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Endoglucanase is an enzyme initiating hydrolysis of cellulose. The enzyme has been applied in various fields, such as medicine and food. Previous research has been isolated cellulolytic bacteria that are capable to produce endoglucanase, i.e. Bacillus brevis and Bacillus cereus from bekatul. Determination of bacterial isolate capability to produce endoglucanase can be conducted by gene detection. The detection method involves PCR using a universal designed primer to amplify the targeted gene. The aim of study was to design primers which amplify endoglucanase gene. The steps involved collecting data through NCBI, multiple sequence alignment using MUSCLE, design primer, in silico PCR and primer characteristics checking. Primer was designed by online program and literature. Template for primer design was Bacillus sp. (LN680001) consisting of 1500 bp. Primer design used three kinds of online program, i.e. Primer3plus, Primerquest tool, and primer BLAST. Every program resulted different length of product. Primer3plus and Primerquest tool produced 1124-1209 bp and 225-534 bp of product length, while primer BLAST produced 14031451 bp of product length. Primer based online program could not isolated endoglucanase gene completely. Based on literature study, primer CelF and CelR has been isolated endoglucanase gene, 1500bp. Analysis result showed literature studied primer produce length of product longer than online program primer. Primer endoglucanase gene was obtained 5’ ATGAAACGGTCAATCTC for primer forward and 5’ CTAATTTGGTTCTGTTCCC for primer reverse. Prediction of primer characterization based on annealing temperature, 41.2-42.1°C, and percentage of GC 46.3-48.1%. In silico PCR amplification analysis showed that primer proved specific to amplify the endoglucanase gene from Bacillus genus to produce complete gen, 1500 bp.

Primer design and in silico analysis of endoglucanase gene for Bacillus genus