Increase In Xylanase Activity Research Articles

Hyper xylanase production and potential of xylooligosaccharides formation from a novel Bacillus australimaris KS2

The study has been designed to isolate xylanase-producing bacteria and parametric optimization for maximum production. The xylanase was utilized for enzymatic hydrolysis of wheat bran (WB) and wheat bran extracted xylan (WBEx) to obtain xylooligosaccharides. The research was planned for value addition in the agriculture sector with low-cost enzyme production and its utilization in prebiotic production from agro-residue. The xylanase obtained from the new isolate Bacillus australimaris KS2 was optimized with 2.0% of 24 h inoculum supplemented with 2.0% wheat bran at pH 7, 35 °C for 48 h with an agitation rate of 150 rpm. There was a 2.96-fold increase in xylanase activity from its initial (2997.07 IU/mL) activity to the optimized condition (8873.07 IU/mL). The maximum reducing sugar production over enzymatically hydrolyzed WB was found after 8 h of reaction time at pH 5, 55 °C with 375 units of enzyme. In the case of enzymatically hydrolyzed WBEx, the maximum production of 588 mg/g was achieved after 16 h at pH 6 and 55 °C of reaction temperature at the same amount of enzyme dose. The results demonstrated that the new isolate Bacillus australimaris KS2 was an efficient xylanase producer. Xylooligosaccharides produced through enzymatic hydrolysis of WBEx showed good prebiotic potential over Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus rhamnosus.

Correlation Between Kidney Function and Intestinal Biological Characteristics of Adenine and Folium SennaeInduced Diarrhea Model in Mice.

This study investigated the correlation among kidney function, intestinal enzyme activities, and microbial activity of adenine and Folium sennae-induced diarrhea model in mice, which provided a basis for clinical treatment of kidney-intestinal correlation. We performed different doses of adenine combined with Folium sennae intragastric administration to establish the animal model of diarrhea. We assessed thymus and spleen indexes, serum creatinine, urea nitrogen and uric acid contents, intestinal contents and mucosal enzyme activities, and microbial activity. After modeling, mice presented increased serum creatinine and decreased urea nitrogen. Uric acid showed different changes in the different model groups. The thymus index in the model mice was trending downward, whereas the spleen index was the opposite. Moreover, model mice induced a non-significant increase in xylanase activity of the intestinal contents and mucosa compared to the control performance. Sucrase content of the intestinal contents increased considerably in the model groups but decreased in the intestinal mucosa. Lactase and amylase induced different trends in the different modeling methods. As well, the microbial activity of intestinal contents increased significantly, while that of intestinal mucosa decreased. Adenine combined with Folium sennae successfully replicated diarrhea in mice models. Using 50 mg/ (kg/day) adenine for 14 days in combination with 10 g/(kg/day) Folium sennae decoction for 7 days caused kidney function injury in diarrhea mice. In addition, kidney function injury was accompanied by changing in intestinal functional enzyme activity and microbial activity.

Effects of coated copper sulphate and coated folic acid supplementation on growth, rumen fermentation and urinary excretion of purine derivatives in Holstein bulls

The study investigated the impacts of coated copper sulphate (CCS) and coated folic acid (CFA) on growth performance, nutrient digestibility and rumen fermentation in bulls. Fourty Holstein bulls were blocked by individual initial body weight and randomly allocated to one of four groups with a 2 × 2 factor design. The CCS at 0 (CCS-) or 8 mg Cu/kg (CCS+) was added to diets with CFA at 0 (CFA-) or 4 mg FA/kg (CFA+), respectively. In the rumen characteristics an interaction between CCS and CFA was observed. The decrease in rumen ammonia N and increase in xylanase activity, Ruminococcus albus population and urinary total purine derivatives were greater for supplementing CCS in CFA- diets than in CFA + diets. Dry matter intake increased with CFA inclusion and tended to be increased with CCS addition. Inclusion of CFA or CCS elevated average daily gain and apparent digestibility of dry matter, organic matter, crude protein, neutral detergent fibre and acid detergent fibre and decreased feed conversion ratio. Rumen pH was unaltered but total volatile fatty acids concentration and the ratio of acetate to propionate increased with addition of CFA or CCS. Dietary inclusion of CCS increased activities of cellulase and protease and copies of total bacteria, Butyrivibrio fibrisolvens and R. flavefaciens, but decreased α-amylase activity and copies of Prevotella ruminicola and Ruminobacter amylophilus. Rumen enzyme activity and bacteria population increased with CFA addition. The results showed that CFA and/or CCS had stimulatory impacts on growth and feed efficiency in bulls.

