Cell growth and cellulase production inTrichoderma viride on microcrystalline cellulose

The production of CM and FP cellulases was studied during the growth of a wild strain ofTrichoderma viride on microcrystalline cellulose. Part of the enzymes was found to be released into the medium while another part remained bound to the cell. Bound cellulases are released into the medium at the stage of cell lysis which takes place in the post-stationary phase. In this period extracellular CM and FP cellulases attain maximum activities. When the hyphae are subjected to a cold shock, maximum cellulase activity is detected already at the beginning of the stationary phase. An indirect method of dry cell mass determination showed that during exponential growth of cells on microcrystalline cellulose the μmax was 0.23 and the yield coefficient was 41 %.     

Adsorption of cellulase fromTrichoderma viride on microcrystalline cellulose

Summary The adsorption behaviour of cellulase fromTrichoderma viride on microcrystalline celluloses with different specific surface areas was studied. The adsorption was found to fit a Langmuir isotherm. There was an increase in the maximum adsorption amount (A_max) as the specific surface area of microcrystalline cellulose increased. The values of A_max and adsorption equilibrium constant (K) decreased with increasing temperature. Thermodynamic parameters in adsorption were calculated from K. It was found from the enthalpy of adsorption, that van der Waals-Type interaction was responsible for adsorption of cellulase on microcrystalline cellulose. The adsorption process was exothermic and an adsorption enthalpy-controlled reaction.     

Kinetics of solka floc cellulose hydrolysis by trichoderma viride cellulase

A product inhibition model is developed to describe the hydrolysis of cellulose by the Trichoderma viride enzyme system. It is assumed that noncompetitive inhibition by cellobiose dominates the reaction kinetics. Experiments show that this is indeed a reasonable assumption for initial cellulose concentrations of up to 15 g/liter and at hydrolysis extents up to 65′. Kinetic parameters were determined for the noncompetitive inhibitionmodel in batch experiments with durations of up to 1.5 hr. These parameterswere then used in predicting reaction progress for up to 10 hr. Cellobiose was added to the reaction mixture at the onset of some runs and againreliable predictions were obtained for up to 8 hr of hydrolysis. Finally reaction was carried out in a membrane reactor whereby the product cellobiose was being continuously removed and again reasonable predictability was obtained with a higher net reaction rate.     

Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

Conversion of cellulose to glucose with cellulase of Trichoderma viride ITCC-1433

Trichoderma viride ITCC-1433 secretes a cellulase complex that is rich in β-glucosidase and therefore well suited for the saccharification of cellulosic materials. The cellulase was investigated with respect to optimum conditions of reaction and enzyme stability. Avicelase, CMCase, and β-glucosidase differed considerably in their physicochemical properties. At temperatures above 50°C, β-glucosidase is not very stable. Therefore, as a compromise the conditions of hydrolysis were chosen to be 50°C and pH 4.5. With the crude culture filtrate of T. viride ITCC-1433 a nearly pure glucose solution of 4% is reached from a 10% cellulose suspension. Wood pulp and newsprint are hydrolyzed to a much smaller extent. With an enzyme concentrate up to 8% glucose accumulated in the reaction fluid within 48 hr. At this time the glucose-cellobiose ratio was 75:1. Glucose was demonstrated to be the most potent inhibitor of total hydrolysis. The addition of glucose to the enzyme-substrate solution at zero time completely stopped its own formation and cellobiose and reducing groups (oligosaccharides) accumulated. By removing glucose through an ultrafilter device about 90% saccharification of cellulose to glucose was achieved in 48 hr without any accumulation of cellobiose.     

Saccharification of native and degraded cotton cellulose and commercial microcrystalline cellulose by Trichoderma viride cellobiohydrolase I

The degree of polymerization of samples of acid degraded cotton cellulose has no appreciable influence on the saccharification by cellobiohydrolase I from Trichoderma viride. The increase in the number of cellulose molecule ends, achieved by a 30-fold decrease in molecular weight, does not produce the effect which could be expected for a pure end-wise mode of action of this exoglucanase. Microcrystalline celluloses saccharified by the same enzyme yield considerably more reducing sugars than cotton cellulose, either with a similar degree of polymerization or one of about 7000. It appears, therefore, that the difference in the susceptibility of the commercial substrates is not a consequence of their low degree of polymerization.     

