Cellulase production and activity in a species ofCladosporium

ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO₃ was better than NH₄Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C.     

Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.     

Cellulase production and activity by Trichoderma sp. A-001

Trichoderma species A-001 was grown on various carbon and nitrogen sources supplemented with surfactants on shake cultures. Although the degree of growth was variable, the organism grew on all carbon substrates. Large amounts of the cellulase enzyme components were released into the growth medium during growth on filter paper. In the filter paper containing medium, the organism produced 167 U/ml of carboxymethylcellulase (CMCase), 18 U/ml of filter paper activity (FPase) and 49 U/ml of beta-glucosidase activity (BGDase). Wheat straw and grass were better carbon sources than cotton or barley husks. Nitrogen in the form of KNO₃ was better than NH₄Cl or urea in facilitating the production of cellulase. Of the surfactants used, Tween-80 at 0.2% concentration in the medium increased the production of cellulase several-fold. All the cellulase components were optimally active in the assay at pH 5.5 and 60°C. CMCase and FPase lost 20–33% of their activities when kept at 60°C for 4 h before assaying. On the other hand, BGDase was moderately stable; it lost only 37% of its activity when maintained at 70°C for 4 h.     

The effect of temperature on growth and cellulase (β-1,4-endoglucanase) production in the compost fungusChaetomium thermophile var.dissitum

Chaetomium thermophile var.dissitum, isolated from an experimental urban refuse compost, had the following growth characteristics: Minimum temperature, 27±1°C; optimum, 45–50°C; maximum, 57±1°C; pH optimum 5.5–6.0.A number of carbohydrates could be used for growth, but cellulase formation measured with carboxymethylcellulose as substrate was initiated only on cellulose or xylan. With cellulose as the carbon source, cellulase accumulation in the culture filtrate followed closely that of growth, when the temperature was varied. pH optimum for the cellulase system was 5.0.The optimum temperature for cellulase activity with carboxymethylcellulose as substrate varied between 77°C with 1/2 h incubation time and 58°C with 10 h incubation time.With cotton as substrate, the optimum temperature was 58°C regardless of incubation time. Carboxymethylcellulose had a higher stabilizing effect on the enzyme than cotton. The temperature stability of the cellulase was highest at pH 6.0.     

Cellulolytic Activity of Thermomonospora curvata: Optimal Assay Conditions, Partial Purification, and Product of the Cellulase

Thermomonospora curvata produces cellulases active against both cotton fibers (designated C(1) activity) and carboxymethylcellulose (C(x) activity). In reaction systems employing optimal substrate concentration, pH, and temperature, hydrolysis rates (measured by the release of soluble reducing sugars) were initially linear and decreased on prolonged incubation, although only a small amount of substrate (1 to 2%) had been hydrolyzed. Persistence of this lower rate, even after addition of fresh enzyme (in the C(1) assay system), indicated alteration of cellulose susceptibility to hydrolysis rather than enzyme inactivation. Partial purification by (NH(4))(2)SO(4) precipitation and exclusion chromatography resolved cellulase activity into two fractions. The sole product of purified cellulase activity on ground cotton fibers appears to be cellobiose.     

Polysaccharide-hydrolyzing enzymes ofFrankia (Actinomycetales)

Studies were made of the polysaccharide-hydrolyzing activity inFrankia (Actinomycetales) grown in synthetic media using modifications of three standard assay procedures. In screening five different strains ofFrankia for cellulase activity, based on the method of utilization of cellulose in liquid culture, only one strain, CcI3, degraded filter paper cellulose to complete disintegration and only under very specific conditions of pH and primary carbon source. When carboxymethylcellulose (CMC) at 1% was used as substrate, all five strains showed the capacity to produce reducing sugars as hydrolytic products. Microcystalline cellulose, xylans and gum arabic were hydrolyzed to a lesser extent. Optimum activity depended upon pH and primary carbon source with pH 5.0 and pyruvate or propionate producing highest activities. In fractionation studies of culturedFrankia, assays for hydrolysis of 1% CMC in liquid medium showed that highest activity was in the enzyme preparation supernatant with lesser activity in the cell-free extract and cell wall fractions.Frankia strain CpI1 showed the greatest total hydrolytic activity against CMC after 2 weeks of culture. Strains ArI3 and CcI3 also showed good activity. The agar plate method for direct dye-polysaccharide interaction proved to be the least sensitive assay method with only ArI3 showing significant activity using CMC as substrate. It appears that theFranka strains grown in synthetic media all showed hydrolytic activity but the degree of hydrolysis of polysaccharides to reducing sugars depends upon strain of bacteria and very specific cultural conditions.     