Gel microdroplet–based high-throughput screening for directed evolution of xylanase-producing Pichia pastoris

Xylanases have useful applications in a wide range of industries. In this regard, Pichia pastoris has become one of the most attractive host platforms for large-scale production of xylanases. However, genomic engineering is still required for overexpression and efficient secretion. In this paper, we applied droplet-based method to screen directed evolved extracellular xylanase producing P.pastoris strain. Xylanase-producing P.pastoris cells were encapsulated in gel microdroplets with a fluorogenic reporter substrate. Improved production of xylanase increases fluorescence intensity of gel microdroplets, enabled accurate selection of evolved clones by droplet sorting. The screening strategy was validated by identifying yeast with improved xylanase production from a mixed sample with a positive selection accuracy of up to 98%. After three rounds of mutagenesis and selection, approximately 108 variants were screened, and a P.pastoris clone with more than 1.3-fold increase in xylanase activity was identified, representing cellular functions improvement of the production host. The throughput of this approach was at least 103-fold higher than that of the robot-assisted microtiter plate reader, and reagent consumption was reduced by ∼106-fold. Furthermore, the greatly shortened incubation time prior screening significantly accelerated the process of directed evolution.

GH10 XynF1 and Xyn11A: the predominant xylanase identified in the profiling of extracellular proteome of Aspergillus oryzae LC1

Advanced techniques of enzyme production and purification have become prerequisite due to their diverse industrial applications. There is an utmost requirement for screening of new strains capable of synthesising industrially useful enzymes. The present study reports the production and profiling of extracellular proteins expressed by the newly isolated strain of a filamentous fungus, Aspergillus oryzae LC1. The extracellular enzyme production was done by submerged fermentation using Mendel’s and Sternberg’s medium (MSM), and its optimisation was done using one factor at a time (OFAT). The presence of xylanase was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. In addition, the profiling of extracellular proteome of Aspergillus oryzae LC1 was carried out by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS). In this study, media optimisation showed 5.7-fold increase in xylanase activity. The multiple bands observed in zymography revealed the presence of various forms of xylanase. A total of 73 proteins were identified in LC-MS/MS analysis. Functional classification showed that the hydrolytic enzymes consisted of 48% glycoside hydrolase, 11% proteases, 1% polysaccharide lyase and esterase’s, 9% oxidoreductases and 30% other proteins. A total of 26 families of glycosidic hydrolase were detected with other protein families such as serine peptidase, S, LysM, G-D-S-L, M35, carboxyl esterase (CE1), pectate lyase (PL) and oxidoreductases. Among the huge diversity of synergistically acting biomass cleaving enzymes, endo-1, 4-β xylanase with isoforms: xyn F1, xyn B, β xylanase and xyn 11A belonging to GH10 family covered the major portion of the total percentage of identified proteins. As per our knowledge, this is the first report of extracellular proteome analysis of Aspergillus oryzae LC1 suggesting its capability for recombinant expression and evaluation in hemicellulose deconstruction applications.

Construction of enhanced transcriptional activators for improving cellulase production in Trichoderma reesei RUT C30