A method for increasing cellulase production by Trichoderma viride

A doubling of cellulase production by Trichoderma viride (QM9414) is possible by increasing the cellulose concentration in the medium from 0.75 to 2%, increasing the nitrogen concentration, and controlling pH during growth. A four-to fivefold increase in β -glucosidase is found with the higher cellulose concentration. Culture filtrates from 2% cellulose cultures can reduce the hydrolysis time in a practical saccharification to one-half that required by culture filtrates from 0.75% cellulose cultures.     

Strain improvement for enhanced production of cellulase in Trichoderma viride

The filamentous fungi Trichoderma species produce extracellular cellulase. The current study was carried out to obtain an industrial strain with hyperproduction of cellulase. The wild-type strain, Trichoderma viride TL-124, was subjected to successive mutagenic treatments with UV irradiation, low-energy ion beam implantation, atmospheric pressure non-equilibrium discharge plasma (APNEDP), and N-methyl-N'-nitro-N-nitrosoguanidine to generate about 3000 mutants. Among these mutants, T. viride N879 strain exhibited the greatest relevant activity: 2.38-fold filter paper activity and 2.61-fold carboxymethyl cellulase, 2.18-fold beta-glucosidase, and 2.27-fold cellobiohydrolase activities, compared with the respective wild-type activities, under solid-state fermentation using the inexpensive raw material wheat straw as a substrate. This work represents the first application of APNEDP in eukaryotic microorganisms.     

Properties of cellulase fromTrichoderma viride

Cellulase produced by Trichoderma viride acted on carboxymethyl cellulose with a Km of 4.9 g substrate per litre, showing a pH optimum at 4.5 and a temperature optimum at 55 degrees C. Ag+, Hg2+, Zn2+, Cu2+ and N3- were inhibitory..     

pH值对绿色木霉(Trichoderma viride)产纤维素酶的影响

采用微晶纤维素为唯一诱导性碳源,对绿色木霉(Trichoderma viride)在摇瓶发酵过程中控制与不控制pH产纤维素酶进行比较.控制pH时胞外蛋白浓度为0.72 mg/mL比不控制pH时提高43%;FPA、EG、GB和CBH酶活为15.0U/mL,120.0U/mL,1.75U/mL,0.85U/mL分别是不控制pH时的2.1、2.3、11.7和1.7倍.在不同pH下测定纤维素酶液各酶活,表明pH值显著影响纤维素酶各单酶酶活.在pH2.7时,β-葡萄糖苷酶酶活仅为pH4.8时酶活的4%;pH回调试验结果表明β-葡萄糖苷酶对pH敏感,并在催化功能上发生不可逆变化.对纤维素酶液添加分离得到的各单酶,当添加β-葡萄糖苷酶时最多可以提高FPA酶活20%.因此β-葡萄糖苷酶是影响综合酶活的关键酶.通过拉曼光谱检测出β-葡萄糖苷酶在pH5.0有活性状态下,酶蛋白主链结构主要为a-螺旋和无规则卷曲;在pH2.0没有活性状态下,酶蛋白主链结构的无规则卷曲发生较大变化,a-螺旋也受到一定影响.这说明pH对β-葡萄糖苷酶构象的改变是造成其活性变化的主要原因.     

Enhancement of cellulase production by Trichoderma viride using carbon/nitrogen double-fed-batch

Summary The production of cellulase by Trichoderma viride has been remarkably raised by means of a fed batch fermentation with the addition of cellulose as the carbon source and ammonia as nitrogen source during cultivation.     

