Enhanced cellulase production byAspergillus fumigatus fresenius

The compost isolate,Aspergillus fumigatus, produces the exoglucanase, endoglucanase, and Β-glucosidase enzymes required for the breakdown of crystalline cellulose. Cellulose breakdown and extracellular enzyme levels in liquid culture can be affected by low pH values attained during fungal growth. During growth ofA. fumigatus on modified Czapeck-Dox medium containing 1% (w/v) Avicel, it was found that Β-glucosidase activity was lost and endoglucanase activity, reduced, when pH values fell below 3. The effect of buffering (0.2M phosphate, pH 6.15) was examined and compared with the unbuffered medium. Beta-glucosidase activity could be detected throughout the incubation period in the buffered medium and endoglucanase activity was approximately tenfold greater. Exoglucanase activity also showed an increase in the buffered system. Concentrations of phosphate buffer ranging from 0.05 to 0.8M were incorporated into the medium and optimum cellulose breakdown and extracellular enzyme production occurred between 0.1 and 0.2M. Reports suggest that increasing substrate concentration does not improve upon the levels of extracellular cellulase produced because of enzyme inactivation resulting from rapid decreases in pH. Using the buffered medium described previously,A. fumigatus was grown on concentrations of Avicel ranging from 0.5 to 10% (w/v). Cellulose breakdown and extracellular enzyme production was compared with that achieved by a similar nonbuffered system. Endoglucanase and Β-glucosidase activity increased with time and with substrate concentration up to 5% (w/v) in the buffered medium. Beta-glucosidase was negligible at all concentrations of Avicel in the unbuffered medium and endoglucanase activity decreased with increasing substrate concentration with maximum levels approximately eightfold lower than in the buffered system. Extracellular exoglucanase activity was lower in the buffered medium and only increased to levels comparable with those achieved by the unbuffered medium towards the end of the incubation period. In the unbuffered system, exoglucanase activity decreased with increasing substrate concentration, but no such effect was observed in the buffered medium. Negligible growth occurred in both media at 10% (w/v) substrate. The percentage weight loss recorded in the Czapeck-Dox medium also decreased with increasing substrate concentration, while in the buffered medium, over 95% weight loss was recorded in up to 5% (w/v) Avicel. It appeared that cellulose breakdown was more rapid in the buffered medium and a time-course carried out to determine the rate of cellulolysis showed 97% cellulose breakdown after 12 d, corresponding to a plateau and a subsequent decrease in extracellular cellulase levels.     

Xylanase production byAspergillus tamarii

Aspergillus tamarii has been found to grow well and to produce high cellulase-free xylanase activity when growing on corn cob powder as the principal substrate. Maximum xylanase production (285-350 U/mL) was obtained when the strain was grown in media supplemented with high corn cob concentration (5-8%, w/v) for 5 d. The presence of constitutive levels of xylanase was detected in cultures with glucose as the carbon source. Zymogram analysis for detection of xylanase activity after electrophoresis in polyacrylamide gels has shown thatA. tamarii produces at least two xylanases under the conditions utilized. The hydrolysis patterns of xylan demonstrated that the xylanases were endoenzymes, yielding mainly xylobiose, xylotriose, and higher xylooligosaccharides with traces of xylose.     

Cellulase and xylanase production byAspergillus sp. G-393

Applied Biochemistry and Biotechnology - The production of cellulase and xylanase can be achieved byAspergillus sp. G-393 using agricultural wastes. Enzymes produced by the strain G-393 were stable...     

The effects of agitation and aeration on the production of gluconic acid byAspergillus niger

Applied Biochemistry and Biotechnology - The effects of agitation and aeration in the production of gluconic acid byAspergillus niger from a glucose medium were investigated. Experiments were...     

Whole broth cellulase production for use in simultaneous saccharification and fermentation

Applied Biochemistry and Biotechnology - Cellulase, an enzyme that catalyzes the breakdown of cellulose into glucose, is produced inside fungal cells and secreted into the surrounding media....     