Enhancing cellulase production in Trichoderma reesei is of great interest for an economical biorefinery. Artificial transcription factors are a potentially powerful molecular strategy for improving cellulase production in T. reesei. In this study, enhanced transcriptional activators XYR1VP, ACE2VP, and ACE1VP were constructed by linking the C terminus of XYR1, ACE2, or ACE1 with an activation domain of herpes simplex virus protein VP16. T. reesei transformants TXYR1VP, TACE2VP, and TACE1VP showed improved cellulase and/or xylanase production. TXYR1VP has a cellulase-free phenotype but with significantly elevated xylanase production. Xylanase I and xylanase II activities [U/(mg biomass)] increased by 51% and 80%, respectively, in TXYR1VP in comparison with parental strain RUT C30. The filter paper activity of TACE2VP in the Avicel-based medium increased by 52% compared to that of RUT C30. In the Avicel-based medium, TACE1VP manifested an 80% increase in FPase activity and a 50% increase in xylanase activity as compared to those of RUT C30. Additionally, when pretreated corn stover was hydrolyzed, crude enzymes produced from TACE1VP yielded a greater glucose release than did the enzymes produced by parental strain RUT C30.

Valorization of Lignocellulosic Residues for Cost-Effective Production of Thermo-Alkali-Stable Xylanase by Geobacillus thermodenitrificans X1 of Indian Himalayan Hot Spring

Thermo-alkali-stable and cellulase-free xylanases finds vital applications in pulp bleaching and biomass conversion processes. The objective of the study was to optimize xylanase production by a novel isolate, Geobacillus thermodenitrificans X1. Different physico-chemical parameters were tested for xylanase production by one-factor-at-a-time (OFAT) approach, followed by statistical optimization using response surface methodology (RSM). Various lignocellulosic residues such as wheat straw, rice straw, corn cob and wheat bran were compared with the commercial xylan for xylanase production. The maximum xylanase activity production of 18.5 U/mL was achieved after OFAT optimization at 50 °C and pH 7–8, 200 rpm, after 96 h of incubation time. Subsequently, RSM optimization resulted in 1.3-fold increase in xylanase activity, i.e. 24 ± 2 U/mL. The present study reports the highest xylanase production on lignocellulosic residue (wheat straw) compared to other reported G. thermodenitrificans isolates.

Peach-palm (Bactris gasipaes Kunth.) waste as substrate for xylanase production by Trichoderma stromaticum AM7

ABSTRACTThis work investigated the xylanase production by fungi isolates from tropical agroforestry systems using peach-palm industrial waste as a substrate. Trichoderma stromaticum AM7 was the best xylanase producer and there was a 160% increase in xylanase activity after optimizing by the Box–Behnken statistical design. The optimization process demonstrated that the maximum activity occurred at 0.95% nitrogen concentration after 6 days of cultivation at 32°C, which achieved a yield of 1440 U g−1 of dry initial substrate. Analysis by scanning electron microscopy showed degradation of the fibers after cultivation. The optimum pH and temperature for xylanase activity were 5.0 and 50°C, respectively. The extensive degradation of the peach-palm waste and xylanase production by T. stromaticum AM7 was due to the combination of a good physicochemical composition of the culture medium and the characteristics of the selected fungus.

Overproduction of Xylanase from Mutants of Bacillus subtilis with Barley Husk as the Prime Carbon Source under Submerged Fermentation after Random Mutagenesis Using Ethyl Methane Sulfonate (EMS) and Acridine Orange (AO)

