Specific Activity Of Xylanase Research Articles

Produksi Enzim Spesifik Xilanase pada Variasi Suhu Konsorsium Trikultur Bakteri Termofilik

Xylanase is an extracellular enzyme that is capable of hydrolyzing xylan (hemicellulose) into xylose and xylo-oligosaccharides. Xylanase can be produced by microbes through a fermentation process, the microbes used are thermophilic bacteria. Thermophilic bacteria are microorganisms that are capable of producing thermostable enzymes through fermentation. The bacteria used are MSS11, MS 16 and MS 18. These thermophilic bacteria can live at temperatures above 45°C-80°C. To determine specific enzyme activity, enzyme and protein activity values were tested using a 540 nm (enzyme) and 750 nm (protein) septovotometer. The method used in this research is descriptive in triplicate. The research was carried out at the Microbiology Laboratory, Faculty of Mathematics and Natural Sciences, Padang State University on December 23 2023. Beechwood medium which had been mixed with MSS 11, MS 16, and MS 18 bacteria was then incubated at temperatures of 55℃, 65℃ and 75℃ so that specific enzyme activity was obtained. Of these three temperatures, the optimum specific enzyme activity of xylanase is 65℃, where the specific enzyme value obtained at 65℃ is 3.31 U/mg. Meanwhile, at low temperatures, namely at 55℃, the enzymatic reaction is slow, namely it has an enzyme activity value of 1.02 U/mg. Finally, at a temperature of 75, there was a decrease in specific enzyme activity, where the specific enzyme activity value was 3.12 U/mg.

Global characterization of GH11 family xylanases genes in Neostagonosporella sichuanensis and functional analysis of Nsxyn1 and Nsxyn2.

Rhombic-spot disease, caused mainly by Neostagonosporella sichuanensis, significantly impacts the yield and quality of fishscale bamboo (Phyllostachys heteroclada). Xylanases are essential for pathogenic fungi infection, yet their specific functions in the physiology and pathogenicity of N. sichuanensis remain unclear. Here, we characterized three xylanase proteins with glycosyl hydrolase 11 domains from the N. sichuanensis SICAUCC 16-0001 genome and examined the function of Nsxyn1 and Nsxyn2. Purified Nsxyn1 and Nsxyn2 proteins displayed specific xylanase activity in vitro and induced cell death in Nicotiana benthamiana, independent of their enzymatic function. Both proteins possessed signal peptides and were confirmed as secreted proteins using a yeast secretion system. Subcellular localization revealed that Nsxyn1 and Nsxyn2 localized in both the cytoplasm and nucleus and can trigger cell death in N. benthamiana through Agrobacterium tumefaciens-mediated transient transformation. qRT-PCR results showed notable upregulation of Nsxyn1 and Nsxyn2 during infection, with Nsxyn1 exhibiting an 80-fold increase at 15 days post-inoculation. Deletion of Nsxyn1 and Nsxyn2 in N. sichuanensis impaired xylan degradation, adaptation to osmotic and oxidative stress, and pathogenic full virulence. Deletion of Nsxyn1 notably slowed fungal growth and reduced spore production, whereas only a reduction in microconidial production was observed in Nsxyn2 mutants. Complementation of Nsxyn1 and Nsxyn2 only partially restored these phenotypic defects in the ∆Nsxyn1 and ∆Nsxyn2 mutants. These findings suggest that Nsxyn1 and Nsxyn2 contribute to N. sichuanensis virulence and induced plant defense responses, providing new insights into the function of xylanases in the interaction between fishscale bamboo and N. sichuanensis.

Effect of multiple nonthermal plasma treatments of filamentous fungi on cellular phenotypic changes and phytopathogenicity

The effect of multiple sublethal doses of non-thermal plasma treatments on fungal cells phenotypical changes and the reduction in phytopathogenicity of Fusarium oxysporum, Botrytis cinerea, and Alternaria alternata was examined. The intensity of these changes depended on the species of fungus and the number of exposures of the mycelia to the DBD plasma. Microscopic observations showed that the plasma damaged the surface of the hyphae, increased their thickness and decreased overall dry biomass of the organisms. A decrease in pectinolytic activity was found in F. oxysporum and A. alternata, in contrast to B. cinerea, where an increase in pectinolytic activity was observed after the fifth plasma treatment. Changes in specific xylanase activity varied and were dependent on the species of fungus. The percentage of cucumber seeds germinated artificially infected with mycelium after multiple plasma treatments increased compared to those that were mycelium infected prior to plasma exposure. Plants that developed from seeds after plasma exposure were characterized by a higher biomass and longer roots and stems. Multiple treatments of the studied fungi with plasma, followed by seed infection, increased the SWVI and SWVI indexes of cucumber seedlings, but they did not reach the characteristic value of the control seeds (not infected with fungi). The reduced phytopathogenicity of the tested fungi was confirmed by artificial infestation of tomato fruits.