Penicillium verruculosum WA 30 a new source of cellulase

Summary Of fungi 110 strains were screened for extracellular cellulase production in shake flask experiments. Twelve strains produced the enzyme in significant quantity. Since the enzyme activity was assayed by different methods (liberation of reducing sugar from cotton, filter paper, carboxymethylcellulose and cellobiose), the estimation of the productivity of the strains differed according to the substrate used. The best cotton degrading activity per fermentation volume as well as per mg of secreted soluble protein was achieved by Penicillium verruculosum WA 30, a wild-type strain, for which the cellulase productivity has not yet been published. The cotton degrading (so-called C₁) activity was successfully enhanced nearly threefold in medium experiments. Analyses of saccharification digests showed that glucose was the predominant product, with negligible amounts of cellobiose. The pH and temperature optima for WA 30 cellulase complex were pH 4.2 and 60°C.     

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.     

Cellulolytic activity of Actinomycetes isolated from termites (Termitidae) gut

Abstract Cellulolytic actinomycetes were isolated from the hindgut of four different termites: Macrotermes, Armitermes, Odontotermes and Microcerotermes spp.
The isolated actinomycetes ( Streptomyces sp. and Micromonospora sp.) were grown on cellulosic substrates and their extracellular cellulase (C_l, C_x and cellobiase) activity evaluated; using filter paper as a substrate for C_l, carboxymethylcellulose (CMC) for C_x and d -cellobiose for cellobiase, all strains were shown to degrade soluble and insoluble cellulose; optimum pH for growth was 6.2–6.7 at 28°C; three strains could grow at 48°C on cellulosic substrates.
Some strains exhibited high cellulase activity, constant for 5–7 days, but inhibition by glucose was a common feature for almost all isolates.     

Induction of Cellulolytic and Xylanolytic Enzyme Systems in Streptomyces spp

Extracellular enzyme preparations from Streptomyces flavogriseus and Streptomyces olivochromogenes cultures grown on cellulose contained primarily cellulase activities, but similar preparations from cultures grown on xylan-containing materials possessed high levels of both cellulase and xylanase activities. Growth conditions that gave high endoxylanase levels also resulted in the production of enzymes involved in the hydrolysis of the nonxylose components of xylan. Specific acetyl xylan esterase activities were identified in enzyme preparations from both organisms. Both organisms also produced alpha-l-arabinofuranosidase activity that was not associated with endoxylanase activity. Other activities produced were alpha-l-O-methylglucuronidase and ferulic acid esterase. The latter enzyme was produced only by S. olivochromogenes and is an activity which has not previously been identified as a component of hemicellulase preparations.     

Isolation and partial properties of cellulose-decomposing strain of Cytophaga sp. LX-7 from soil

A new facultatively anaerobic, Gram-negative bacterium, Cytophaga sp. LX-7, degrading crystalline cellulose completely, was isolated from soil by dilution plating on cellodextrin agarose plates. This strain could excrete extracellularly all three types of cellulase and cellulosic substrates were the strongest inducer of endocellulase with CMC-liquefying activity production. No reducing sugar was found in cultures of cellulose during incubation. An enzyme which degrades crystalline cellulose was detected in cultures of cellulose by measuring the formation of soluble carbohydrate but was not detected by determining the reducing sugar released. This strain also synthesized cell-bound cellobiose oxidizing enzyme which was previously noted only in fungi. Both cellulose and soluble sugars could promote the production of cellobiose oxidizing enzyme.     

降解棉秸秆耐热菌株的鉴定及降解条件优化

【目的】鉴定从新疆棉花秸秆高温堆肥中分离出的两株耐热真菌Z1、Z2的属种,并通过优化影响菌株产生纤维素酶的因素来提高菌株对秸秆的降解率。【方法】经形态学和菌株的ITS区克隆与序列分析确定属种,以液体摇瓶发酵产滤纸酶活性(FPA)变化为衡量指标,对Z1、Z2以及二者混合菌(MS)的纤维素酶产生条件进行优化。【结果】菌株Z1为曲霉属烟曲霉(Aspergillus fumigatus Fresen),Z2为蚀丝霉属(Myceliophthora Cost.)。确定Z1以棉秸秆为碳源、以NaNO3为氮源、起始pH 9.5、接种量11%、50°C摇床培养10 d,对棉秸秆降解率为10.19%;Z2以麦秸秆为碳源、以NaNO3为氮源、起始pH 5.5、接种量9%、50°C摇床培养10 d,对麦秆降解率为27.50%;MS以棉花秸秆为碳源、以蛋白胨为氮源、起始pH 5.5、接种量11%、50°C摇床培养10 d,对棉秸秆的降解率为53.45%。【结论】实验表明,MS(Z1、Z2混合)对秸秆的降解效果优于单株菌,降解率达到一半以上,本研究中的两株耐热真菌在降解棉花秸秆、小麦秸秆等农作物废弃秸秆中具有较高的应用价值。     