Continuous cultivation of Trichoderma viride on cellulose

Using ball milled cellulose as the only carbon source Trichoderma viride was grown in a continuous flow culture at pH = 5.0 and T = 30°C. Steady-state values for cell protein, cellulose, and cellulase for different substrate concentrations (4–11 g/liter) and dilution rates (0.033–0.080 hr^?1) were obtained. Under steady-state conditions, 50–75% of the cellulose was consumed indicating a critical dilution rate on 0.17 hr^?1. Cellulase activity (U/ml) in the fermentation broth increased slightly with increasing substrate concentration and decreased with increasing dilution rate, while the specific cellulase productivity (U/mg cell protein·hr) was fairly independent of the dilution rate, with a maximum around D = 0.05 hr^?1. Following step changes in substrate concentration and dilution rate, new steady-state values were reached after three to five residence times (cell protein and cellulose) and four to six residence times (celullase activity).     

Kinetics of the hydrolysis of cellulose by beta-1,4-glucan cellobiohydrolase of Trichoderma viride

The cellulolytic enzyme beta-1,4-glucan cellobiohydrolase (CBH) has been isolated from the crude mixture of cellulase enzymes of Trichoderma viride by gel filtration and ion-exchange methods, and some aspects of its kinetic behaviour have been examined. Studies of the initial rates of the CBH-catalyzed production of cellobiose from fibrous alpha-cellulose show that (i) the dissociation constant for cellobiose competitive product inhibition of the reaction is Ki = (1.13 +/- 0.37) X 10(-3) M, (ii) the adsorption of CBH on fibrous alpha-cellulose and its subsequent reaction conform to kinetic equations developed in conjunction with the Langmuir adsorption isotherm, (iii) the rate-pH curve has a maximum at pH 5.2 and decreases at higher and lower pH values, exhibiting enzyme pK values of 3.8 and 6.5, and (iv) the energy of activation of the overall reaction between 5 and 60 degrees C is 5.3 +/- 0.3 kcal mol-1 at pH 5.2. Studies of the time course of the reaction over extended periods of time up to 40% hydrolysis of the cellulose show that (v) the data fit better to a competitive product inhibition model than to models of anticompetitive product inhibition or noncompetitive product inhibition.     

Cultivation of yeasts on hydrolysate nutrient medium obtained from the production of microcrystalline cellulose

Studies of the biomass production during a continuous cultivation of yeasts on a nutrient medium, prepared from a hydrolysate from the production of microcrystalline cellulose, have been carried out. A new strain of yeasts has been used. Its cultivation has been achieved without addition of biostimulators to the nutrient medium in spite of their absence in the initial hydrolysate. Practically a complete assimilation of sugars has been achieved at high dilution rates (D = 0.25 to 0.50 h⁺¹). The yield of biomass achieved is above 50% compared to the initial sugars and it contains 48.89% true protein. The results obtained offer the possibility of a complex utilization of the products of cellulose hydrolysis in the production of microcrystalline cellulose with a realization of a waste free technology.     

Detrimental effect of cellulose oxidation on cellulose hydrolysis by cellulase

To effectively convert complex and recalcitrant biomass carbohydrates to simple platform sugars useful for fuel and chemicals production, mechanical or chemical pre-treatments are often required to make the carbohydrates more accessible for enzymatic hydrolysis. Due to their harsh conditions, some pre-treatments might negatively affect enzymatic hydrolysis because of events such as cellulose oxidation. To study how oxidative modification may impact cellulose's reactivity toward hydrolysis by cellulases, we prepared three cellulose substrates by cupric ion and hypochlorite oxidations, and subjected the derived celluloses to hydrolysis by various cellobiohydrolases from glycoside hydrolase families 6 and 7, and one cellulolytic Hypocrea jecorina extracellular enzyme mixture. We observed a profound decrease of enzymatic hydrolysis on the oxidized celluloses. The effect was attributed to the interference, from oxidized functional groups in cellulose, on its binding/activation in the active pocket/tunnel of cellobiohydrolases. Potential implication of the observed effect from cellulose oxidation on pre-treatment optimization and cellulase improvement was discussed.