Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium.
Wejse, P. L., Ingvorsen, K. & Mortensen, K. K. (2003). Extremophiles, 7(5), 423-431.
The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca+2, Mn+2, Mg+2, Ba+2, Li+2, NaN3, and isopropanol was observed. The K m and V max values determined for Xyl 1 with 4-O-methyl-D-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.
Genes encoding xylan and β-glucan hydrolysing enzymes in Bacillus subtilis: characterization, mapping and construction of strains deficient in lichenase, cellulase and xylanase.
Wolf, M., Geczi, A., Simon, O. & Borriss, R. (1995). Microbiology, 141(2), 281-290.
The gene encoding extracellular xylanase (xynA) was amplified as a 770 bp DNA fragment Bacillus subtilis 168 chromosomal DNA by PCR. The genes encoding endo-β-1,4-glucanase (eglS) and endo-β-1,3-1,4-glucanase (bglS) were isolated from a genomic library of B. subtilis 168. The sequences of xynA and eglS were identical to those of the xylanase and cellulase genes from B. subtilis PAP115. Integrative plasmids containing DNA fragments with deletions in the coding region of the genes were constructed and used to replace the chromosomal eglS, bglS and xynA genes of B. subtilis 168. Strains without any detectable activity against xylan (Xyn-), carboxymethylcellulose (Egl-) or mixed linked β-1,3-1,4-glucan (Egl- Bgl-) were obtained. The genes were mapped at 170° (eglS), 175° (xynA) and 340° (bglS) on the B. subtilis chromosome.
Identification and characterization of a new xylanase from Gram-positive bacteria isolated from termite gut (Reticulitermes santonensis).
Mattéotti, C., Bauwens, J., Brasseur, C., Tarayre, C., Thonart, P., Destain, J., Francis, F., Haubruge, E., De Pauw., E., Portetelle., D. & Vandenbol, M. (2012). Protein Expression and Purification, 83(2), 117-127.
Termites are world champions at digesting lignocellulosic compounds, thanks to cooperation between their own enzymes and exogenous enzymes from microorganisms. Prokaryotic cells are responsible for a large part of this lignocellulolytic activity. Bacterial enzyme activities have been demonstrated in the higher and the lower termite gut. From five clones of Gram-positive bacteria isolated and identified in a previous work, we constructed a genomic DNA library and performed functional screening for alpha-amylase, beta-glucosidase, and xylanase activities. One candidate, Xyl8B8, showed xylanase activity. Sequence analysis of the genomic insert revealed five complete ORFs on the cloned DNA (5746 bp). Among the encoded proteins were a putative endo-1,4-beta-xylanase (XylB8) belonging to glycoside hydrolase family 11 (GH11). On the basis of sequence analyses, genomic DNA organization, and phylogenetic analysis, the insert was shown to come from an actinobacterium. The mature xylanase (mXylB8) was expressed in Escherichia coli and purified by affinity chromatography and detected by zymogram analysis after renaturing. It showed maximal xylanase activity in sodium acetate buffer, pH 5.0 at 55°C. Its activity was increased by reducing agents and decreased by Cu2+, some detergents, and chelating agents. Its substrate specificity appeared limited to xylan.
Xylanase production by a novel halophilic bacterium increased 20-fold by response surface methodology.
Wejse, P. L., Ingvorsen, K. & Mortensen, K. K. (2003). Enzyme and Microbial Technology, 32(6), 721-727.
Medium optimisation for a novel halophilic eubacterium, strain SX15, resulted in a 20-fold increase of extracellular xylanase activity. This facilitates the purification of xylanase produced by this strain. Prior experiments revealed that xylan concentration, source and concentration of nitrogen and salinity were important variables for xylanase production. Based on this, we adopted a fractional factorial design to determine the best combinations and approximate levels of these values. Subsequently, response surface (RS) methodology was applied to locate the optimal levels of xylan, NH4Cl and salinity.
Endo-1, 4-beta-xylanase B from Aspergillus cf. niger BCC14405 isolated in Thailand: Purification, characterization and gene isolation.
Krisana, A., Rutchadaporn, S., Jarupan, G., Lily, E., Sutipa, T. & Kanyawim, K. (2005). Journal of Biochemistry and Molecular Biology, 38(1), 17-23.
During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of 55°C. When tested using xylan from birchwood, it showed Km and Vmax values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by CuSO4, EDTA, and by FeSO4. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1, 4-β-xylanase. The full-length gene encoding endo-1, 4-β-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1, 4-â-xylanase B previously identified in A. niger.
An internal cellulose-binding domain meidates adsorption of an engineered bifunctional xylanase/cellulase.
Tomme, P., Gilkes, N. R., Miller Jr, R. C., Warren, A. J. & Kilburn, D. G. (1994). Protein Engineering, 7(1), 117-123.
A chimeric xylanase/endoglucanase (XynCenA) with an internal cellulose-binding domain was constructed by fusing the Bacillus subtilis xyn gene fragment to the 5'-end of the Cellulomonas fimi cenA. A polyhistidine-encoding sequence was also fused to the 5'-end of the xyn gene. The gene fusion was overexpressed in Escherichia coli and the fusion poly-peptide purified from the cell extracts using the polyhistldine tail. The hybrid protein behaved like the parental endo-glucanase or xylanase when assayed on a number of soluble and insoluble cellulosic substrates or xylans. The presence of two distinct active sites and the internal cellulose-binding domain did not significantly affect the hydrolysis of any of these substrates. However, the fusion protein exhibited a strong affinity for both mkrocrystaUine cellulose (Avicel) and regenerated chitin. Like the parental endoglucanase, bound XynCenA could not be duted from these porysaccharides with either low or high salt buffer or distilled water. More stringent conditions, such as 1% SDS or 8 M guanidinium hydro-chloride, fully desorbed the protein. The fusion protein did not adsorb significantly to insoluble xylan.
