• Food Science and Biotechnology
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• v.18 no.3
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• pp.776-781
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• 2009

A putative cyclomaltodextrinase (BHCD) gene was found from the genome of Bacillus halodurans C-125, which encodes 578 amino acids with a predicted molecular mass of 67,279 Da. It shares 42-59% of amino acid sequence identity with common cyclomaltodextrinase (CDase)-family enzymes. The corresponding gene was cloned by polymerase chain reaction (PCR) and the dimeric enzyme with C-terminal 6-histidines was successfully overproduced and purified from recombinant Escherichia coli. BHCD showed the highest activity against ${\beta}-CD$ at pH 7.0 and $50^{\circ}C$. Due to its versatile hydrolysis and transglycosylation activities, BHCD has been confirmed as a member of CDases. However, BHCD can be distinguished from other typical CDases on the basis of its novel multisubstrate specificity. While typical CDases have over 10 times higher activity on ${\beta}-CD$ than starch or pullulan, the CD-hydrolyzing activity of BHCD is only 2.3 times higher than pullulan. In particular, it showed significantly higher activity ratio of maltotriose to acarbose than other common CDase-family enzymes.

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.155-162
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• 2020

Acetyl xylan esterase (AXE; E.C. 3.1.1.72) is one of the accessory enzymes for xylan degradation, which can remove the terminal acetate residues from xylan polymers. In this study, two genes encoding putative AXEs (LaAXE and BhAXE) were cloned from Lactobacillus antri DSM 16041 and Bacillus halodurans C-125, and constitutively expressed in Escherichia coli. They possess considerable activities towards various substrates such as p-nitrophenyl acetate, 4-methylumbelliferyl acetate, glucose pentaacetate, and 7-amino cephalosporanic acid. LaAXE and BhAXE showed the highest activities at pH 7.0 and 8.0 at 50℃, respectively. These enzymes are AXE members of carbohydrate esterase (CE) family 7 with the cephalosporine-C deacetylase activity for the production of antibiotics precursors. The simultaneous treatment of LaAXE with Thermotoga neapolitana β-xylanase showed 1.44-fold higher synergistic degradation of beechwood xylan than the single treatment of xylanase, whereas BhAXE showed no significant synergism. It was suggested that LaAXE can deacetylate beechwood xylan and enhance the successive accessibility of xylanase towards the resulting substrates. The novel LaAXE originated from a lactic acid bacterium will be utilized for the enzymatic production of D-xylose and xylooligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.29-36
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• 2007

The xynA gene encoding the xylanase A of Paenibacillus sp. DG-22 was isolated with a DNA probe obtained by PCR amplification, using degenerated primers deduced from the amino acid residues of the known N-terminal region of the purified enzyme and the conserved region in the family 11 xylanases. The positive clones were screened on the LB agar plates supplemented with xylan, by the Congo-red staining method. The xynA gene consists of a 630-bp open reading frame encoding a protein of 210 amino acids, and the XynA preprotein contains a 28-residues signal peptide whose cleavage yields a l82-residues mature protein of a calculated molecular weight of 20,000Da and pI value of 8.77. The cloned DNA fragment also has another ORF of 873 nucleotides that showed 76% identity to the putative transcriptional activator of Bacillus halodurans C-125. Most of the xylanase activity was found in the periplasmic space of E. coli. The xynA gene was subcloned into pQE60 expression vector to fuse with six histidine-tag. The recombinant xylanase A was purified by heating and immobilized metal affinity chromatography. The optimum pH and temperature of the purified enzyme were 6.0 and $60^{\circ}C$, respectively. This histidine-tagged xylanase A was less thermostable than the native enzyme.