Optimization of annealing temperature for detection of lipase gene in Bacillus subtilis using polymerase chain reaction (PCR) method

Abstract

Enzyme lipase (EC 3.1.1.3, triacylglycerol acylhydrolase) is a group of enzymes that hydrolyzes fat into fatty acids and glycerol. In addition, the lipase enzyme plays a role in various reactions, such as esterification, interesterification, and transesterification reactions. Lipase can be obtained from microbes, plants, and animals. The most important source of lipase is microorganisms that are produced by fermentation of different bacteria. Microbial Lipases are easy to produce and cost of production is low. Bacteria like Bacillus are commercially used for lipase production. Recombinant lipase enzyme can be expressed by lipase gene (lip gene) and secreted in the bacterial Bacillus subtilis. Polymerase Chain Reaction (PCR) is in vitro amplification Deoxyribonucleic acid (DNA) method. The purity and yield of the reaction products depend on several parameters, one of which is the annealing temperature. The aim of this research was to optimize an annealing temperature for lip gene amplification using PCR. The success in amplifying a gene by PCR technique using a specially designed primer is determined by the precision of the primary attachment temperature with the DNA. Two Primer pairs are designed to successfully amplify DNA sequence fragment, namely xynlip_for_41_nt_and xynlip_rev_38_nt_. The primer combination produced the best results regarding band intensity and relative absence of unspecific bands, producing the expected amplicons of approximately 792 bp. Regarding the annealing temperatures, the best amplification was obtained at 60 °C.