Aims: Xylanase (EC 3.2.1.8) also known as endo-1,4-β-xylanohydrolase is a type of hydrolytic enzyme participated in the hydrolysis of hemicelluloses particularly in xylan to generate xylose and xylo-oligosaccharides. Due to its enormous potentials, xylanase is frequently used in biobleaching of kraft pulp, clarification of fruit juice, extraction of plant oils, processing of animal feeds, softening Original Research Article Ho and Chinonso; BMRJ, 14(1): 1-17, 2016; Article no.BMRJ.22959 2 of fruits, degradation of agricultural wastes and plant fibers and manufacturing of chemicals including biofuel, ethanol and xylitol. These applications of xylanase avoid the use of chemicals that are expensive, mutagenic and highly non-biodegradable. Interestingly, in recent years, the applications of xylanase in biobleaching and bioprocessing of paper pulp have gained numerous attentions and interests in the industry over the world. Therefore, couple of lignocellulolytic substrate as the alternative cheap carbon source and strain improvement for overproduction of microbial xylanase is implemented as a more potent approach in improving its yield and productivity in submerged fermentation. As a result, the main aim of the present study was primarily involved in the overproduction of xylanase by five mutant strains of Bacillus subtilis subsp. spizizenii ATCC 6633 designated as the MXB 1, MXB 2, MXB 3, MXB 4 and MXB 5 in submerged fermentation using barley husk as the prime carbon source. Methodology: In order to attain the mutants, B. subtilis was subjected to random mutagenesis using ethyl methane sulfonate (EMS) and acridine orange (AO) in the earlier study before screened for the overproduction of xylanase in the present investigation. Results: Based on the present investigation, mutant strains of B. subtilis ATCC 6633 were identified as the potent xylanase producers using cheap agro-industrial residue of barley husk as the sole carbon source under submerged fermentation. Furthermore, extracellular protein production and profile of medium pH during growth of wild type and mutants of B. subtilis under submerged fermentation were also elucidated. Based on the result findings, the time course of xylanase biosynthesis by the mutants of B. subtilis revealed that the enzyme production was initiated from the logarithmic to stationary growth phase whereby the maximum xylanase activity was achieved after 24 h of fermentation. In fact, all mutant strains of B. subtilis were successfully synthesized relatively higher production of xylanase than their parental wild type in submerged fermentation using barley husk as the prime carbon source. Notably, the maximum xylanase activity of 1.76±0.02 U/mL was attained by the mutant MXB 4 of B. subtilis which was approximately 29.4% increase in xylanase activity than the wild type with 1.36±0.003 U/mL. Furthermore, MXB 1, MXB 2, MXB 3 and MXB 5 also exhibited comparatively higher maximum xylanase activity of 1.64±0.009 U/mL, 1.73±0.05 U/mL, 1.74±0.02 U/mL and 1.66±0.02 U/mL compared to their parental wild type. Indeed, the statistical single factor analysis of variance (ANOVA) on xylanase production revealed there was significant difference in the mean of the xylanase production by the wild type and mutant strains of B. subtilis (p<0.05). On the other hand, the total maximum extracellular protein production was achieved by mutant strains of MXB 2 and MXB 4 with 0.82±0.02 mg/mL and 0.82±0.03 mg/mL, respectively. Both demonstrated increment of 49.1% in protein production than the wild type which possessed relatively lower concentration of 0.55±0.01 mg/mL. Besides that, the profile of medium pH on xylanase activity by mutants and wild type of B. subtilis was also elucidated in the present study. The highest xylanase activity was attained at slight acidic pH of 6.1±0.2 as shown by the mutant MXB 4 in comparison with wild type at pH 6.47±0.3. Conclusion: In a nutshell, the result findings suggested the mutant strains of B. subtilis ATCC 6633 particularly MXB 4 as the most potent xylanase producer under submerged fermentation using barley husk as the prime carbon source. Mutant MXB 4 of B. subtilis is anticipated to be beneficial in various xylanase applications especially in the processing of animal feeds and food industry.

Optimization of xylanase production by filamentous fungi in solid-state fermentation and scale-up to horizontal tube bioreactor.

Five microorganisms, namely Aspergillus niger CECT 2700, A. niger CECT 2915, A. niger CECT 2088, Aspergillus terreus CECT 2808, and Rhizopus stolonifer CECT 2344, were grown on corncob to produce cell wall polysaccharide-degrading enzymes, mainly xylanases, by solid-state fermentation (SSF). A. niger CECT 2700 produced the highest amount of xylanases of 504 ± 7 U/g dry corncob (dcc) after 3 days of fermentation. The optimization of the culture broth (5.0 g/L NaNO3, 1.3 g/L (NH4)2SO4, 4.5 g/L KH2PO4, and 3 g/L yeast extract) and operational conditions (5 g of bed loading, using an initial substrate to moistening medium of 1:3.6 (w/v)) allowed increasing the predicted maximal xylanase activity up to 2,452.7 U/g dcc. However, different pretreatments of materials, including destarching, autoclaving, microwave, and alkaline treatments, were detrimental. Finally, the process was successfully established in a laboratory-scale horizontal tube bioreactor, achieving the highest xylanase activity (2,926 U/g dcc) at a flow rate of 0.2 L/min. The result showed an overall 5.8-fold increase in xylanase activity after optimization of culture media, operational conditions, and scale-up.

