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%.     

Penicillium brasilianum as an enzyme factory; the essential role of feruloyl esterases for the hydrolysis of the plant cell wall

The production of arabinoxylan-degrading enzymes by the fungus Penicillium brasilianum, grown on different carbon and nitrogen sources as well as different environmental conditions was investigated. Highest feruloyl esterase (225 mU/ml) and alpha-L-arabinofuranosidase (211 mU/ml) activities were obtained when P. brasilianum was grown on sugar beet pulp, whereas maximum xylanase (17 U/ml) activity was found during growth on oat spelt xylan. Yeast extract was the preferable nitrogen source for the production of all the three enzymes. Further optimization of the production of the crude enzyme mixture was examined by experimental design using a D-optimal quadratic model. Investigation of the microbial regulation of enzyme production showed that the presence of free ferulic acid further stimulated the production and pointing to that the fungal regulatory mechanism involved a coordinated production and secretion of feruloyl esterase, xylanase and alpha-L-arabinofuranosidase. Since agroindustrial by-products are a potential source of phenolic acids, crude enzyme mixtures of P. brasilianum were tested for their hydrolysis abilities against eight complex or model substrates. While total release of phenolic acids and pentoses was not observed, the synergistic enhancement of hydrolysis in the presence of feruloyl esterase was clearly demonstrated.     

Brewer's spent grain and corn steep liquor as substrates for cellulolytic enzymes production by Streptomyces malaysiensis

Aims:  To evaluate cellulase production by Streptomyces malaysiensis in submerged fermentation using brewer's spent grain (BSG) and wheat bran (WB) as carbon source, and corn steep liquor (CSL) as nitrogen source, as compared to yeast extract (YE), and partial characterization of the crude enzyme.
Methods and Results:  Maximum cellulase production by Streptomyces malaysiensis (720 U l⁻¹) occurred within 4 days incubation when using a growth medium containing BSG 0·5% (w/v) and CSL1·2% (w/v). CMCases activity showed to be stable over an acidic pH range (2·0–7·0) and in temperatures of 40–60°C. Zymogram indicated three bands of CMCase activity, with different molecular masses.
Conclusion:  S. malaysiensis was able to grow and produce good levels of CMCases using solely brewer's spent grain and corn steep liquor as low-cost substrates, making this strain and these low cost by-product worthy for further investigation, and potentially feasible for biotechnological applications in different areas.
Significance and Impact of the Study:  To our knowledge, this is the first study reporting the use of the low-cost by-products brewer's spent grain and corn steep liquor, as sole substrates for microbial enzyme production.     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

Production of xylanase and protease by Penicillium janthinellum CRC 87M-115 from different agricultural wastes

Five agricultural wastes were evaluated in submerged fermentation for xylanolytic enzymes production by Penicillium janthinellum. The wastes were hydrolyzed in acid medium and the liquid fraction was used for cultivation. Corn cob (55.3 U/mL) and oat husk (54.8 U/mL) were the best inducers of xylanase. Sugar cane bagasse (23.0 U/mL) and corn husk (23.8 U/mL) were moderately good, while cassava peel was negligible. Protease production was very low in all agro-industrial residues. The maximum biomass yields were 1.30 and 1.17 g/L for cassava peel and corn husk after 180 h, respectively. Xylanolytic activity showed a cell growth associated profile.     

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

Production and characterization of xylanase from Thielaviopsis basicola

Summary The black rot fungus Thielaviopsis basicola has the ability to grow on cellulosic biomass, producing xylanase. Of the four cellulosic substrates tested, rice straw was found to be the best for production of xylanase. A xylanase activity of 34 U/ml was obtained with rice straw which was more than three times that obtained with larchwood xylan. The -xylosidase activities obtained with these two substrates were 0.05 U/ml and 0.016 U/ml respectively. Both enzymes are active at pH 5 but the temperature optima of xylanase and -xylosidase activities are 60°C and 40°C respectively. The xylanase activity is stable over a pH range of 4–8 but the stability towards temperature falls sharply above 50°C.     

Induction of ferulic acid esterase and xylanase activities in Streptomyces avermitilis UAH30

Ferulic acid esterase activity (FAE) was detected, along with xylanase activity, in culture supernatants from Streptomyces avermitilis UAH30 grown in the presence of the lignocellulosic substrates, oat spelt xylan, wheat bran without starch and sugar cane bagasse. The maximum activity was detected with wheat bran (1.75 mU ml⁻¹). No correlation between FAE activity and the amount of esterified ferulic acid present in the substrate was observed. The addition of either glucose, mannitol or glycerol to the culture medium containing oat spelt xylan resulted in a reduction of 40–75% in the xylanase activity detected in culture supernatants. FAE activity could only be detected in supernatants from cultures grown in the presence of glycerol and mannitol, when commercially available xylanases were added to the assay. These results highlight the importance of assaying for FAE activity in the presence of high levels of xylanase activity.     

