• Journal of Microbiology and Biotechnology
• /
• 제3권4호
• /
• pp.224-231
• /
• 1993

The nucleotide sequence of the xylanase gene (xynY) from alkalophilic Bacillus sp. YC-335 was determined and analyzed. An open reading frame of 1, 062 base pairs for xynY gene was observed and encoded for a protein of 354 amino acids with a molecular weight of 38, 915. S1 nuclease mapping showed that the transcription initiation sites of the xynY gene were different in Bacillus sp. YC-335 and Escherichia coli HB101 (pYS55). S1 mapping also showed that -10 region of the xynY gene recognized by RNA polymerases of E. coli and Bacillus sp. YC-335 were TACAGT and TATGAT , respectively. A ribosome binding site sequence with the free energy of -17.0 Kcal/mol was observed 9 base pairs upstream from the unusual initiation codon, TTG. The proposed signal sequence consisted of 27 amino acids, 2 of which were basic amino acid residues and 21 were hydrophobic amino acid residues. When the amino acid sequences of xylanases were compared, Bacillus sp. YC-335 xylanase showed more than 50% homology with xylanases from B. pumilus, B. subtilis, and B. circulans.

• Journal of Microbiology and Biotechnology
• /
• 제23권3호
• /
• pp.397-404
• /
• 2013

Paenibacillus xylanilyticus KJ-03 was isolated from soil samples obtained from a field with Amorphophallus konjac plants. A gene encoding xylanase was isolated from KJ-03 and cloned using a fosmid library. The xynA gene encodes xylanase; it consists of 1,035 bp and encodes 345 amino acids. The amino acid sequence deduced from the P. xylanilyticus KJ-03 xylanase showed 81% and 69% identities with those deduced from the P. polymyxa E681 and Paenibacillus sp. HPL-001 xylanases, respectively. The xynA gene comprises a single domain, consisting of a catalytic domain of the glycosyl hydrolase (GH) 10 family. The xynA gene was expressed in Escherichia coli BL21 (trxB), and the recombinant xylanase was purified by Niaffinity chromatography. The purified xylanase showed optimum activity with birchwood xylan as a substrate at $40^{\circ}C$ and pH 7.4. Treatment with $Mg^{2+}$ and $Li^+$ showed a slight decrease in XynA activity; however, treatment with 5 mM $Cu^{2+}$ completely inhibited its activity. The results of the thin layer chromatography analysis indicated that the major hydrolysis product was xylobiose and small amounts of xylose and xylotriose. XynA showed increased activity with oat spelt xylan and birchwood xylan, but showed only slight activity with locust bean gum.

• Journal of Industrial Technology
• /
• 제29권A호
• /
• pp.169-176
• /
• 2009

The gene encoding endoxylanase (xylS) was isolated from a genomic library of Bacillus licheniformis NBL420. Two positive clones, which harbor 1.5 kb and 0.8 kb inserts respectively, were screened on RBB dyed-xylan plates and the recombinant plasmids were named as pBX3 and pBX5. The nucleotide sequencings of two inserts revealed the existence of common 639 bp of open reading frame which encode 232 amino acids. The xylS gene was successfully subcloned into pET22b(+) vector and overexpressed. Enzymatic properties including optimum pH, optimum temp, thermostability and pH stability were investigated. Activity staining of XylS was identical with that of original Bacillus licheniformis NBL420.

• Microbiology and Biotechnology Letters
• /
• 제17권2호
• /
• pp.154-159
• /
• 1989

Chromosomal DNA fragments of Bacillus sp. YA-14 isolated from soil as a potent xylan hydrolyzing bacterium, were ligated to a vector plasmid, pBR322, and used to transfer Escherichia coli HB101 cells. The recombinant plasmid pYDC21 was found to enable the transformants to produce xylanase. pYDC21 was found to contain the 3 kb HindIII fragment originated from the Bacillus sp. YA-14 chromosomal DNA by southern hybridization. The optimum temperature and pH for the reaction of xylanse produced by E. coli (pYDC21) were appeared at 50$_7$C and pH 7.0, respectiveiy. the xylanase enzyme was stable between pH 5.0 and 7.0 and maintained stably up to 4$0^{\circ}C$.

