Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-β-1,4-xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase.

Optimization of production and partial characterization of xylanase from a newly isolated Bacillus amyloliquefaciens

This paper reports the process of production optimization and partial characterization of xylanase from a newly isolated Bacillus amyloliquefacies VR002, isolated from local soil. The microorganism exhibited maximum xylanase production when 1.0% (v/v) of inoculum size was added to culture medium with initial pH 6, 1.0% (w/v) birchwood xylan, at 35 °C after 48h of incubation. Xylanase production in different carbon sources apart from birchwood xylan and xylose did not show high production levels. Optimum pH for xylanase activity was 6.0. The enzyme was alkali-stable and retained 100% of residual activity over the pH range from 6.0 to 10.0 for 24 h at 25°C. Optimum temperature for enzyme activity was 55°C. Xylanase was 100% stable at 4°C and 25°C even after 24h of incubation, a desirable characteristic for enzyme storage. Moreover, best crude extract volume and time reaction were found to be 10 µL and 5 min, respectively. After optimization of production and activity parameters, an increase of nearly 60-fold in xylanase activity (44.12 ± 4.36 U/mL) was achieved. Characteristics of B. amyloliquefaciens VR002 xylanase are particularly desirable for biotechnological applications

Xylanase SMXL1 from Stenocarpella maydis: Purification and biochemical characterization

This study aimed to develop a method for the purification of a xylanase called SMXL1 produced by Stenocarpella maydis and its biochemical characterization. The enzyme was purified using a Rotofor preparative chamber and one chromatographic step in an ion exchange column coupled to equipment FPLC. Posteriorly the protein was characterized, and its effect on the birchwood xylan degradation was determine by HPLC. The purified enzyme showed a molecular weight of 55 kDa calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification process obtained a yield of 6.5  0.3 %. The activity was stable at a pH range of 4 to 10 and temperatures of 45 to 60 °C. The optimum values of temperature and pH were 55 °C and 4, respectively. The Michaelis constant (Km) value was 2.61 mg/mL and the Vmax was 3.02 µmol/mL/min using birchwood xylan as substrate and the Michaelis-Menten equation. The enzyme is inhibited by the cations Mn2+ and by Fe3+ and degrades the birchwood xylan being the principal products the xylobiose and the xylose. This work is the first report of the purification and biochemical characterization of a xylanase called SMXL1 produced by S. maydis.

Improving the catalytic efficiency of thermostable Geobacillus stearothermophilus xylanase XT6 by single-amino acid substitution

Directed evolution using error-prone polymerase chain reaction was employed in the current study to enhance the catalytic efficiency of a thermostable Geobacillus stearothermophilus xylanase XT6 parent. High-throughput screening identified two variants with enhanced activity. Sequencing analysis revealed the presence of a single-amino acid substitution (P209L or V161L) in each variant. The maximum activity of mutant V161L and P209L was at 85°C and 70°C, respectively. Both mutants exhibited maximum activity at pH 7. The thermal and alkaline tolerance of mutant V161L only were markedly improved. The two mutants were more resistant to ethanol inhibition than the parent. Substrate specificity of the two mutants was shifted from beechwood xylan to birchwood xylan. The potential of the two mutants to hydrolyze rice straw and sugarcane bagasse increased. Both turnover number (kcat) and catalytic efficiency (kcat/kM) increased 12.2- and 5.7-folds for variant P209L and 13- and 6.5-folds for variant V161L, respectively, towards birchwood xylan. Based on the previously published crystal structure of extracellular G. stearothermophilus xylanase XT6, V161L and P209L mutation locate on βα-loops. Conformational changes of the respective loops could potentiate the loop swinging, product release and consequently result in enhancement of the catalytic performance.

Exploiting xylan as sugar donor for the synthesis of an antiproliferative xyloside using an enzyme cascade

Background Currently, industrial societies are seeking for green alternatives to conventional chemical synthesis. This demand has merged with the efforts to convert lignocellulosic biomass into value-added products. In this context, xylan, as one of main components of lignocellulose, has emerged as a raw material with high potential for advancing towards a sustainable economy. Results In this study, the recombinant endoxylanase rXynM from the ascomycete Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris and used as one of the catalysts of an enzyme cascade developed to synthesize the antiproliferative 2-(6-hydroxynaphthyl) β-d-xylopyranoside, by transglycosylation of 2,6-dihydroxynaphthalene. The approach combines the use of two fungal xylanolytic enzymes, rXynM and the β-xylosidase rBxTW1 from the same fungus, with the cost-effective substrate xylan. The reaction conditions for the cascade were optimized by a Central Composite Design. Maximal productions of 0.59 and 0.38 g/L were reached using beechwood xylan and birchwood xylan, respectively. For comparison, xylans from other sources were tested in the same reaction, suggesting that a specific optimization is required for each xylan variety. The results obtained using this enzyme cascade and xylan were similar or better to those previously reported for a single catalyst and xylobiose, an expensive sugar donor. Conclusions Beechwood and birchwood xylan, two polysaccharides easily available from biomass, were used in a novel enzyme cascade to synthetize an antiproliferative agent. The approach represents a green alternative to the conventional chemical synthesis of 2-(6-hydroxynaphthyl) β-d-xylopyranoside using a cost-effective substrate. The work highlights the role of xylan as a raw material for producing value-added products and the potential of fungal xylanolytic enzymes in the biomass conversion.