Cloning, expression, characterization, and high cell-density production of recombinant endo-1, 4-β-xylanase from Aspergillus niger in Pichia pastoris.
Ruanglek, V., Sriprang, R., Ratanaphan, N., Tirawongsaroj, P., Chantasigh, D., Tanapongpipat, S., Pootanakit, K. & Eurwilaichitr, L. (2007). Enzyme and Microbial Technology, 41(1), 19-25.
A recombinant gene XylB (564 bp) encoding endo-1, 4-β-xylanase, obtained from Aspergillus niger BCC14405, was successfully cloned and secreted as a 21 kDa in Pichia pastoris under the control of AOX1 promoter. The activity of the recombinant xylanase was highest at 55°C which was 5°C higher than native xylanase. In addition, the recombinant xylanase was active over the range of pH 3.6–6.5 with maximal activity at pH 5 (8007 U/mg). When compared to a commercial enzyme in vitro digestibility of the recombinant enzyme was 1.8- and 2.4-folds higher digesting rates of rice bran and soybean meal fibers, respectively. Two-liter production of xylanase was performed with BSM medium which increased cell concentration up to 84.5 gdry-weight/L via the 80% µmax exponential feed strategy. This process provided maximum xylanase production (3676 U/mL) with highest specific activity (7352 U/mgprotein) and volumetric productivity (22,832 U/L/h) at 3.0% (v/v) methanol induction. By far, this was the highest xylanase expression in P. pastoris host system being reported. Thus, this BCC14405 recombinant xylanase could be produced and used effectively as a feed additive for animals.
Salinity and temperature effects on accessibility of soluble and cross‐linked insoluble xylans to endo‐xylanases.
Wejse, P. L., Ingvorsen, K. & Mortensen, K. K. (2005). IUBMB Life, 57(11), 761-763.
Different responses to salinity were observed for an extremely halotolerant endo-xylanase when assayed with soluble birchwood glucoronoxylan and cross-linked dyed insoluble birchwood glucoronoxylan. Shrinking of insoluble xylan particles due to increased ionic strength is proposed as the explanation. Temperature affected the xylanase activity measurement on the insoluble xylan greatly, likely due to increased enzyme accessible surface of the substrate at high temperatures.
Evaluation of three automated genome annotations for Halorhabdus utahensis.
Bakke, P., Carney, N., DeLoache, W., Gearing, M., Ingvorsen, K., Lotz, M., McNair, J.,Penumetcha, P., Simpson, S., Voss, L., Win, M., Heyer, L. J. & Campbell, A. M. (2009). PLoS One, 4(7), e6291.
Genome annotations are accumulating rapidly and depend heavily on automated annotation systems. Many genome centers offer annotation systems but no one has compared their output in a systematic way to determine accuracy and inherent errors. Errors in the annotations are routinely deposited in databases such as NCBI and used to validate subsequent annotation errors. We submitted the genome sequence of halophilic archaeon Halorhabdus utahensis to be analyzed by three genome annotation services. We have examined the output from each service in a variety of ways in order to compare the methodology and effectiveness of the annotations, as well as to explore the genes, pathways, and physiology of the previously unannotated genome. The annotation services differ considerably in gene calls, features, and ease of use. We had to manually identify the origin of replication and the species-specific consensus ribosome-binding site. Additionally, we conducted laboratory experiments to test H. utahensis growth and enzyme activity. Current annotation practices need to improve in order to more accurately reflect a genome's biological potential. We make specific recommendations that could improve the quality of microbial annotation projects.
Production of β-xylanase and β-xylosidase by the extremely halophilic archaeon Halorhabdus utahensis.
Wainø, M. & Ingvorsen, K. (2003). Extremophiles, 7(2), 87-93.
The extremely halophilic archaeon, Halorhabdus utahensis, isolated from the Great Salt Lake, Utah, produced β-xylanase and β-xylosidase activities. Both enzymes were active over a broad NaCl range from near zero to 30% NaCl when tested with culture broth. A broad NaCl optimum was observed for β-xylanase activity between 5% and 15% NaCl, while β-xylosidase activity was highest at 5% NaCl. Almost half of the maximum activities remained at 27%–30% NaCl for both enzyme activities. When dialyzed culture supernatant and culture broth were employed for determination of β-xylanase and β-xylosidase stabilities, approximately 55% and 83% of the initial β-xylanase and β-xylosidase activities, respectively, remained after 24 h incubation at 20% NaCl. The enzymes were also shown to be slightly thermophilic; β-xylanase activity exhibiting two optima at 55° and 70°C, while β-xylosidase activity was optimal at 65°C. SDS-PAGE and zymogram techniques revealed the presence of two xylan-degrading proteins of approximately 45 and 67 kDa in culture supernatants. To our knowledge, this paper is the first report on hemicellulose-degrading enzymes produced by an extremely halophilic archaeon.