• Journal of Microbiology and Biotechnology
• /
• 제11권1호
• /
• pp.131-137
• /
• 2001

Previous work has identified that only the catabolite responsive element A (creA; previously called cre-2) out of two potential cre sequences (cre-1: nucleotide +160 to +173 and cre-2: +173 to +186), recognized within the coding region of the xylanaseA gene (xynA) of Bacillus stearothermophilus No.236, was actually, was actually involved in the carbon catabolite repression(CCR) of xynA expression in B. subtilis. However, the level of CCR of xynA expression in the original B.stearothermophilus No.236 strain (70-fold repression). Therefore, to search for an additional cre element in the promoter region, the upstream region of the xynA gene was subcloned by chromosome walking, and as a result, another potential cre element (nucleotide -124∼-137; designated creB) was recognized in this region. The cre-like sequence revealed a high homology to the cre consensus sequence. The xylanase activity of B. subtilis MW15 bearing pWPBR14 (containing creA and creB) cultured in a medium containing xylose as the sole carbon source was about 7.7 times higher than that observed for the same culture containing glucose. B. subtilis MW15 bearing pWPBR23 (containing only creA) produced an activity about 2.4 times higher. This pattern of CCR was confirmed using derivatives of xynA::aprA fusion plasmids. Furthermore, a measurement of the amounts of the xynA transcript showed a similar pattern as that for the production of xylanase. In addition, the synthesis of xylanase in B. subtilis QB7115 [a catabolite control protein A (ccpA) mutant strain] carrying pWPBR14 was almost completely relieved from glucose repression. Together, these results lead to a conclusion that the CCR of the expression of the xynA gene is mediated by CcpA binding at creA and creB sites in B. subtilis.

• Microbiology and Biotechnology Letters
• /
• 제48권2호
• /
• pp.158-166
• /
• 2020

A gene coding for the xylanase was cloned from Paenibacillus woosongensis, followed by determination of its complete nucleotide sequence. This xylanase gene, designated as xyn10A, consists of 1,446 nucleotides encoding a polypeptide of 481 amino acid residues. Based on the deduced amino acid sequence, Xyn10A was identified to be a modular enzyme composed of a catalytic domain highly homologous to the glycosyl hydrolase family 10 xylanase and a putative carbohydrate-binding module (CBM) in the C-terminus. By using DEAE-sepharose and phenyl-sepharose column chromatography, Xyn10A was purified from the cellfree extract of recombinant Escherichia coli carrying a P. woosongensis xyn10A gene. The N-terminal amino acid sequence of the purified Xyn10A was identified to exactly match the sequence immediately following the signal peptide predicted by the Signal5.0 server. The purified Xyn10A was a truncated protein of 33 kDa, suggesting the deletion of CBM in the C-terminus by intracellular hydrolysis. The purified enzyme had an optimum pH and temperature of 6.0 and 55-60℃, respectively, with the kinetic parameters V_max and K_m of 298.8 U/mg and 2.47 mg/ml, respectively, for oat spelt xylan. The enzyme was more active on arabinoxylan than on oat spelt xylan and birchood xylan with low activity for p-nitrophenyl-β-xylopyranoside. Xylanase activity was significantly inhibited by 5 mM Cu²⁺, Mn²⁺, and SDS, and was noticeably enhanced by K⁺, Ni²⁺, and Ca²⁺. The enzyme could hydrolyze xylooligosaccharides larger than xylobiose. The predominant products resulting from xylooligosaccharide hydrolysis were xylobiose and xylose.

