiency of the N63D mutant was lower than the wild type, respectively 16.4537 (mg/mL) and 16.7394 (mg/mL). kcat N63D is larger than the wild type, respectively 1.0949x10-5 (sec-1) and 3.39531x10-6 (sec-1).

Keywords: enzyme activity- enzymatic reaction kinetics- site-directed mutagenesis- xylanase

Topic: Biochemistry