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.
The optimization of some extracellular enzymes biosynthesis by Aspergillus niger 377-4.
Wikiera, A., Mika, M., Janiszewska, A. S. & Zyla, K. (2015). Journal of Scientific & Industrial Research, 74, 145-149.
The effect of initial solid and moisture contents, temperature and time of incubation on the production of polygalacturonase, phytase, acid phosphatase, xylanase and β-glucanase by Aspergillus niger 377-4 during solid state fermentation was studied. Parameters of enzyme synthesis were optimized using statistical experimental designs. It was shown that the capacity of strain to synthesize the aforementioned enzymes could be modified within a wide range by culture parameters selection. The optimal polygalacturonase production efficiency was achieved with the initial medium mass of 19.9 g and humidity of 59.9%, after 77.7 h of incubation at 28.9°C. The best combination of culture parameters for phytase synthesis was: initial medium mass 19.9 g, moistures 50%, temperature 33°C and incubation time 83.9 h. The highest activity of acid phosphatase was obtained after 81.3 h of incubation at 27°C, with initial substrate mass of 17.8 g and moistness content of 60%. The initial solid and moisture contents to synthesize xylanase were 19.9 g and 50%, respectively, with incubation time of 73 h at 29.6°C. The highest efficiency of β-glucanase biosynthesis was obtained when A. niger 377-4 was cultivated for 80.4 h at 27°C on a initial medium mass of 20 g and initial level of moistness 59.9%.
Structural and functional characterization of a novel family GH115 4-O-methyl-α-glucuronidase with specificity for decorated arabinogalactans.
Aalbers, F., Turkenburg, J. P., Davies, G. J., Dijkhuizen, L. & van Bueren, A. L. (2015). Journal of Molecular Biology, 427(24), 3935-3946.
Glycoside hydrolases are clustered into families based on amino acid sequence similarities, and belonging to a particular family can infer biological activity of an enzyme. Family GH115 contains α-glucuronidases where several members have been shown to hydrolyze terminal α-1,2-linked glucuronic acid and 4-O-methylated glucuronic acid from the plant cell wall polysaccharide glucuronoxylan. Other GH115 enzymes show no activity on glucuronoxylan, and therefore, it has been proposed that family GH115 may be a poly-specific family. In this study, we reveal that a putative periplasmic GH115 from the human gut symbiont Bacteroides thetaiotaomicron, BtGH115A, hydrolyzes terminal 4-O-methyl-glucuronic acid residues from decorated arabinogalactan isolated from acacia tree. The three-dimensional structure of BtGH115A reveals that BtGH115A has the same domain architecture as the other structurally characterized member of this family, BoAgu115A; however the position of the C-terminal module is altered with respect to each individual enzyme. Phylogenetic analysis of GH115 amino sequences divides the family into distinct clades that may distinguish different substrate specificities. Finally, we show that BtGH115A α-glucuronidase activity is necessary for the sequential digestion of branched galactans from acacia gum by a galactan-β-1,3-galactosidase from family GH43; however, while B. thetaiotaomicron grows on larch wood arabinogalactan, the bacterium is not able to metabolize acacia gum arabinogalactan, suggesting that BtGH115A is involved in degradation of arabinogalactan fragments liberated by other microbial species in the gastrointestinal tract.
Aspergillus hancockii sp. nov., a biosynthetically talented fungus endemic to southeastern Australian soils.
Pitt, J. I., Lange, L., Lacey, A. E., Vuong, D., Midgley, D. J., Greenfield, P., Bradbury, M. I., Lacey, E., Busk, P. K., Pilgaard, B., Chooi, Y. H. & Piggott, A. M. (2017). PloS One, 12(4), e0170254.
Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.
Cell separation in kiwifruit without development of a specialised detachment zone.
Prakash, R., Hallett, I. C., Wong, S. F., Johnston, S. L., O’Donoghue, E. M., McAtee, P. A., Seal, A. G., Atkinson, R. G. & Schröder, R. (2017). BMC Plant Biology, 17(1), 86.
