Effect of additional carbon source and moisture level on xylanase production by Cochliobolus sativus in solid fermentation

The fungus Cochliobolus sativus has been shown to be an efficient producer ofxylanase from an industrial point ofview. The addition of extra carbon sources and the initial moisture content of the solid-state fermentation were found to have a marked influence on the xylanase production by C. sativus Cs6 strain. Xylan and starch resulted in an increased xylanase production (1469.4 and 1396.56 U/g, respectively) after 8 days of incubation. Optimal initial moisture content for xylanase production was 80%. The cultivation systems can easily be modified to enhance the productivity of the enzyme formation by C. sativus Cs6, which will facilitate the scale up processes for mass production.     

Correlative Analysis of Cochliobolus sativus Pathogenicity and in vitro Xylanase Activity

Spot blotch (SB) caused by Cochliobolus sativus has been the major yield‐reducing factor for barley production during the last decade. In this study, the correlation between aggressiveness and in vitro xylanase production of 29 isolates of C. sativus was investigated. Isolate aggressiveness was evaluated in term of lesion form in barley leaves. Additionally, the isolates were compared for their ability to produce in vitro significant levels of xylanase activities when grown in a liquid medium. Aggressive isolates released more xylanase of weakly aggressive isolates. Correlation tests analysis revealed a significant relationship (r = 0.84, r = 0.50; P < 0.01) between the xylanase (per unit fungal mass) and aggressiveness on the two barley cultivars Arabi Abiad and Bowman, respectively. Correlation between the production of this enzyme and the origin of the isolates was not found. The results indicate that the production of xylanase influences the aggressiveness of the isolates of C. sativus towards barley seedlings.     

Solid-state fermentation for xylanase production by Thermoascus aurantiacus using response surface methodology

We investigated xylanase production by Thermoascus aurantiacus using semisolid fermentation. Multivariant statistical approaches were employed to evaluate the effects of several variables (initial moisture in the medium, cultivation time, inoculum level, and bagasse mass) on xylanase production. The initial moisture content and bagasse mass were the most important factors affecting xylanase activity. The xylanase activity produced by the fungus under the optimized conditions (81% moisture content and 17 g bagasse) was found to be 2700 U per gram of initial dry matter, whereas its value predicted by a polynomial model was 2400 U per gram of initial dry matter. Received: 4 December 1998 / Received revision: 15 March 1999 / Accepted: 16 May 1999     

Xylanase production under solid-state fermentation and its characterization by an isolated strain of <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> in India

Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like K_m and V_max were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl₂, xylose and Tween 80, while significantly inhibited by Hg⁺⁺, Cu⁺⁺ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca⁺⁺. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C.     

Comprehensive studies on optimization of cellulase and xylanase production by a local indigenous fungus strain via solid state fermentation using oil palm frond as substrate

The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 10⁶ spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.     

Improved xylanase production using apple pomace waste by Aspergillus niger in koji fermentation

Xylanase production by Aspergillus niger NRRL‐567 in solid‐state fermentation (koji fermentation) was optimized using 2⁴ factorial design and response surface methodology. The evaluated variables were the initial moisture level and concentration of inducers [veratryl alcohol (VA), copper sulphate (CS), and lactose (LAC)], leading to the response of xylanase production. Initial moisture level and LAC were found to be the most significant variable for xylanase production (p<0.05). The highest xylanase production was observed with 3578.8 ± 65.3 IU/gds (gram dry substrate) under optimal conditions using initial moisture of 85% (v/w), pH 5.0 and inducers VA (2 mM/kg), LAC 2% (w/w), and CS (1.5 mM/kg) after 48 h of incubation time. Higher xylanase activity of 3952 ± 78.3 IU/gds was attained during scale‐up of the process in solid‐state tray fermentation under optimum conditions after 72 h of incubation time. The present study demonstrates that A. niger NRRL‐567 can efficiently be used to achieve xylanase production with an economical and environmental benefit in solid‐state tray fermentation. The developed process can be used to develop an effective process for commercially feasible bioproduction of xylanases for speciality applications, such as conversion of lignocellulosic biomass to biofuels and other value‐added products.     