Enzymatic depolymerization of wheat straw polysaccharides

The purpose of this study is to develop a technology for enzymatic processing for depolymerization of polysaccharides in wheat straw to obtain the maximum yield of glucose and sorbitol. Cellulolytic enzymes endo-1,4-β-glucanase (EC 3.2.1.4) and cellobiose (1,3-β-glucosidase) (CF 3.2.1.21) were isolated and studied in local strains Tr. viride 121, which are grown under deep cultivation conditions. A technology has been developed for obtaining a complex preparation “Cellozyme G20x” with a high yield and specific activity of cellulase, xylanase, β-glucanase and pectinase, and a scheme for purification from cellulases by precipitation, ultrafiltration, and freeze drying is not inferior in efficiency to commercial preparations. The physicochemical properties of the preparation “Cellozyme G20x” have been studied, the optimal parameters of the action and stability of the enzyme preparation have been established. The efficiency of Cellozyme G20x for hydrolysis of straw polysaccharides was 35-40% in terms of glucose yield.

Production and Characterization of Thermoalkaliphilic Xylanase from Bacillus halodurans Cm1, and Its Application on Degumming Process of Ramie (Boehmeria nivea L.Gaud) Fiber as Textile Raw Material

Ramie fiber is a potential raw material to substitute imported raw materials such as cotton. Due to its higher hemicellulose content, ramie fiber required hydrolysis in a process called degumming. Enzymatic degumming is environmentally friendly compared to traditional process which using chemicals. Alkalithermophilic xylanase have high ability in hemicellulose hydrolysis. The production of xylanase was conducted by submerged fermentation of Bacillus halodurans CM1 in 20L bioreactor using Mamo and corncob medium with optimum conditions at 50°C, pH 9, 150 RPM and 1 vvm. The optimum specific activity of xylanase measured by Bailey method at 70°C and pH 9 is 475.41 U/mg. Xylanase was stable at 50°C, pH 9 and relatively stable to K+, Na2+, Co2+ and Ca2+ metal ions and Triton-X, Saba dan Tween-80 surfactants. Degumming process was carried out by immersing ramie fibers in formulated degumming solution with vlot 1:20 at 50°C, 150 RPM and 180 minutes. The enzymatic degumming process may substitute or reduce the use of chemicals due to its significant effect on ramie fiber quality. Enzymatic and chemical degumming process reduce the weight of Ramie Fiber to 7.23 %, and 7.72 %, slightly higher than enzymatic degumming 7.15%. Enzymatic degumming maintains tensile strength at 27.51 %. Whiteness index enhanced to 2.99% enzymatically and 3.49% chemically.
 Keywords: Bacillus halodurans CM1, enzymatic degumming, ramie fiber, textile industry, thermoalkaliphilic xylanase

Expression of the Xylanase Gene from Pyromyces finnis in Pichia pastoris and Characterization of the Recombinant Protein

The heterologous expression and characteristics of a new xylanase from Pyromyces finnis are described. The endo-1,4-β-xylanase XylP (EC 3.2.1.8) consists of 223 amino acids and 19 residues of a putative signal peptide in the N-terminal region. The amino-acid sequence of the mature protein has the greatest homology (84%) with the sequence of the native catalytic N-terminal domain of Neocallimastix patriciarum endo-1,4-β-xylanase. A synthetic nucleotide sequence encoding the mature XylP protein was expressed in Pichia pastoris. The purified recombinant enzyme was active with birch xylan and arabinoxylan as substrates. The optimal pH and temperature for enzyme activity were established as 5.0 and 50°C, respectively, with the use of birch xylan. The specific activity of xylanase was 4700 U/mg protein, and KM and Vmax were equal to 0.51 mg/mL and 7395.3 μmol/(min mg), respectively. The recombinant XylP protein showed moderate thermal and high pH stability, as well as resistance to digestive enzymes and protein xylanase inhibitors from cereals. It was also shown that Mg2+, Co2+ and Li+ ions have a positive effect on enzyme activity.