An Innovative Biocatalyst for Continuous 2G Ethanol Production from Xylo-Oligomers by Saccharomyces cerevisiae through Simultaneous Hydrolysis, Isomerization, and Fermentation (SHIF)

Many approaches have been considered aimed at ethanol production from the hemicellulosic fraction of biomass. However, the industrial implementation of this process has been hindered by some bottlenecks, one of the most important being the ease of contamination of the bioreactor by bacteria that metabolize xylose. This work focuses on overcoming this problem through the fermentation of xylulose (the xylose isomer) by native Saccharomyces cerevisiae using xylo-oligomers as substrate. A new concept of biocatalyst is proposed, containing xylanases and xylose isomerase (XI) covalently immobilized on chitosan, and co-encapsulated with industrial baker’s yeast in Ca-alginate gel spherical particles. Xylo-oligomers are hydrolyzed, xylose is isomerized, and finally xylulose is fermented to ethanol, all taking place simultaneously, in a process called simultaneous hydrolysis, isomerization, and fermentation (SHIF). Among several tested xylanases, Multifect CX XL A03139 was selected to compose the biocatalyst bead. Influences of pH, Ca2+, and Mg2+ concentrations on the isomerization step were assessed. Experiments of SHIF using birchwood xylan resulted in an ethanol yield of 0.39 g/g, (76% of the theoretical), selectivity of 3.12 gethanol/gxylitol, and ethanol productivity of 0.26 g/L/h.

Nghiên cứu biểu hiện và tinh chế -glucuronidase có nguồn gốc vi khuẩn dạ cỏ dê trong tế bào Esccherichia coli

α-1,2-Glucuronidase GH67 là enzyme có khả năng phân cắt chuỗi bên 4-O-methylglucuronic acid ((Me)GlcA) trong glucuronoarabinoxylan để làm tăng chuyển hóa lignocellulose thành đường đơn cho lên men sản xuất cồn và các chất có giá trị từ sinh khối thực vật. Trong nghiên cứu này, gen Gglc1 dài 1908 nucleotide mã hóa cho enzyme α-1,2-glucuronidase GH67 trưởng thành có nguồn gốc từ vi khuẩn trong dạ cỏ dê đã được biểu hiện thành công trong chủng E. coli Roseta. Hầu hết enzyme được biểu hiện dưới dạng tan khi chủng tái tổ hợp được nuôi cấy trong môi trường TB, PE có 0,05 mM IPTG ở 25oC, 30oC. Enzym được tinh chế thành công bằng sắc ký ái lực với độ tinh sạch 90%. Hoạt tính của enzyme đã được đánh giá sơ bộ dựa trên sự chuyển hóa (Me)GlcA trong birchwood xylan thành 4-O-methyl-D-glucuronic acid và D-glucuronic acid làm thay đổi pH dung dịch và được phát hiện bằng bromothymol blue. Enzyme sẽ được tiếp tục đánh giá tính chất để xem xét khả năng bổ sung vào hỗn hợp enzyme cho chuyển hóa hiệu quả sinh khối thực vật thành đường.

Alkaline pretreatment for practicable production of ethanol and xylooligosaccharides

The economics for production of secondgeneration (2G) ethanol from sugarcane bagasse in large scale, competing with the cogeneration of electric energy, is still not consolidated. In this scenario, the key for feasibility may be the biorefinery concept, a multiproduct industry using biomass fractions to produce energy, chemicals and by-products. Xylooligosaccharides (XOS) are oligomers of xylose often used as additives in food, animal feeds, and drugs. The effect of NaOH pretreatment on the recovery of xylan for XOS production from sugarcane bagasse under different conditions, namely 121°C, 4-7% NaOH loading, was investigated. The best condition was 4% NaOH and 60 min of reaction, achieving 55% of xylan extraction, without monomer production. In order to produce XOS, soluble and immobilized xylanases were used to hydrolyze commercial birchwood xylan (as control) and the sugarcane bagasse xylan. The immobilized endoxylanase produced XOS with 37% of xylobiose and 20% of xylotriose (w/w). The small production of xylose clearly indicated the purity of the xylan extracted from sugarcane bagasse. The biocatalyst had more than 90% of its activity preserved after 5 reaction cycles. The results showed the suitability of sugarcane bagasse as a raw material for production of ethanol and of XOS using immobilized xylanase.