Enhanced glucose production from cellulose using coimmobilized cellulase and β-glucosidase

β-Glucosidase was covalently immobilized alone and coimmobilized with cellulase using a hydrophilic polyurethane foam (Hypol®FHP 2002). Immobilization improved the functional properties of the enzymes. When immobilized alone, the K_m for cellobiose of β-glucosidase was decreased by 33% and the pH optimum shifted to a slightly more basic value, compared to the free enzyme. Immobilized β-glucosidase was extremely stable (95% of activity remained after 1000 h of continuous use). Coimmobilization of cellulase and β-glucosidase produced a cellulose-hydrolyzing complex with a 2.5-fold greater rate of glucose production for soluble cellulose and a four-fold greater increase for insoluble cellulose, compared to immobilized cellulase alone. The immobilized enzymes showed a broader acceptance of various types of insoluble cellulose substrates than did the free enzymes and showed a long-term (at least 24 h) linear rate of glucose production from microcrystalline cellulose. The pH optimum for the coimmobilized enzymes was 6.0. This method for enzyme immobilization is fast, irreversible, and does not require harsh conditions. The enhanced glucose yields obtained indicate that this method may prove useful for commercial cellulose hydrolysis.     

Xylanase production by Trichoderma reesei rut C-30 on rice straw

Xylanase production of Trichoderma reesei Rut C-30 was examined at different initial pH values (4.8, 5.9, and 7.0) on rice straw in shake flasks, and in a fermentor, for the best pH condition. Enzyme performance was tested on ammonia-treated dwarf elephant grass. The maximum xylanase activities, 92 and 122 IU/mL, were obtained at pH 4.8 in the shake flasks and fermentor, respectively, in which good growth of the fungus was observed during the first 24 h and consumption of proteins dissolved from the rice straw caused the pH to rise later to values between 6.4 and 6.7 (optimal for xylanase production). The xylanases from T. reesei were as effective as Multifect XL, a commercial enzyme preparation, in hydrolyzing ammonia-treated elephant grass.     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Liquid fluidized bed starter culture ofTrichoderma reesei for cellulase production

A starter culture ofTrichoderma reesei (Rut-C30) prepared in a liquid fluidized bed reactor (LFBR) gave better growth and greater cellulase production in submerged fermentation than a conventional shake flask inoculum. The LFBR starter was prepared by first coatingT. reesei spores to 0.25 mm size corncob (1.0x10⁸g^-1) in a medium containing 1.0% corncob, 0.5 gL^-1 xylose and 0.1 gL^-1 lactose in a balanced salt solution, then fluidizing the particles in the LFBR for 36 h to allow germination of the spores, and covering the particles with an approx 30 μm thick biofilm. This biofilm that developed in constant adherence to the lignocellulosic carrier, apparently became well adapted to grow rapidly on insoluble cellulose substrates (Solca Floc), and had the enzymes of the cellulase complex induced for increased cellulase production. The LFBR starter used in a stirred tank reactor (STR) gave 15 gL^-1 biomass production and 6.5 IU mL^-1 overall cellulase activity with a volumetric productivity of 64 IU L^-1h^-1 in a 5 d fermentation, compared with a 7 d shake flask inoculum that gave 11 gL^-1 biomass and 3.2 IU mL^-1 cellulase activity, with a volumetric productivity of 31IU L^-1h^-1. The LFBR starter culture retained its viability in dry storage for 6–9 mo.     