Fusion expression of β-mannanase Man5A and xylanase Tlxyn11B in Pichia pastoris

Mannanase and xylanase, the main hemicellulolytic enzymes, are widely used in food, feed, textile and papermaking industries, and usually they are used in combination. Mannanase Man5A from Talaromyces leycettanus JCM12802 consist of the carbohydrate binding module (CBM), linker region and catalytic domain. The CBM coding region of Man5A was removed and fused to C-terminal of the xylanase gene Tlxyn11B. The fusion gene Tlxyn11B-linker-man5A was successfully expressed in Pichia pastoris and the fusion protein Tlxyn11B-Man5A was purified and characterized. The theoretical molecular weights of Tlxyn11B, Man5A without CBM region, and Tlxyn11B-Man5A are 21.6 kDa, 41.0 kDa, and 62.6 kDa, respectively. The fusion protein had high xylanase and mannanase activities. The optimal temperature of the fused xylanase is 70 °C, which is 5 °C higher than Tlxyn11B-w (xylanase before fusion). The fused mannanase exhibited maximal activity at 90 °C, which is similar to Man5A-w (mannanase before fusion). More than 48% of xylanase activity of Tlxyn11B-Man5A was residual after the condition of 60 °C with 1 h, which is significantly higher than Tlxyn11B-w (only 20% of activity was left at 60 °C for 20 minutes). The optimal pHs of Tlxyn11B-Man5A for xylanase and mannanase activity are 4.0 and 5.0, respectively, which are 0.5 and 1.0 units higher than those of Tlxyn11B-w and Man5A-w. The pH range of fused enzymes got wider and the pH stability is improved. The specific activities of xylanase and mannanase of Tlxyn11B-Man5A are 1 784.3 U/mg and 1 639.6 U/mg, respectively, which is lower than those of Tlxyn11B and Man5A (8 300.0 U/mg and 1 979.0 U/mg). It may be due to of the high molecular weight of fusion enzyme. The Km and Vmax of the fused xylanase and mannanase are 1.2 mg/mL and 1.7 mg/mL, 2 000.0 μmol/(min·mg) and 2 831.6 μmol/(min·mg), respectively. Tlxyn11B and Man5A were successfully fusion expressed in P. pastoris, and the good properties of fusion of xylanase and mannanase make it has great application potential in animal feed, food and other industrial production, and it provided new ideas for the improvement of enzyme performance.

Construction of a new lipase- and xylanase-producing oleaginous yeast consortium capable of reactive azo dye degradation and detoxification

A new oleaginous yeast consortium Y-BC-SH which stands for molecularly identified species Yarrowia sp., Barnettozyma californica and Sterigmatomyces halophilus was successfully constructed in this study. This multipurpose oleaginous yeast consortium was developed based on its higher ability to accumulate large amounts of lipids in the form of triacylglycerol, grow on xylose, produce lipase and xylanase and it could rapidly decolorize and degrade commonly-used textile reactive azo dyes. The specific enzyme activities of lipase, xylanase, xylan esterase, β-xylosidase, CMCase, β-glucosidase and cellobiohydrolase produced by Y-BC-SH were significantly higher than that of individual strains. As chemical oxygen demand reduction had occurred in the dye mixture solutions, it was evidence of their color removal and mineralization by Y-BC-SH. The significant induction of oxidoreductive enzymes by Y-BC-SH was probably due to the coordinated metabolic interactions of the individual strains. Phytotoxicity assay confirmed that metabolites generated after dye degradation by Y-BC-SH are non-toxic.

