Peningkatan termostabilitas enzim alkalifilik xilanase dengan site directed mutagenesis

Abstract

Application of xylanase in the pulp and paper industries required enzyme that is resistant to alkaline and high temperatures conditions. A suitable enzyme for that purpose is an alkalo-thermostable xylanase. The objective of this research is mutated gene alkxynaq1cmu by Site Directed Mutagenesis (SDM) method to increase thermostability of the xylanase. pGEMalkxynaq1cmu recombinant plasmids were mutated separately using 5 pairs of mutagenic primers, namely Q53K, Q62L, E146V, T194I, and S330I. Restriction result, 2 out of 5 mutated clones (2.2 and M3) has a XbaI restriction site that produce 3 DNA fragments. Mutan clones have xylanase activity that were varied from 22.7 U/ml to 49.3 U/ml. The results of sequencing from 3 clones showed 2 clones (2.2 & 3.9) contain a single nucleotide mutation and one clone (M3) contains a double nucleotides mutation. Clones 2.2 has successfully exchanged by Q62L primer that changed the amino acid glutamine to lysine, 3.9 clone has successfully exchanged by E146V primer, amino acid glutamic acid to valine, and M3 has successfully exchanged by E146V and Q62L primers.