Simplified feeding strategies for the fed-batch cultivation of Kluyveromyces lactis GG799 for enhanced recombinant xylanase production

A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20gL(-1) and 80% dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53UmL(-1) was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13UmL(-1)) was measured at a C/N ratio of 50.08. These conditions led to a 45.8% increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500gL(-1) glucose led to a 6.2-fold increase (465.07UmL(-1)) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.

Auto-inducible expression system based on the SigB-dependent ohrB promoter in Bacillus subtilis

The reliable production of heterologous proteins is important in the field of industrial biotechnology. This can be achieved by applying auto-inducible gene expression systems. The development of a Bacillus subtilis expression plasmid harboring SigB-dependent ohrB promoter was reported. The expression system was subjected to high cell density cultivation to produce xylanase as a stable model protein. The recombinant strain was cultured in a synthetic medium containing glucose as the carbon source. The exponential fed-batch feeding strategy was applied to prevent substrate inhibition. A sharp increase of xylanase activity (about 6-fold) at the end of the fermentation was observed as a result of sigma factor B (SigB) protein activation, supporting auto-inducibility of the expression system. For the control strain a specific induction of the xylanase activity was not observed. The recombinant strain was capable to offer a 5-fold increase in xylanase activity in comparison with the control strain. In addition, the constructed system displayed catabolite repression resistance ability. This SigB-dependent expression system can be considered as a biotechnology tool and an alternative to eliminate the cost of conventional inducers, e.g. isopropyl-β-galactopyranoside.

Co-production of xylanase and xylooligosaccharides from lignocellulosic agricultural wastes

Lignocellulosic biomass including agricultural residues and by-products, and woody biomass are produced worldwide in large amounts every year. With the exception of limited-usage areas, these materials are regarded as waste. Therefore, renewable lignocellulosic biomass is gaining considerable attention because of its potential for use in the production of biofuels and other value-added products. In this study, the co-production of two value-added products, xylanase (enzyme) and xylooligosaccharides (XO), was investigated by utilizing lignocellulosic biomass residues within a multi-product biorefinery framework. Box–Behnken design-based response surface methodology was employed to optimize culture parameters for xylanase production from a thermophilic fungus, Scytalidium thermophilum, and the enzyme was biochemically characterized. Optimized media conditions resulted in an approximately 2-fold increase in xylanase activity compared to the initial conditions. Crude enzyme solution was capable of producing XO from the hemicellulosic fraction of various biomass residues. The highest hemicellulose extraction yield was achieved from corn cob (37.7 ± 1.5%), followed by wheat bran (26.6 ± 0.8%) and cotton stalks (22.2 ± 0.6%). The highest reducing end concentration was obtained from sunflower seed shell (4.89 ± 0.02 mg mL−1), followed by sunflower stalks (4.41 ± 0.03 mg mL−1) and corn cob (3.49 ± 0.09 mg mL−1). Accordingly, corn cob yielded the highest XO production (172.1 mg XO per g raw biomass residue) with the main products of xylose (X1) and xylobiose (X2). In terms of the hemicellulosic fraction, beechwood had the maximum XO yield with 793 mg XO per g hemicellulose. Sunflower seed shell hydrolysis yielded mainly X1 and xylotetrose (X4) without X2 and xylotriose (X3). After 1 h of reaction time, X2 and higher sugars were obtained from the commercial beechwood xylan, while only X1 and X4 were obtained after 6 h. Xylose-free xylobiose was obtained from sugar beet bagasse and wheat bran, which are regarded as potential XO sources because XO with degrees of polymerization from 2–4 are preferred in food applications and XO without xylose are important in the food industry from a prebiotic point of view.

Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide

Microalgae have recently received attention as a potential low-cost host for the production of recombinant proteins and novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expression of heterologous genes from the nuclear genome. Here we describe a nuclear expression strategy using the foot-and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistance in Chlamydomonas reinhardtii. We demonstrate that strains transformed with ble-2A-GFP are zeocin-resistant and accumulate high levels of GFP that is properly ‘cleaved’ at the FMDV 2A peptide resulting in monomeric, cytosolic GFP that is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expression vector to engineer the heterologous expression of the industrial enzyme, xylanase. We demonstrate that linking xyn1 expression to ble2A expression on the same open reading frame led to a dramatic (∼100-fold) increase in xylanase activity in cells lysates compared to the unlinked construct. Finally, by inserting an endogenous secretion signal between the ble2A and xyn1 coding regions, we were able to target monomeric xylanase for secretion. The novel microalgae nuclear expression strategy described here enables the selection of transgenic lines that are efficiently expressing the heterologous gene-of-interest and should prove valuable for basic research as well as algal biotechnology.

Optimization of Xylanase production from Penicillium sp.WX-Z1 by a two-step statistical strategy: Plackett-Burman and Box-Behnken experimental design.

The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in the medium for xylanase production by Penicillium sp.WX-Z1 and subsequent use of the response surface methodology (RSM) was further optimized for xylanase production by Box-Behnken design. The important nutrient sources in the culture medium, identified by the initial screening method of Placket-Burman, were wheat bran, yeast extract, NaNO3, MgSO4, and CaCl2. The optimal amounts (in g/L) for maximum production of xylanase were: wheat bran, 32.8; yeast extract, 1.02; NaNO3, 12.71; MgSO4, 0.96; and CaCl2, 1.04. Using this statistical experimental design, the xylanase production under optimal condition reached 46.50 U/mL and an increase in xylanase activity of 1.34-fold was obtained compared with the original medium for fermentation carried out in a 30-L bioreactor.

Rumen microbial and fermentation characteristics are affected differently by bacterial probiotic supplementation during induced lactic and subacute acidosis in sheep

BackgroundRuminal disbiosis induced by feeding is the cause of ruminal acidosis, a digestive disorder prevalent in high-producing ruminants. Because probiotic microorganisms can modulate the gastrointestinal microbiota, propionibacteria- and lactobacilli-based probiotics were tested for their effectiveness in preventing different forms of acidosis.ResultsLactic acidosis, butyric and propionic subacute ruminal acidosis (SARA) were induced by feed chalenges in three groups of four wethers intraruminally dosed with wheat, corn or beet pulp. In each group, wethers were either not supplemented (C) or supplemented with Propionibacterium P63 alone (P) or combined with L. plantarum (Lp + P) or L. rhamnosus (Lr + P). Compared with C, all the probiotics stimulated lactobacilli proliferation, which reached up to 25% of total bacteria during wheat-induced lactic acidosis. This induced a large increase in lactate concentration, which decreased ruminal pH. During the corn-induced butyric SARA, Lp + P decreased Prevotella spp. proportion with a concomitant decrease in microbial amylase activity and total volatile fatty acids concentration, and an increase in xylanase activity and pH. Relative to the beet pulp-induced propionic SARA, P and Lr + P improved ruminal pH without affecting the microbial or fermentation characteristics. Regardless of acidosis type, denaturing gradient gel electrophoresis revealed that probiotic supplementations modified the bacterial community structure.ConclusionThis work showed that the effectiveness of the bacterial probiotics tested depended on the acidosis type. Although these probiotics were ineffective in lactic acidosis because of a deeply disturbed rumen microbiota, some of the probiotics tested may be useful to minimize the occurrence of butyric and propionic SARA in sheep. However, their modes of action need to be further investigated.

Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation

This paper reports the production of a cellulase-free and alkali-stable xylanase in high titre from a newly isolated Bacillus pumilus SV-85S using cheap and easily available agro-residue wheat bran. Optimization of fermentation conditions enhanced the enzyme production to 2995.20 +/- 200.00 IU/ml, which was 9.91-fold higher than the activity under unoptimized basal medium (302.2 IU/ml). Statistical optimization using response-surface methodology was employed to obtain a cumulative effect of peptone, yeast extract, and potassium nitrate (KNO(3)) on enzyme production. A 2(3) central composite design best optimized the nitrogen source at the 0 level for peptone and yeast extract and at the -alpha level for KNO(3), along with 5.38-fold increase in xylanase activity. Addition of 0.1% tween 80 to the medium increased production by 1.5-fold. Optimum pH for xylanase was 6.0. The enzyme was 100% stable over the pH range from 5 to 11 for 1 h at 37 degrees C and it lost no activity, even after 3 h of incubation at pH 7, 8, and 9. Optimum temperature for the enzyme was 50 degrees C, but the enzyme displayed 78% residual activity even at 65 degrees C. The enzyme retained 50% activity after an incubation of 1 h at 60 degrees C. Characteristics of B. pumilus SV-85S xylanase, including its cellulase-free nature, stability in alkali over a long duration, along with high-level production, are particularly suited to the paper and pulp industry.

Enhanced production of xylanase by Aspergillus carneus M34 in solid-state fermentation with agricultural waste using statistical approach

The production of a low molecular weight xylanase by Aspergillus carneus M34 was investigated in solid-state fermentation using agricultural waste as the substrate. Experimental designs were practiced for optimisation of the medium composition. When agricultural wastes of coba husk and corn steep liquor were used in the ratio of 4.5:0.5, a 22.5% increase of xylanase activity was observed compared with the medium containing only coba husk. The incubation time for xylanase production can be reduced from 12 days to six days by increasing the inoculum size to 2 x 10(7) sporesmL(-1). The optimal media compositions were as follows by using statistical approach: coba husk/corn steep liquor (4.5/0.5); NH(4)NO(3), 32.4 gL(-1); CaCl(2), 1.34 gL(-1); MnSO(4).7H(2)O, 0.0124 gL(-1). Xylanase activity of 1721 Ug(-1) substrate was obtained by A. carneus M34 in a six-day period at 30 degrees C, which was a 227% increase compared with that obtained before applying the Plackett-Burman and response surface methodology experimental design.

Potential of solid-state fermentation enzymes of Aspergillus oryzae in biobleaching of paper pulp

Production of xylan-degrading enzymes, endo-β-xylanase (EC 3.2.1.8), β-xylosidase (EC 3.2.1.37), α- l-arabinofuranosidase (EC 3.2.1.55 and α-galactosidase (EC 3.2.1.22) by Aspergillus oryzae NRRL 1808 was investigated in solid-state fermentation using eucalyptus soda-anthraquinone and bagasse soda pulps as carbon feedstock. The impact of different nitrogen sources and initial fermentation pH on enzyme production was studied in medium optimisation experiments using fractional factorial design. The use of ammonium nitrate and corn steep liquor favoured xylanase production on eucalyptus pulp, whereas potassium nitrate and corn steep liquor increased xylanase yields on bagasse pulp. Xylanase production on eucalyptus and bagasse pulps under optimised conditions peaked on day 4 of cultivation (3200 and 2675 IU g −1 dry matter, respectively) which represents an increase in xylanase activity of 60% and 37%, respectively, over the non-opimised conditions. Biobleaching of eucalyptus and bagasse pulps was carried out using the whole fermented material (residual pulp, fungal biomass and in situ enzymes) from the cultivation of A. oryzae without a prior down streaming of the in situ enzymes from these pulps. Overall, the bagasse pulp was more susceptible to enzyme bleaching than eucalyptus pulp. In all instances, a greater brightness gain was induced with the in situ solid-state enzymes (0.9–3.0 points brightness increase) than a commercial enzyme control (0.8–2.5 points). At the same bleaching costs (US$ 1 or 3 t −1 pulp), the solid-state enzymes produced under optimised conditions were 20–36% more efficient in improving the brightness of paper pulp than the commercial enzyme. However, the presence of increased levels of side chain xylan degrading enzymes in the optimised medium did not improve the bleaching abilities of xylanase. It was demonstrated that bagasse pulp could successfully be used as carrier of A. oryzae enzymes in biobleaching of eucalyptus pulp without sacrificing the bleaching efficiency of xylanase.

The methylotrophic yeast as a host for the expression and production of thermostable xylanase from the bacterium

A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3gl(-1) xylanase with a maximum activity of 3130Ul(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T(m)) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.