Rational engineering of the Trichoderma reesei RUT-C30 strain into an industrially relevant platform for cellulase production

BackgroundThe path for the development of hypersecreting strains of Trichoderma reesei capable of producing industrially relevant enzyme titers remains elusive despite over 70 years of research and industrial utilization. Herein, we describe the rational engineering of the publicly available T. reesei RUT-C30 strain and a customized process for cellulase production based on agroindustrial by-products.ResultsA CRISPR/Cas9 system was used to introduce six genetic modifications in RUT-C30. Implemented changes included the constitutive expression of a mutated allele of the cellulase master regulator XYR1, the expression of two heterologous enzymes, the β-glucosidase CEL3A from Talaromyces emersonii and the invertase SUC1 from Aspergillus niger, and the deletion of genes encoding the cellulase repressor ACE1 and the extracellular proteases SLP1 and PEP1. These alterations resulted in a remarkable increase of protein secretion rates by RUT-C30 and amended its well described β-glucosidase deficiency while enabling the utilization of sucrose and eliminating the requirement of inducing sugars for enzyme production. With a developed sugarcane molasses-based bioprocess, the engineered strain reached an extracellular protein titer of 80.6 g L−1 (0.24 g L−1 h−1), which is the highest experimentally supported titer so far reported for T. reesei. The produced enzyme cocktail displayed increased levels of cellulase and hemicellulase activities, with particularly large increments being observed for the specific activities of β-glucosidase (72-fold) and xylanase (42-fold). Notably, it also exhibited a saccharification efficiency similar to that of a commercially available cellulase preparation in the deconstruction of industrially pretreated sugarcane straw.ConclusionThis work demonstrates the rational steps for the development of a cellulase hyperproducing strain from a well-characterized genetic background available in the public domain, the RUT-C30, associated with an industrially relevant bioprocess, paving new perspectives for Trichoderma research on cellulase production.

Selection of Microorganisms Possessing Thermostable Lignocellulolytic Enzymes and Application of the Enzymes for Saccharification of Pretreated Palm Oil Mill Wastes

This study attempted to find potential effective thermotolerant microorganisms producing complex enzymes for use in the hydrolyzing empty fruit bunch (EFB) to reduce cost of enzyme and enhance the efficiency of saccharification. The enrichment process at 45 °C was employed as a strategy to obtain four effective thermotolerant microorganisms. Streptomyces thermocarboxydus ME742, Bacillus subtilis ME751 and Bacillus amyloliquefaciens ASB/TRE produced the highest activity of xylanase (226.2 U/mL), CMCase (3.84 U/mL) and FPase (69.55 U/mL), respectively, while Aspergillus fumigatus A4112 exhibited the highest specific activity of xylanase (637.9 U/mg), CMCase (5.55 U/mg) and FPase (21.58 U/mg). Xylanase of isolated ME742 and A4112, CMCase of isolated ASB/TRE, FPase of isolated ME742, ME751 and ASB/TRE possessed thermostability with 80% remaining activity at 60 °C after 1 h incubation. These four strains were capable to reduce 49–78% (w/w) lignin in raw EFB with simultaneous enzyme production. The EFB residue was reused as substrate for saccharification with the highest amount of reducing sugar using the crude enzymes from S. thermocarboxydus ME742 (9.24 mg/g EFB). The sugar was 3.76 and 3.61 fold higher than that obtained from saccharification of acid- and alkaline-pretreated EFB, respectively. Moreover, the crude enzymes from A. fumigatus A4112 and B. amyloliquefaciens ASB/TRE hydrolyzed palm oil mill effluent (POME) to generate high yield of reducing sugar (61.01–64.63 mg/g TS-POME). Therefore, these selected strains were considered as the potent biological tool applicable in the bioconversion of oil palm biomass to fermentable reducing sugars.

Chemical composition and in vitro ruminal fermentation of common tree forages in the semi-arid range lands of India

The objective of study was to look for the promising tree leaves for feeding livestock particularly the small ruminants. Ten tree leaves were collected from semiarid region of Rajasthan and evaluated for their nutritional quality in terms of chemical composition as well as in vitro dry matter and organic matter digestibility (IVDMD and IVOMD). Most of the tree leaves were rich in CP content. OM, CP and ADF content of these collected tree leaves varied from 87.1 to 92.5%, 9.4 to 19.8% and 22.7 to 47.9% on DM basis, respectively. Rumen protozoal number decreased due to inclusion of Sapindus mukorossi, Azadirachta indica and Prosopis cineraria tree leaves in the incubation media. IVDMD, IVOMD, TVFA and propionate production significantly higher for Ailanthus excelsa tree leaves followed by Acacia arabica and Acacia senegal tree leaves. All the tested tree leaves had no effect on β-glucosidase and amylase enzyme activity. However, specific activity of carboxymethyl cellulase and xylanase reduced significantly due to addition of Sapindus mukorossi, Azadirachta indica and Prosopis cineraria tree leaves in the incubation medium. The results indicated that among the tree leaves tested in the present study Ailanthus excelsa, Acacia arabica and Acacia senegal are good tree fodder for feeding to the ruminants.