Production and partial characterization of arabinoxylan-degrading enzymes by Penicillium brasilianum under solid-state fermentation

The production of a battery of arabinoxylan-degrading enzymes by the fungus Penicillium brasilianum grown on brewer’s spent grain (BSG) under solid-state fermentation was investigated. Initial moisture content, initial pH, temperature, and nitrogen source content were optimized to achieve maximum production of feruloyl esterase, xylanase, and α-l-arabinofuranosidase. Under the optimum growth conditions (80% moisture, pH 6, 26.5°C, and 5 g/l nitrogen source), the maximum level of feruloyl esterase (1,542 mU/g BSG) was found after 196 h, whereas xylanase (709 U/g BSG) and ArabF activity (3,567 mU/g BSG) were maximal after 108 h and 96 h, respectively. Based on substrate utilization data, the feruloyl esterases produced by P. brasilianum was anticipated to subclass B. A crude enzyme (CE) preparation from P. brasilianum culture grown on BSG was tested for the release of hydroxycinnamic acids and pentoses from BSG. The P. brasilianum CE produced in this work contains a balance of cell wall-modifying enzymes capable of degrading arabinoxylan of BSG by more than 40%.     

Production of xylanase under solid-state fermentation by <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> JP-1 and its application

The production of extracellular xylanase by a locally isolated strain of Aspergillus tubingensis JP-1 was studied under solid-state fermentation. Among the various agro residues used wheat straw was found to be the best for high yield of xylanase with poor cellulase production. The influence of various parameters such as initial pH, moisture, moistening agents, nitrogen sources, additives, surfactants and pretreatment of substrates were investigated. The production of the xylanase reached a peak in 8 days using untreated wheat straw with modified MS medium, pH 6.0 at 1:5 moisture level at 30 °C. Under optimized conditions yield as high as 6,887 ± 16 U/g of untreated wheat straw was achieved. Crude xylanase was used for enzymatic saccharification of agro-residues like wheat straw, rice bran, wheat bran, sugarcane bagasse and industrial paper pulp. Dilute alkali (1 N NaOH) and acid (1 N H₂SO₄) pretreatment were found to be beneficial for the efficient enzymatic hydrolysis of wheat straw. Dilute alkali and acid-pretreated wheat straw yielded 688 and 543 mg/g reducing sugar, respectively. Yield of 726 mg/g reducing sugar was obtained from paper pulp after 48 h of incubation.     

Production and biochemical characterization of a novel cellulase-poor alkali-thermo-tolerant xylanase from <Emphasis Type="Italic">Coprinellus disseminatus</Emphasis> SW-1 NTCC 1165

Fungi producing xylanases are plentiful but alkali-thermo-tolerant fungi producing cellulase-poor xylanase are rare. Out of 12 fungal strains isolated from various sources, Coprinellus disseminatus SW-1 NTCC 1165 yielded the highest xylanase activity (362.1 IU/ml) with minimal cellulase contamination (0.64 IU/ml). The solid state fermentation was more effective yielding 88.59% higher xylanase activity than that of submerged fermentation. An incubation period of 7 days at 37°C and pH 6.4 accelerated the xylanase production up to the maximum level. Among various inexpensive agro-residues used as carbon source, wheat bran induced the maximum xylanase titres (469.45 IU/ml) while soya bean meal was the best nitrogen source (478.5 IU/ml). A solid substrate to moisture content ratio of 1:3 was suitable for xylanase production while xylanase titre was repressed with the addition of glucose and lactose. The xylanase and laccase activities under optimized conditions were 499.60 and 25.5 IU/ml, respectively along with negligible cellulase contamination (0.86 IU/ml). Biochemical characterization revealed that optimal xylanase activity was observed at pH 6.4 and temperature 55°C and xylanase is active up to pH 9 (40.33 IU/ml) and temperature 85°C (48.81 IU/ml). SDS–PAGE and zymogram analysis indicated that molecular weight of alkali-thermo-tolerant xylanase produced by C. disseminatus SW-1 NTCC 1165 was 43 kDa.     

Rice straw fermentation by Schizophyllum commune ARC-11 to produce high level of xylanase for its application in pre-bleaching

Rice straw is valuable resource that has been used as substrate for cost effective production of xylanase under solid-state fermentation by a newly isolated white rot fungi, S. commune ARC-11. Out of eleven carbon sources tested, rice straw was found most effective for the induction of xylanase that produced 4288.3?IU/gds of xylanase by S. commune ARC-11. Maximum xylanase production (6721.9?IU/gds) was observed on 8^th day of incubation at temperature (30?°C), initial pH (7.0) and initial moisture content (70.0%). The supplementation of ammonium sulphate (0.08% N, as available nitrogen) enhanced the xylanase production up to 8591.4?IU/gds. The xylanase production by S. commune ARC-11 was further improved by the addition of 0.10%, (w/v) of Tween-20 as surfactant. The maximum xylanase activities were found at pH 5.0 and temperature 55?°C with a longer stability (180?min) at temperature 45, 50 and 55?°C. This xylanase preparation was also evaluated for the pre-bleaching of ethanol-soda pulp from Eulaliopsis binata. An enzyme dosage of 10?IU/g of xylanase resulted maximum decrease in kappa number (14.51%) with a maximum improvement 2.9% in ISO brightness compared to control.     

