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Journal : Microbiology Indonesia

Cloning of α-L-arabinofuranosidase Genes and Its Expression in Escherichia coli: A Comparative Study of Recombinant Arabinofuranosidase Originatingin Bacillus subtilis DB104 and Newly Isolated Bacillus licheniformis CW1 MOCHAMAD NURCHOLIS; NIKNIK NURHAYATI; IS HELIANTI; MARIA ULFAH; BUDIASIH WAHYUNTARI; AGUSTIN KRISNA WARDANI
Microbiology Indonesia Vol. 6 No. 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.6.1.1

Abstract

Arabinofuranosidase (Abfa) is one of the most important enzymes involved in degradation of lignocelullose biomass.  Two genes encoding α-L-Arabinofuranosidase (abfA), each from Bacillus subtilis DB104 (abfAa1) and an indigenous Indonesian B. licheniformis CW1 (abfAb3), were cloned by the PCR approach  and expressed in Escherichia coli. Sequences analysis of abfAa1 and abfAb3 revealed that each consists of 1721 and 1739 base pairs long DNA, respectively. Each clone contains a hypothetical open reading frame of 1503 and 1509 bp that encode an Abfa protein of 500 and 502 amino acids for abfAa1 and abfAb3, respectively. The deduced amino acid sequence of AbfaB3 shares 75% identity to that of AbfaA1. The recombinant enzymes were expressed constitutively in E. coli. Partial characterization of those enzymes revealed that the AbfaA1 and AbfaB3 were optimally active at 50 ºC and 60 ºC at pH 6, respectively. Thermostability studies of the recombinant enzymes with p-nitrophenyl α-L-arabinofuranoside at their optimal conditions showed that up to 50% AbfaA1 activity was lost after 5 h incubation at 50  ºC, whereas the AbfaB3 retained its activity over 75% after 12 h pre-incubation oat 60 ºC. This thermostability study of recombinant AbfaB3 showed for the first time that the arabinofuranosidase from B. licheniformis is a thermostable enzyme. The recombinant enzyme showed a higher optimal reaction temperature (60 ºC) in comparison to the previously reported thermostable arabinofuranosidase. The thermostable AbfaB3 has a potential to be applied to the degradation of lignocellulose biomass synergistically with thermostable xylanases, for instance in the production of xylo-oligosaccharides.
Gene Cloning of Xylanase Glycoside Hydrolase Family 11 from Bacillus halodurans CM1 in Escherichia coli DH5α Muhamad Taufiqul Naufal; Agustin Krisna Wardani; IS HELIANTI
Microbiology Indonesia Vol. 13 No. 4 (2019): December 2019
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.13.4.3

Abstract

Xylanase is an enzyme that can break down xylan into xylose and xylooligosaccharide that is widely used in industry. Seeing the many applications of this enzyme, researchers conducted many studies on how to increase the productivity and effectiveness of the xylanase enzyme. One of the method that can be used to increase the xylanase enzyme production process is by using recombinant DNA technology such as cloning. Bacillus halodurans CM1 is a local alkalothermophilic bacterium that potential producer for xylanase and other industrial enzymes. This research was conducted to clone the GH11 xylanase coding gene from B. halodurans CM1 using pJET 1.2 / blunt plasmid as vector into Escherichia coli DH5α as cell host and  determine the nucleotide base sequence of the GH11 xylanase coding gene from B. halodurans CM1. The results showed the GH11 xylanase gene from B. halodurans CM1 was successfully cloned in  E. coli DH5α and based on the results of BLAST nucleotides had 99% similarities with that of endo-1,4-beta -xylanhydrolase (xyn11A) from B. halodurans C-125. Key words: Bacillus halodurans CM1, cloning, xylanase glycoside hydrolase family 11