Enhanced methane production from wheat straw with the assistance of lignocellulolytic microbial consortium TC-5

The major obstacle of methane production from lignocellulose lies in the inefficient deconstruction of biomass. In this study, an anaerobic microbial consortium TC-5 was enriched with high lignocellulose-degradation capacity to enhance methane production from wheat straw. High degradation ratio of 45.7% of un-pretreated wheat straw was achieved due to a multi-species lignocellulolytic enzyme presented in the crude culture supernatant. The specific activity of xylanase, xylan esterase and β-xylosidase reached the highest level of 4.23, 0.15 and 0.48 U/mg, while cellobiohydrolase, endoglucanase and β-glucosidase showed the highest specific activity of 0.36, 0.22 and 0.41 U/mg during 9 days’ degradation. Inoculation of TC-5 in digestion sludge during anaerobic digestion of wheat straw resulted in remarkable enhancement of 22.2% and 36.6% in methane yield under mesophilic and thermophilic conditions, respectively. This work demonstrates the potential of TC-5 for enhancing the production of biogas and other chemicals through biomass based biorefinery.

Effect of CBM1 and linker region on enzymatic properties of a novel thermostable dimeric GH10 xylanase (Xyn10A) from filamentous fungus Aspergillus fumigatus Z5

Xylanase with a high thermostability will satisfy the needs of raising the temperature of hydrolysis to improve the rheology of the broth in industry of biomass conversion. In this study, a xylanase gene (xyn10A), predicted to encode a hydrolase domain of GH10, a linker region and a CBM1 domain, was cloned from a superior lignocellulose degrading strain Aspergillus fumigatus Z5 and successfully expressed in Pichia pastoris X33. Xyn10A has a specific xylanase activity of 34.4 U mg−1, and is optimally active at 90 °C and pH 6.0. Xyn10A shows quite stable at pHs ranging from 3.0 to 11.0, and keeps over 40% of xylanase activity after incubation at 70 °C for 1 h. Removal of CBM1 domain has a slight negative effect on its thermostability, but the further cleavage of linker region significantly decreased its stability at high temperature. The transfer of CBM1 and linker region to another GH10 xylanase can help to increase the thermostability. In addition, hydrolase domains between the two Xyn10A proteins naturally formed a dimer structure, which became more thermostable after removing the CBM1 or/and linker region. This thermostable Xyn10A is a suitable candidate for the highly efficient fungal enzyme cocktails for biomass conversion.

Bi-functional fusion enzyme EG-M-Xyn displaying endoglucanase and xylanase activities and its utility in improving lignocellulose degradation

In this study, the gene fusion of endoglucanase (EG, one of cellulases) from Teleogryllus emma and xylanase (Xyn, one of hemicellulases) from Thermomyces lanuginosus was constructed to generate a fusion enzyme (EG-M-Xyn). Through the expression and purification by ultrafiltration and size-exclusion chromatography, the purified EG-M-Xyn had a molecular weight of 75.5 kDa and exhibited the specific activity of CMCase and xylanase as 306.8 U/mg and 1227.3 U/mg, respectively. The Km values (CMC and beechwood xylan) were 6.8 and 60.6 mg mL−1 while catalytic efficiency (kcat/Km) values of CMCase and xylanase were 3280 and 38,797 min−1 mg−1 mL, respectively.EG-M-Xyn exerted great properties for its great potential in improving the enzymatic hydrolysis of lignocellulosics to produce fermentable sugars. First, EG-M-Xyn showed mild reaction pH and temperature of 5.5 and 50 °C, respectively. Secondly, EG-M-Xyn exhibited great heat tolerance of T1/2 values of 173 (CMCase) and 693 min (xylanase). Lastly and most importantly, application of EG-M-Xyn in combination with Ctec2 (commercial enzyme) in the saccharification led to a 10–20% net increase in fermentable sugars liberated from pretreated rice straw in comparison to the Ctec2 alone group. In conclusion, EG-M-Xyn had great potential in generating fermentable sugars from renewable agro-residues for biofuel and fine chemical industry.

