Asian Journal of Microbiology, Biotechnology & Environmental Sciences Paper

Vol 19, Issue 3, 2017; Page No.(531-539)

AN EXTRACELLULAR α-L-ARABINOFURANOSIDASE IN ESCHERICHIA COLI USING SECRETORY EXPRESSION SYSTEM

LAURA NAVIKA YAMANI, MOHD. ANUAR JONET, AFAF BAKTIR, ROSLI MD. ILLIAS AND NI NYOMAN TRI PUSPANINGSIH1

Abstract

Libraries of modified genes are often screened during the process of genetic engineering. It is important, therefore, to create expression systems which allow for the rapid screening of many clones for obtaining desired mutant. The expression of recombinant enzyme using secretory system which will secrete enzyme into the growth medium is more considered for screening of clones. We describe the first report showing the expression of extracellular α-L-arabinofuranosidase (Abfa) in E. coli. Abfa gene expressed intracellularly by E. coli DH5a/pTP510 was cloned into pBM5 (vector containing M5 signal peptide) as secretory expression system and expressed in E. coli BL21 (DE3), producing an extracellular α-L-arabinofuranosidase (E. coli BL21(DE3) /pBM5Abf). The activity of enzyme secreted into growth medium was measured by reacting with 4-methylumbelliferyl-α-L-arabinofuranoside (MUAF) as substrate to produce easily visualized blue fluorescence. Our results thus indicated that expression of Abfa induced by adding 2,5 mM IPTG, 10 mM Glycine and incubated at 30º C for 36 hours were conditions capable of providing the maximum secretion level. The optimal temperature and pH value of extracellular Abfa were 70°C and 8.0, respectively. The stability of enzyme was active at a broad pH range (pH 4-8). Our result showed that Abfa expressed extra cellularly by E. coli BL21 (DE3)/pBM5Abf with pH optimum 8 is more active in alkaline condition when compared with expressed intracellular by E. coli DH5a/pTP510 with pH optimum 6. To our knowledge, this is the first Abfa to be expressed extracellular in E. coli BL21 (DE3) using pBM5 before the improvement of Abfa activity by genetically engineering.

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