Background: Unlike in abscission or dehiscence, fruit of kiwifruit Actinidia eriantha develop the ability for peel detachment when they are ripe and soft in the absence of a morphologically identifiable abscission zone. Two closely-related genotypes with contrasting detachment behaviour have been identified. The ‘good-peeling’ genotype has detachment with clean debonding of cells, and a peel tissue that does not tear. The ‘poor-peeling’ genotype has poor detachability, with cells that rupture upon debonding, and peel tissue that fragments easily. Results: Structural studies indicated that peel detachability in both genotypes occurred in the outer pericarp beneath the hypodermis. Immunolabelling showed differences in methylesterification of pectin, where the interface of labelling coincided with the location of detachment in the good-peeling genotype, whereas in the poor-peeling genotype, no such interface existed. This zone of difference in methylesterification was enhanced by differential cell wall changes between the peel and outer pericarp tissue. Although both genotypes expressed two polygalacturonase genes, no enzyme activity was detected in the good-peeling genotype, suggesting limited pectin breakdown, keeping cell walls strong without tearing or fragmentation of the peel and flesh upon detachment. Differences in location and amounts of wall-stiffening galactan in the peel of the good-peeling genotype possibly contributed to this phenotype. Hemicellulose-acting transglycosylases were more active in the good-peeling genotype, suggesting an influence on peel flexibility by remodelling their substrates during development of detachability. High xyloglucanase activity in the peel of the good-peeling genotype may contribute by having a strengthening effect on the cellulose-xyloglucan network. Conclusions: In fruit of A. eriantha, peel detachability is due to the establishment of a zone of discontinuity created by differential cell wall changes in peel and outer pericarp tissues that lead to changes in mechanical properties of the peel. During ripening, the peel becomes flexible and the cells continue to adhere strongly to each other, preventing breakage, whereas the underlying outer pericarp loses cell wall strength as softening proceeds. Together these results reveal a novel and interesting mechanism for enabling cell separation.
Diversity of microbial carbohydrate-active enzymes in Danish anaerobic digesters fed with wastewater treatment sludge.
Wilkens, C., Busk, P. K., Pilgaard, B., Zhang, W. J., Nielsen, K. L., Nielsen, P. H. & Lange, L. (2017). Biotechnology for Biofuels, 10(1), 158.
Background: Improved carbohydrate-active enzymes (CAZymes) are needed to fulfill the goal of producing food, feed, fuel, chemicals, and materials from biomass. Little is known about how the diverse microbial communities in anaerobic digesters (ADs) metabolize carbohydrates or which CAZymes that are present, making the ADs a unique niche to look for CAZymes that can potentiate the enzyme blends currently used in industry. Results: Enzymatic assays showed that functional CAZymes were secreted into the AD environments in four full-scale mesophilic Danish ADs fed with primary and surplus sludge from municipal wastewater treatment plants. Metagenomes from the ADs were mined for CAZymes with Homology to Peptide Patterns (HotPep). 19,335 CAZymes were identified of which 30% showed 50% or lower identity to known proteins demonstrating that ADs make up a promising pool for discovery of novel CAZymes. A function was assigned to 54% of all CAZymes identified by HotPep. Many different α-glucan-acting CAZymes were identified in the four metagenomes, and the most abundant family was glycoside hydrolase family 13, which contains α-glucan-acting CAZymes. Cellulytic and xylanolytic CAZymes were also abundant in the four metagenomes. The cellulytic enzymes were limited almost to endoglucanases and β-glucosidases, which reflect the large amount of partly degraded cellulose in the sludge. No dockerin domains were identified suggesting that the cellulytic enzymes in the ADs studied operate independently. Of xylanolytic CAZymes, especially xylanases and β-xylosidase, but also a battery of accessory enzymes, were present in the four ADs. Conclusions: Our findings suggest that the ADs are a good place to look for novel plant biomass degrading and modifying enzymes that can potentiate biological processes and provide basis for production of a range of added-value products from biorefineries.
Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter.
Huang, Y., Yi, Z., Jin, Y., Huang, M., He, K., Liu, D., Luo, H., Zhao, D., He, H., Fang, Y. & Zhao, H. (2017). Frontiers in Microbiology, 8, 1747.
Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.