Ozonation Pretreatment Evaluation for Xylanase Crude Extract Production from Corncob under Solid-State Fermentation

Xylanases are highly exploited enzyme in industries, including food and chemical industry. Xylanases can be utilized in catalyzing the endohydrolysis of 1,4-β-xylosidic linkages in xylan, lignocellulosic component to produce xylose-monomer. This research aims to optimize xylanase production from alternative substrate, corncob. Corncob contains 41.17% of hemicellulose, polymer of xylan. Xylanases are produced through solid state fermentation by Trichoderma viride. Ratio between substrate and moistening solution was 0.63 g/mL with fermentation temperature 32,8OC. Variables varied include incubation time and pretreatment (using autoclave, ozonation, combination of ozonation and autoclave, also without pretreatment). Xylanase activity was measured by DNS method using 1% of xylan as substrate standard. The result showed that the best incubation time is 36 h with 14403.8707 U/mg protein for specific xylanase activity by using autoclave as pretreatment. Ozonation pretreatment process can increase the enzyme activity of xylanase.

EXPLORATION OF BROTH CHICKEN GUT AS GROWTH MEDIA OF Escherichia coli BL21 pET-Endo FOR ENDO-Β-1,4-D-XYLANASE PRODUCTION

<p>Endo-β-1,4-D-xylanase is a hydrolytic enzyme that breakdown the 1.4 chain of xylan polysaccharide. We have succes to transform the plasmid pET-Endo gene encoding endo-1,4-β-D-xylanase from Bacillus sp. originally from termites abdominal to E. coli BL21. The clone was ready for large scale of enzyme production. To reduce production cost, we look for subtitute media for the expensive Luria Berthani broth. Chicken guts broth is good alternative while rich of protein and very cheap. The content of N dissolved chicken guts broth reaches 87 % of LB broth. Growth of E. Coli BL21 in Chicken guts broth and LB broth (as control) was observed by Optical Density (OD) using spectrofotometer. Concentration of glucose added in broth and incubation temperature was varied. The result showed that optimal growth was in addition of 1.5 % glucose and incubated at 37 <sup>o</sup>C for 16 h. This optimal condition was used to grow E. coli BL21 pET-Endo for xylanase production. Enzyme purification was done by Ni-NTA affinity chromatography. Highest protein yield was 0.076 mg/mL obtained in 100 mM imidazole elucidation. The activity and specific activity of xylanase were estimated as 0.042 U/mL and 0.556 U/µg, respectively. The purification factor was 3.16 time and the molecular weight of enzyme was ± 30, 000 Dalton</p>

Oligomerization triggered by foldon: a simple method to enhance the catalytic efficiency of lichenase and xylanase

BackgroundEffective and simple methods that lead to higher enzymatic efficiencies are highly sough. Here we proposed a foldon-triggered trimerization of the target enzymes with significantly improved catalytic performances by fusing a foldon domain at the C-terminus of the enzymes via elastin-like polypeptides (ELPs). The foldon domain comprises 27 residues and can forms trimers with high stability.ResultsLichenase and xylanase can hydrolyze lichenan and xylan to produce value added products and biofuels, and they have great potentials as biotechnological tools in various industrial applications. We took them as the examples and compared the kinetic parameters of the engineered trimeric enzymes to those of the monomeric and wild type ones. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the trimeric lichenase and xylanase increased 4.2- and 3.9- fold. The catalytic constant (kcat) of the trimeric lichenase and xylanase increased 1.8- fold and 5.0- fold than their corresponding wild-type counterparts. Also, the specific activities of trimeric lichenase and xylanase increased by 149% and 94% than those of the monomeric ones. Besides, the recovery of the lichenase and xylanase activities increased by 12.4% and 6.1% during the purification process using ELPs as the non-chromatographic tag. The possible reason is the foldon domain can reduce the transition temperature of the ELPs.ConclusionThe trimeric lichenase and xylanase induced by foldon have advantages in the catalytic performances. Besides, they were easier to purify with increased purification fold and decreased the loss of activities compared to their corresponding monomeric ones. Trimerizing of the target enzymes triggered by the foldon domain could improve their activities and facilitate the purification, which represents a simple and effective enzyme-engineering tool. It should have exciting potentials both in industrial and laboratory scales.