Response surface optimization of fermentation conditions for producing xylanase by <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">niger</Emphasis> SL-05

Fermentation conditions were statistically optimized for producing extracellular xylanase by Aspergillus niger SL-05 using apple pomace and cotton seed meal. The primary study shows that culture medium with a 1:1 ratio of apple pomace and cotton seed meal (carbon and nitrogen sources) yielded maximal xylanase activity. Three significant factors influencing xylanase production were identified as urea, KH(2)PO(4), and initial moisture content using Plackett-Burman design study. The effects of these three factors were further investigated using a design of rotation-regression-orthogonal combination. The optimized conditions by response surface analysis were 2.5% Urea, 0.09% KH(2)PO(4), and 62% initial moisture content. The analysis of variance indicated that the established model was significant (P < 0.05), "while" or "and" the lack of fit was not significant. Under the optimized conditions, the model predicted 4,998 IU/g dry content, whereas validation experiments produced an enzymatic activity of xylanase at 5,662 IU/g dry content after 60 h fermentation. This study innovatively developed a fermentation medium and process to utilize inexpensive agro-industrial wastes to produce a high yield of xylanase.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

Production and partial characterization of xylanase by Sporotrichum thermophile under solid-state fermentation

A number of factors affecting production of xylanase, by the thermophilic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimized. Solid state fermentation in a laboratory horizontal bioreactor using the optimized medium allowed the production of 320 U g^–1 of carbon source which compared favourably with those reported for other microorganisms. Optimal xylanase activity was observed at pH 5 and 70 °C. Chromogenic (fluorogenic) 4-methylumbelliferyl -glycoside of xylobiose (MUX₂) was used to characterize the xylanase multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major xylanase fraction exhibiting pI and molecular mass values 4 and 90–120 kDa, respectively.     

Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation

The feasibility of using grape pomace for the production of xylanase and exo-polygalacturonase by Aspergillus awamori in solid state fermentation has been evaluated. Solid state fermentation experiments indicated that the particle size did not influence the enzyme production. The addition of extra carbon sources and the initial moisture content of the grape pomace were found to have a marked influence on the enzymes yields. Xylanase and exo-PG activities were high at 65% (w/w) initial moisture content and glucose supplementation.     

樟绒枝霉(Malbranchea cinnamomea)固体发酵产木聚糖酶的发酵条件优化

[目的] 研究樟绒枝霉(Malbranchea cinnamomea) CAU521利用农业废弃物固体发酵产木聚糖酶的发酵条件.[方法]采用单因素试验法优化影响菌株产酶的各个条件,包括碳源种类、氮源种类、初始pH、初始水分含量、培养温度及发酵时间共6个因素.[结果]获得的最佳产酶条件为:稻草为发酵碳源、2％(W/W)的酵母提取物为氮源、初始pH 7.0、初始水分含量80％和发酵温度45℃.在此条件下发酵6d后木聚糖酶的酶活力达到13 120 U/g干基碳源.[结论]樟绒枝霉固体发酵产木聚糖酶的产酶水平高,生产成本低,具有潜在的工业化应用前景.     

Xylanase production in solid-state fermentation: a study of its properties

Summary Xylanase production by Aspergillus niger van Tieghem was studied in solid-state cultivation. The screening of substrates was carried out in column incubators aerated with humidified air at 30°C. Results of physiological studies showed that the best yield of xylanase was 2500 U/g dry matter on a mixture of straw+bran 1:1 at 70% of moisture content.In order to compare some properties of the xylanase produced in both liquid and solid cultures, A. niger was also grown on xylan in submerged cultures. The enzymes produced in solid and liquid cultures have an optimum pH of about 3.8 and 4.5, respectively. Xylanase synthetized in solid fermentation is a little more thermostable than that from liquid culture and is maximally active at 50° C, compared to 45° C for enzyme from liquid culture.     