STATISTICAL OPTIMIZATION OF A NOVEL LOW-COST MEDIUM BASED ON REGIONAL AGRO-INDUSTRIAL BY-PRODUCTS FOR THE PRODUCTION OF PROTEOLYTIC ENZYMES BY Bacillus cereus

Bacillus sp. are specific producers of peptidase amongst bacteria and peptidase enzymes and are of significant ones due to their multifarious applications. Advances in industrial biotechnology offer potential opportunities for economic utilization of agro-industrial by-products for many biochemical reactions. Due to their rich organic nature, they can serve as an ideal substrate for the production of different value added products like peptidases. In the present work, an attempt was made to optimize different variables by Taguchi methodology for the production of peptidase using agro-industrial by-products hydrolyzed by a Bacillus cereus strain, resulting in brewer's spent grain (BSG) being the optimal organic substrate. Subsequently, operative variables for the BSG were investigated using Taguchi methodology in order to maximize the enzyme production. Additionally, the main medium components were optimized using a mixture design. Finally, the production of peptidase by B. cereus was investigated; also the possible interaction with other proteolytic microbial strains was evaluated. A notorious synergistic effect was observed when B. cereus was inoculated with Pseudomonas sp. These brought a triple benefit, first, opening the possibility to produce technical enzymes at low cost, second, giving greater value to a food industry by-product, and third, reducing the environmental impact caused by the product removal directly into the environment.     

STATISTICAL OPTIMIZATION OF A NOVEL LOW-COST MEDIUM BASED ON REGIONAL AGRO-INDUSTRIAL BY-PRODUCTS FOR THE PRODUCTION OF PROTEOLYTIC ENZYMES BY Bacillus cereus

Bacillus sp. are specific producers of peptidase amongst bacteria and peptidase enzymes and are of significant ones due to their multifarious applications. Advances in industrial biotechnology offer potential opportunities for economic utilization of agro-industrial by-products for many biochemical reactions. Due to their rich organic nature, they can serve as an ideal substrate for the production of different value added products like peptidases. In the present work, an attempt was made to optimize different variables by Taguchi methodology for the production of peptidase using agro-industrial by-products hydrolyzed by a Bacillus cereus strain, resulting in brewer's spent grain (BSG) being the optimal organic substrate. Subsequently, operative variables for the BSG were investigated using Taguchi methodology in order to maximize the enzyme production. Additionally, the main medium components were optimized using a mixture design. Finally, the production of peptidase by B. cereus was investigated; also the possible interaction with other proteolytic microbial strains was evaluated. A notorious synergistic effect was observed when B. cereus was inoculated with Pseudomonas sp. These brought a triple benefit, first, opening the possibility to produce technical enzymes at low cost, second, giving greater value to a food industry by-product, and third, reducing the environmental impact caused by the product removal directly into the environment.     

Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.     

Production of thermostable xylanase by a thermophilic fungus,Thermoascus aurantiacus

Twenty-one strains of thermophilic fungi in the Forintek culture collection were screened for their production of xylanolytic (and cellulolytic) enzymes in both solid and aqueous media containing various hemicellulosic and cellulosic substrates. Thermoascus aurantiacus strain C436 was selected as the best producer of extracellular xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) enzymes. High xylanase activity was detected in fungal culture filtrates even when realistic lignocellulosic residues (including steam-exploded aspenwood and untreated aspenwood sawdust) were used as substrates. Maximum xylanase activity (575.9 U ml⁻¹) was detected in cultures grown in Vogel's medium containing oat-spelt xylan. The xylanase activity exhibited a temperature optimum of 75°C and pH optimum around 5.0. The half-lives of the xylanase activity at 70 and 60°C were 1.5 h and 4 days, respectively. Over 90% of the xylanase activity was retained after 12 weeks at 50°C. Crude culture filtrates concentrated by membrane ultrafiltration could effectively hydrolyse xylan and steam-exploded aspenwood hemicellulose to release near theoretical yields of low molecular weight pentose oligomers.     

Optimisation of culture medium and conditions for alpha-l-Arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus

The culture medium for Rhodothermus marinus was optimised on a shake-flask scale by using statistical factorial designs for enhanced production of a highly thermostable alpha-L-arabinofuranosidase (AFase). The medium containing 3.6 g/l birch wood xylan and 8.2 g/l yeast extract yielded a maximum of 110 nkat/ml AFase activity together with 125 nkat/ml xylanase and 65 nkat/ml beta-xylosidase activity. In addition, low levels of beta-mannanase (30 nkat/ml), alpha-galactosidase (0.2 nkat/ml), beta-galactosidase (0.3 nkat/ml), endoglucanase (5 nkat/ml) and beta-glucosidase (30 nkat/ml) were detected in the culture filtrate. Among the various carbon sources tested, birchwood xylan was most effective for the formation of AFase and xylanase activities, followed by oat spelt and beechwood xylans, and xylan-rich lignocelluoses (e.g., starch-free sugar beet pulp and wheat bran). Constitutive levels of enzyme activities were detected when the bacterium was grown on other polysaccharides and low-molecular-weight carbohydrates. A fermentation in a 5-l fermenter (3-l working volume) using the optimised medium yielded 60 nkat/ml AFase associated with 65 nkat/ml xylanase and 35 nkat/ml beta-xylosidase activities. The crude AFase displayed optimal activity between pH 5.5 and 7 and at 85 degrees C. It had half-lives of 8.3 h at 85 degrees C and 17 min at 90 degrees C. It showed high stability between pH 5 and 9 (24 h at 65 degrees C). The combined use of AFase-rich xylanase and mannanase from R. marinus in the prebleaching of softwood kraft pulp gave a brightness increase of 1.8% ISO. To our knowledge, this is the first report on the production of a high AFase activity by an extreme thermophilic bacterium and this enzyme is the most thermostable AFase reported so far.     