Cellulase Enzyme from Aspergillus niger

Cellulase enzyme was produced by a selected strain of Aspergillus niger isolated from deteriorated wood and grown on different carbon sources. Filter paper gave the highest yield, followed by carboxymethyl cellulose (CMC). Cellobiose as well as glucose gave a low yield, while the yield from lactose was negligible. The concentration of filter paper cellulose that induced the maximum yield of the enzyme was 1%. Both soluble cellulose (CMC) and cotton cellulose treated with phosphoric acid (swollen) were easily hydrolyzed by cellulase; an increase in cellulase concentration lead to more hydrolysis of CMC and gave linearity in the reaction velocity. At certain concentrations of the enzyme, increase in CMC concentration, (up to 1%) resulted in more reducing sugar. Beyond this point no more hydrolysis occur.     

棉秸秆降解高温菌株的筛选及产酶分析

从新疆地区分离具有降解棉秸秆纤维素功能的菌株,得到4株耐高温真菌(50°C)。纤维素酶学性质分析表明,该4株菌的纤维素酶具有良好的耐酸性(最适pH为4.5)和耐高温性(最高达60°C)。以羧甲基纤维素钠(CMC-Na)、微结晶纤维素、棉花、滤纸、淀粉、果胶为底物测定酶活力,滤纸酶活力(FPA)最高达2.63 U/mL、淀粉酶活力最高达6.17 U/mL、果胶酶活力最高达5.86 U/mL。4株真菌酶学特性分析表明,该系列菌株在秸秆生物质利用方面有很大的应用潜力。     

Solubilization of native and derived forms of cellulose by cell-free microbial enzymes

1. Cell-free enzymes from Myrothecium verrucaria and Trichoderma koningii hydrolyse native undegraded cellulose, as found in cotton fibres, in a random manner to short insoluble fibres and to minor amounts of soluble products. 2. Enzyme preparations from M. verrucaria fail to attack the short fibres whereas preparations from T. koningii solubilize them completely to sugars at an optimum pH4.2-4.6. 3. The mode of hydrolysis of cotton cellulose by preparations from T. koningii involves from the earliest stages the formation of reducing sugars, followed closely by the appearance of short fibres, until the insoluble and soluble products each constitute about 40-50% of the weight of the initial substrate. After this stage the quantity of sugars increases at the expense of the insoluble short fibres. 4. Depending upon the method of preparation, derived forms of cellulose may be hydrolysed more slowly, much more rapidly, or at the same rate as cotton fibres by enzyme preparations from T. koningii.     

Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

Three different chemical treatments—sulfur dioxide, ozone, and sodium hydroxide—were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganism and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70°C for 72 h) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solublize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO₂ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter), and the release of reducing sugars was determined. The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O₃-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. the SO₂- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent (21 g reducing sugars/100 g organic matter). The rumen fluid digestibility of the enzymatic ally hydrolyzed straw was not increased further over the effect already obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O₃-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification in this study, the absolute result was modest. The limited effect of the combined O₃-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. Based on the differential response of the chemically treated cotton straw to attack by rumen microorganisms on the one hand, and by T. reesei cellulase on the other hand, a hypothesis has been suggested as to the location of lignin and hemicellulose in the cellwall unit of cotton straw.     

Column cellulose hydrolysis reactor: the effect of retention time,temperature, cellulase concentration and exogenously added cellobiase on the overall process

Summary A novel column cellulose hydrolysis reactor with constant enzyme recycling was operated under various conditions to determine the effects of retention time, temperature, cellulase concentration and exogenously added cellobiase on the concentration of the product stream and the productivities of the reactor. Short term (7 days) hydrolysis was best at 42°C while longer term (14 days) hydrolysis was better at 37°C. A retention time of 11 h and reactor cellulase concentration of 30 filter paper units per gram of cellulose gave the best compromise for efficient operation by minimizing product inhibition, maximizing product concentration and minimizing enzyme consumption. The addition of cellobiase to the reactor increased cellulose hydrolysis and raised the proportion of monomeric sugars in the hydrolysate. Continuous cellulose hydrolyses were maintained for 7 and 14 days at 42°C and 37°C, respectively, resulting in volumetric productivities of 6.82 and 4.84 g/l/h and average sugar concentrations of 7.3% and 6.0% (w/v), respectively. Greater than 95% (w/w) of the sugars produced were in the monomeric state. Average cellulase used for the two runs were 8.4 and 5.3 filter paper units per gram of sugar produced, respectively.     

Saccharification of Complex Cellulosic Substrates by the Cellulase System from Clostridium thermocellum

True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of Ca²⁺ and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70°C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70°C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity.     

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

Summary Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.