An efficient production of high-pure xylooligosaccharides from corncob with affinity adsorption-enzymatic reaction integrated approach

Xylooligosaccharides (XOS) are high value-added ingredients for functional foods and they have potential use as prebiotics. In order to reduce the production cost of XOS, we constructed a fusion enzyme consisting of expansin and endo-xylanase from B. subtilis Lucky9 to produce high-pure XOS with affinity adsorption-enzymatic reaction integrated approach. By optimization of inserting linker, the specific xylanase activity of fusion enzyme with R2 linker was increased 1.28-fold than that of native xylanase. Electrophoretic pure fusion enzyme was separated from crude enzyme extracts by affinity adsorption on corncob substrate, with 90.3% recovery in optimized conditions. Then, the fusion enzyme-corncob mixture was directly hydrolyzed. The yield of XOS reached 6.91mg/mL. The production of XOS with high purity of 91.7% was obtained by one step of centrifugation. This affinity adsorption-enzymatic reaction integrated XOS production approach provides a green route to alternative XOS traditional enzymatic production.

Deciphering characteristics of the designer cellulosome from Bacillus subtilis WB800N via enzymatic analysis

Recombinant Bacillus subtilis WB800N harboring pGETS 118 designer cellulosome genes was applied to the artificial cellulosome production. To investigate the characteristics of cellulosome assembly in the supernatant, three free enzymes clones, i.e., endoglucanase, exoglucanase and xylanase, were constructed. The samples of the respective free enzymes and the cellulosome were collected and examined via various enzymatic assays. In comparison of specific activity of anchored cellulosome and the free enzymes on cell pellets, it was found that only anchored cellulosome exhibited the activities. No activity was detected on the cell pellets of the three free enzyme clones. This indicated that the designer cellulosome subunits must be properly self-assembled into a substantial complex capable of the cellulose degradation. To study the cellulose hydrolytic behavior, the best specific activities of endoglucanase and xylanase obtained in the supernatant cellulosome were 3.62±0.02U/mg and 1.97±0.06U/mg, respectively. The end products of the enzyme reaction detected via HPLC analysis showed that oligosaccharide and cellobiose were the major products in the hydrolytic reactions. In the kinetic studies, the Vmax value (8.98mM/h) for endoglucanase in the supernatant cellulosome was higher than pellets (0.603mM/h). However, the endoglucanase of pellets gave a lower Km value of 0.359g/L, indicating a higher substrate affinity than that in the supernatant (11.87g/L).

Induction and catabolite repression of cellulase and xylanase synthesis in the selected white-rot basidiomycetes

Abstract This paper reports regulation of endoglucanase (EC 3.2.1.4) and xylanase (EC 3.2.1.8) production in submerged cultivation of four white-rot basidiomycetes. Among carbon sources tested, the Avicel-based medium provided the highest levels of both hydrolases activities in all fungal cultures. However, the maximum endoglucanase and xylanase activities of the tested basidiomycetes varied from 3.9 U/ml and 7.4 U/ml in Fomes fomentarius to 34.2 U/ml and 29.5 U/ml in Pseudotrametes gibbosa, respectively (P. gibbosa specific cellulase and xylanase activities achieved 8.55 and 7.38 U/mg, respectively). Replacement of Avicel in the medium with carboxymethyl cellulose or xylan significantly lowered the enzyme yield of the tested fungi. Moreover, xylan did not ensure high xylanase activity of these fungi. Lignocellulosic substrates used as a carbon source provided poorer productivity (the specific CMCase activity was 1.12–3.62 U/mg and the specific xylanase activity was 1.95–3.32 U/mg). Expression of endoglucanase and xylanase synthesis in Panus lecometei and P. gibbosa was inducible; supplementation of the glycerol-containing medium with Avicel accompanied with a sharp increase of the fungal specific CMCase and xylanase activities from 0.02–0.04 U/mg to 1.30–8.55 U/mg. Supplementation of the Avicel-induced cultures with glucose or glycerol caused a catabolite repression of the cellulase and xylanase formation by P. gibbosa and P. lecometei. The enzyme synthesis resumed only after depletion of easily metabolizable carbon source, glucose or glycerol, from the medium. The data received suggest that in the tested fungi endoglucanase and xylanase synthesis is under control by a common regulatory mechanism.