• Journal of Microbiology and Biotechnology
• /
• 제12권6호
• /
• pp.890-894
• /
• 2002

A xylanase-producing Trichoderma strain was isolated from soil. Xylanase from Trichoderma strain SY was purified 21-fold to an apparent homogeneity, with a $17.4\%$ yield. The optimum pH and temperature were determined to be 5.5 and $50^{\circ}C$, respectively, and its molecular weight was 21-kDa by SDS-PAGE. The corresponding gene, named xyl, was cloned by RT-PCR. DNA blot analysis of xyl showed that this gene is present as a single copy. The amino acid sequence of the Xyl protein showed similarity to those of other xylanases derived from various fungi. mRNA of xyl was highly expressed when this fungus was grown on cellulose or xylan as a sole carbon source, but undetectable when grown on sucrose. Extracts of Escherichia coli cells expressing xyl were found to have xylanase activity. It was confirmed that xyl from this isolate encodes xylanase.

• Mycobiology
• /
• 제51권4호
• /
• pp.239-245
• /
• 2023

Xylanase has been applied in various sectors, such as biomass conversion, paper, pulp, textiles, and pharmaceutical industries. This study aimed to isolate and screen potential xylanase-producing fungi from the soil of Suphan Buri Province, Thailand. Fifteen fungi were isolated, and their xylanase activities were tested by the qualitative method. The result showed that isolate SP3, SP10 and SP15 gave high xylanase activity with potency index (PI) of 2.32, 2.01 and 1.82, respectively. These fungi were selected for the xylanase quantitative test, isolate SP10 performed the highest xylanase activity with 0.535 U/mL. Through molecular methods using the 𝛽-tubulin gene, isolate SP10 was identified as Penicillium menonorum. The xylanase characteristics from P. menonorum SP10 were determined, including the xylanase isoforms and the optimum pH and temperature. The xylanase isoforms on SDS-PAGE indicated that P. menonorum SP10 produced two xylanases (45 and 54 kDa). Moreover, its xylanase worked optimally at pH 6 and 55 ℃ while reaching 61% activity at 65 ℃. These results proposed P. menonorum SP10 as a good candidate for industrial uses, especially in poultry feed and pulp industries, to improve yield and economic efficiency under slightly acidic and high-temperature conditions.

• Korean Journal of Soil Science and Fertilizer
• /
• 제44권5호
• /
• pp.836-840
• /
• 2011

In order to isolate thermophilic compost-promoting bacteria with high activity of cellulase and xylanase, spent mushroom substrates with sawdust were collected from mushroom cultivation farm, Jinju, Gyeongnam in Korea. Among of the isolates, one strain, designated SJ21 was selected by agar diffusion method. The strain SJ21 was identified as members of the Bacillus lincheniformis by biochemical characteristics using Bacillus ID kit and VITEK 2 system. Comparative 16S rDNA gene sequence analysis showed that strain SJ21 formed a distinct phylogenetic tree within the genus Bacillus and was most closely related to Bacillus subtilis with 16S rDNA gene sequence similarity of 99%. On the basis of its physiological properties, biochemical characteristics and phylogenetic distinctiveness, strain SJ21 was classified within the genus Bacillus, for which the name Bacillus sp. SJ21 is proposed. The cellulase and xylanase activity of Bacillus sp. SJ21 was slightly increased according to bacterial population from exponential phase to stationary phase in growth curve for Bacillus sp. SJ21.

• Journal of Microbiology and Biotechnology
• /
• 제11권3호
• /
• pp.534-538
• /
• 2001

The xynA gene encoding an alikali-tolerant endo-1,4-${\beta}$-xylanase (XYN) was cloned from the alkalophilic Bacillus pumilus A-30. The nucleotide sequence of a 974-bp DNA fragment containing the xynA was determined. An ORF of 684 nucleotides that encoded a protein of 228 amino aicds was detected. Asparagine-71 of XYN from B. Pumilus A-30 showed to be highly conservative in alkaline xylanases of family G/11, upon comparing the amino acid sequences of 17 family G/11 xylanases. Site-directed mutation of N71D of the xynA gene resulted in a decrease of 12.4% in the specific acitivity and a significant decline in the enzyme activity in the alkaline pH range.