Xylanase production by Aspergillus awamori in solid-state fermentation and influence of different nitrogen sources

The use of purified xylan as a substrate for bioconversion into xylanases increases the cost of enzyme production. Consequently, there have been attempts to develop a bioprocess to produce such enzymes using different lignocellulosic residues. Filamentous fungi have been widely used to produce hydrolytic enzymes for industrial applications, including xylanases, whose levels in fungi are generally much higher than those in yeast and bacteria. Considering the industrial importance of xylanases, the present study evaluated the use of milled sugarcane bagasse, without any pretreatment, as a carbon source. Also, the effect of different nitrogen sources and the C∶N ratio on xylanase production by Aspergillus awamori were investigated, in experiments carried out in solid-state fermentation. High extracellular xylanolytic activity was observed on cultivation of A. awamori on milled sugarcane bagasse and organic nitrogen sources (45 IU/mL for endoxylanase and 3.5 IU/mL for β-xylosidase). Endoxylanase and β-xylosidase activities were higher when sodium nitrate was used as the nitrogen source, when compared with peptone, urea, and ammonium sulfate at the optimized C∶N ratio of 10∶1. The use of yeast extract as a supplement to the these nitrogen sources resulted in considerable improvementin the production of xylanases, showing the importance of this organic nitrogen source on A. awamori metabolism.     

Xylanase production by Penicillium canescens 10–10c in solid-state fermentation

Filamentous fungi have been widely used to produce hydrolytic enzymes for industrial applications, including xylanases, whose levels in fungi are generally much higher than those in yeast and bacteria. We evaluated the influence of carbon sources, nitrogen sources, and moisture content on xylanase production by Penicillium canescens 10–10c in solid-state fermentation. Among agricultural wastes tested (wheat bran, untreated wheat straw, treated wheat straw, beet pulp, and soja meal), untreated wheat straw gave the highest production of xylanase. Optimal initial moisture content for xylanase production was 83%. The addition of 0.4 g of xylan or easily metabolizable sugar, such as glucose and xylose, at a concentration of 2% to wheat straw enhanced xylanase production. In solid-state fermentation, even at high concentrations of glucose or xylose (10%), catabolic repression was minimized compared to the effect observed in liquid culture. Yeast extract was the best nitrogen source among the nitrogen sources investigated: peptone, ammonium nitrate, sodium nitrate, ammonium chloride, and ammonium sulfate. A combination of yeast extract and peptone as nitrogen sources led to the best xylanase production.     

Solid-state fermentation of new substrates for production of cellulase and other biopolymer-hydrolyzing enzymes

Applied Biochemistry and Biotechnology -     

Influence of operating conditions and vessel size on oxygen transfer during cellulase production

The production of low-cost cellulase enzyme is a key step in the development of an enzymatic-based process for conversion of lignocellulosic biomass to ethanol. Although abundant information is available on cellulase production, little of this work has examined oxygen transfer. We investigated oxygen transfer during the growth of Trichoderma reesei, a cellulase-producing microorganism, on soluble and insoluble substrates in vessel sizes from 7 to 9000 L. Oxygen uptake rates and volumetric mass transfer coefficients (k _La) were determined using mass spectroscopy to measure off gas composition. Experimentally measured k _La values were found to compare favorably with a k _La correlation available in the literature for a non-Newtonian fermentation broth during the period of heavy cell growth.     

Improvements in titer,productivity, and yield using solka-floc for cellulase production

Researchers studying cellulase enzymes for the economical production of fuel ethanol envision cellulose as the carbon source. However, submerged Trichoderma reesei cultures grown on cellulose exhibit high run-to-run variability. Thus, an investigation of 30 batch cellulase production experiments was instrumental in determining fermentation conditions that improved enzyme titers, yields, and productivities. Eighteen of the 30 batch experiments experienced minimal process upsets and were classified into eight groups based on agitation rate, gas sparge rate, and the use of oxygen supplementation. Comparing corn steep liquor with yeast extract/peptone also tested the effect of different sources of nitrogen in the media. Average 7-d enzyme titers were doubled from 4 to 8 FPU/mL primarily by increasing aeration.     

Effect of pH on cellulase production of Trichoderma ressei RUT C30

Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, β-glucosidase production was increased greatly.     