Extracellular Hemicellulolytic Enzymes from the Maize Endophyte <Emphasis Type="Italic">Acremonium zeae</Emphasis>

Microorganisms that colonize plants require a number of hydrolytic enzymes to help degrade the cell wall. The maize endophyte Acremonium zeae was surveyed for production of extracellular enzymes that hydrolyze cellulose and hemicellulose. The most prominent enzyme activity in cell-free culture medium from A. zeae NRRL 6415 was xylanase, with a specific activity of 60 U/mg from cultures grown on crude corn fiber. Zymogram analysis following SDS-PAGE indicated six functional xylanase polypeptides of the following masses: 51, 44, 34, 29, 23, and 20 kDa. Xylosidase (0.39 U/mg), arabinofuranosidase (1.2 U/mg), endoglucanase (2.3 U/mg), cellobiohydrolase (1.3 U/mg), and β-glucosidase (0.85 U/mg) activities were also detected. Although apparently possessing a full complement of hemicellulolytic activities, cell-free culture supernatants prepared from A. zeae required an exogenously added xylosidase to release more than 90% of the xylose and 80% of the arabinose from corn cob and wheat arabinoxylans. The hydrolytic enzymes from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars. Mention of a trade name or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

Abstract

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2?mM CaCO_3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55?U/ml, 79.22?U/ml, 12.43?U/ml, 64.66?U/ml, and 21.12?U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.     

Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation

AIM: To evaluate the solid-state fermentation (SSF) production of cellulase and hemicellulases (xylanases), by Penicillium echinulatum 9A02S1, in experiments carried out with different concentrations of the pretreated sugar cane bagasse (PSCB) and wheat bran (WB). METHODS AND RESULTS: This study reports the production of xylanolytic and cellulolytic enzymes by P. echinulatum 9A02S1 using a cheap medium containing PSCB and WB under SSF. The highest amounts of filter paper activity (FPA) could be measured on mixtures of PSCB and WB (32.89 +/- 1.90 U gdm(-1)). The highest beta-glucosidase activity was 58.95 +/- 2.58 U gdm(-1) on the fourth day. The highest activity for endoglucanases was 282.36 +/- 1.23 U gdm(-1) on the fourth day, and for xylanases the activity was around 10 U gdm(-1) from the second to the fourth day. CONCLUSIONS: The present work has established the potential of P. echinulatum for FPA, endoglucanase, beta-glucosidase and xylanase productions in SSF, indicating that WB may be partially substituted by PSCB. SIGNIFICANCE AND IMPACT OF THE STUDY: The incorporation of cheap sources, such as sugar cane bagasse, into media for the production of lignocellulose enzymes should help decrease the production costs of enzymatic complexes that can hydrolyse lignocellulose residues for the formation of fermented syrups, thus contributing to the economic production of bioethanol.     

Xylanase production using inexpensive agricultural wastes and its partial characterization from a halophilic <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TPSV 101

A halophilic and alkali-tolerant Chromohalobacter sp. TPSV 101 with an ability to produce extracellular halophilic, alkali-tolerant and moderately thermostable xylanase was isolated from solar salterns. Identification of the bacterium was done based upon biochemical tests and 16S rRNA sequence. The culture conditions for higher xylanase production were optimized with respect to NaCl, pH, temperature, substrates and metal ions and additives. Maximum xylanase production was achieved in the medium with 20% NaCl, pH-9.0 at 40°C supplemented with 1% (w/v) sugarcane bagasse and 0.5% feather hydrolysate as carbon and nitrogen sources. Sugarcane bagasse (250 U/ml) and wheat bran (190 U/ml) were the best inducer of xylanase when used as carbon source as compared to xylan (61 U/ml). The xylanase that was partially purified by protein concentrator had a molecular mass of 15 kDa approximately. The xylanase from Chromohalobacter sp. TPSV 101 was active at pH 9.0 and required 20% NaCl for optimal xylanolytic activity and was active over a broad range of temperature 40–80°C with 65°C as optimum. The early stage hydrolysis products of sugarcane bagasse were xylose and xylobiose, after longer periods of incubation only xylose was detected.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.