Expression of an AT-rich xylanase gene from the anaerobic fungus <Emphasis Type="Italic">Orpinomyces</Emphasis> sp. strain PC-2 in and secretion of the heterologous enzyme by <Emphasis Type="Italic">Hypocrea jecorina</Emphasis>

The catalytic domain encoded by an adenine–thymine (AT)-rich xylanase gene (xynA) of the anaerobic fungus Orpinomyces was expressed in Hypocrea jecorina under the control of the cel7A promoter and terminator. No XynA protein was detected in H. jecorina culture supernatants when the original sequence was fused to the H. jecorina cel5A region coding for its signal peptide, carbohydrate-binding module, and hinge. Replacing the xynA (56% AT content) with a synthetic sequence containing lower AT content (39%) supported the extracellular production (150 mg l⁻¹) of the fusion xylanase by H. jecorina. Northern analysis revealed that successful production after the decrease in AT content was related to higher levels of the xylanase-specific mRNA. Another construct with an RDKR-coding sequence inserted between the cel5A linker and the xynA catalytic domain allowed production of the fully processed active xylanase catalytic domain. Both the fusion (40 kDa) and the fully processed (28 kDa) forms displayed enzymatic properties of family 11 xylanases. Both the R and the Kex2-like KR sites were recognized during secretion, resulting in a mixture of two amino termini for the 28-kDa xylanase. The work demonstrated for the first time that glycoside hydrolases derived from anaerobic fungi can be produced by H. jecorina. The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Solid substrate fermentation for cellulase production using palm kernel cake as a renewable lignocellulosic source in packed-bed bioreactor

Palm kernel cake (PKC), is an agro-industrial residue created in the palm oil industry, and large quantities of PKC are produced in Malaysia. Sustainable development of the palm oil industry in Malaysia demands an economical technology for the environmentally friendly utilization of PKC in industrial utility systems. This research was carried out to evaluate the use of PKC in the production of cellulase by the cultivation of Aspergillus niger FTCC 5003 in a laboratory packed-bed bioreactor for seven days. A central composite design was used to perform eighteen trials of solid substrate fermentation under selected conditions of incubation temperature, initial moisture content of substrate, and airflow rate. Experimental results showed that a cellulase yield of 244.53 U/g of dry PKC was obtained when 100 g of PKC was hydrolyzed at an incubation temperature of 32.5°C, an initial moisture level of 60%, and an aeration rate of 1.5 L/min/g PKC. An empirical second-order polynomial model was adjusted to the experimental data to evaluate the effects of the studied operating variables on cellulase production. The statistical model revealed that the quadratic term for initial moisture content had a significant effect on the production of cellulase (P < 0.01). The regression model also indicated that the quadratic terms for incubation temperature and interaction effects between initial moisture content and aeration rate significantly influenced cellulase production (P < 0.05). The empirical model determined that the optimum conditions for cellulase production were an incubation temperature of 31.0°C, an initial moisture content of 59.0% and an airflow rate of 1.55 L/min/g PKC.     

Production of cellulase‐free xylanase by the recombinant Bacillus subtilis and its applicability in paper pulp bleaching

A metagenomic xylanase gene (Mxyl) was successfully cloned into shuttle vector pWH1520 and expressed in Bacillus subtilis extracellularly. On induction with xylose, recombinant xylanase secretion commenced after 6 h. Identifying critical variables for recombinant xylanase production by one‐variable‐at‐time approach followed by optimization of the selected variables (xylose, inoculum density, incubation density) by response surface methodology (RSM) led to three‐fold enhancement in extracellular xylanase production (119 U mL^?1). When the pulp was treated with recombinant xylanase at 80°C and pH 9.0, kappa number of the pulp was reduced with concomitant increase in brightness and 24% reduction in chlorine consumption. This is the first report on the expression of metagenomic xylanase gene in Bacillus subtilis extracellularly and its utility in developing an environment‐friendly pulp bleaching process. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1441–1447, 2013     

Cloning and Characterization of Xylanase A from the Strain <Emphasis Type="Italic">Bacillus</Emphasis> sp. BP-7: Comparison with Alkaline pI-Low Molecular Weight Xylanases of Family 11

The xynA gene encoding a xylanase from the recently isolated Bacillus sp. strain BP-7 has been cloned and expressed in Escherichia coli. Recombinant xylanase A showed high activity on xylans from hardwoods and cereals, and exhibited maximum activity at pH 6 and 60°C. The enzyme remained stable after incubation at 50°C and pH 7 for 3 h, and it was strongly inhibited by Mn²⁺, Fe³⁺, Pb²⁺, and Hg²⁺. Analysis of xylanase A in zymograms showed an apparent molecular size of 24 kDa and a pI of above 9. The amino acid sequence of xylanase A, as deduced from xynA gene, shows homology to alkaline pI-low molecular weight xylanases of family 11 such as XynA from Bacillus subtilis. Analysis of codon usage in xynA from Bacillus sp. BP-7 shows that the G+C content at the first and second codon positions is notably different from the mean values found for glycosyl hydrolase genes from Bacillus subtilis.