Effect of acetic acid and furfural on cellulase production of Trichoderma reesei RUT C30

Because of the high temperature applied in the steam pretreatment of lignocellulosic materials, different types of inhibiting degradation products of saccharides and lignin, such as acetic acid and furfural, are formed. The main objective of the present study was to examine the effect of acetic acid and furfural on the cellulase production of a filamentous fungus Trichoderma reesei RUT C30, which is known to be one of the best cellulase-producing strains. Mandels’s mineral medium, supplemented with steam-pretreated willow as the carbon source at a concentration corresponding to 10 g/L of carbohydrate, was used. Four different concentration levels of acetic acid (0–3.0 g/L) and furfural (0–1.2 g/L) were applied alone as well as in certain combinations. Two enzyme activities, cellulase and β-glucosidase, were measured. The highest cellulase activity obtained after a 7-d incubation was 1.55 FPU/mL with 1.0 g/L of acetic acid and 0.8 g/L of furfural added to the medium. This was 17% higher than that obtained without acetic acid and furfural. Furthermore, the results showed that acetic acid alone did not influence the cellulase activity even at the highest concentration. However, β-glucosidase activity was increased with increasing acetic acid concentration. Furfural proved to be an inhibiting agent causing a significant decrease in both cellulase and β-glucosidase production.     

Measuring cellulase activity

An approach is presented for obtaining relative filter paper activities for enzyme preparations having activities below that required for application of the traditional International Union of Pure and Applied Chemistry filter paper assay. The approach involves the utilization of protein stabilizers to retard the time-dependent enzyme inactivation that may occur under traditional filter paper assay conditions. Enzyme stabilization allows extended reaction times and the calculation of relative activities based on the time required for saccharification of 3.6% of the traditional substrate, making results proportional to those obtained in the traditional International Union of Pure and Applied Chemistry assay. The assay is demonstrated using a commercial cellulase preparation along with KCl and bovine serum albumin as protein stabilizers.     

Aspergillus fumigatus Thermophilic and Acidophilic Endoglucanases

This study evaluated the production of cellulolytic enzymes by an Aspergillus fumigatus strain, isolated from sugar cane bagasse, according to its ability to grow on microcrystalline cellulose as the sole carbon source. The effect of the carbon source (brewer’s spent grain, sugarcane bagasse, and wheat bran) and of the nitrogen source (corn steep liquor and sodium nitrate) on cellulase production was studied using submerged and solid state cultivations at 30 °C. The highest levels of endoglucanase (CMCase) corresponded to 365 U L^-1 and was obtained using sugarcane bagasse (1%) and corn steep liquor (1.2%) in submerged fermentation within 6 days of cultivation. This supernatant was used to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis that showed six bands with endoglucanase activity. CMCase activity was higher at 65 °C and pH 2.0, indicating that this microorganism produces a thermophilic and acid endoglucanase. Solid state cultivation favored FPase production, that reached 47 U g^-1 of dry substrate (wheat bran and sugarcane bagasse) within 3 days.     

Bubble and foam concentration of cellulase

Applied Biochemistry and Biotechnology - Experiments were conducted to determine the effect of pH and sparging-gas composition on the bubble and foam separation of an aqueous protein solution....     

MnO2纳米粒子固载纤维素酶用于高效水解农业废弃物制备生物乙醇

纤维素酶是一种有效的纤维质类物质水解催化剂,工业应用时可通过固定化纤维素酶来降低其成本。本文将烟曲霉原变种JCF产生的纤维素酶固定在MnO2纳米颗粒上。 MnO2可提高纤维素酶的活性,并充当一个更好的载体。采用扫描电镜表征了所制MnO2纳米粒子及其负载纤维素酶的表面性质,以傅里叶变换红外光谱分析了固定在MnO2纳米粒子上纤维素酶的官能团性质。纤维素酶在MnO2纳米粒子上最大的固定化效率为75%。考察了固定化纤维素酶的活性、操作pH值、温度、热稳定性和重复使用性等性质。结果表明,所制固定化酶的稳定性比游离酶更高。固定于MnO2纳米粒子上的纤维素酶可用于纤维质类物质的水解反应,且能在较宽的温度和pH值范围内使用。表征结果证实了该催化剂具有非常高的催化纤维素类物质水解的活性。