Production and characterization of a highly active cellobiase from Aspergillus niger grown in solid state fermentation

Summary Aspergillus niger produced extracellular cellobiase when grown on different lignocellulosic substrates in solid state fermentation. The enzyme activity and yield were variable according to the carbon source. In Vogel’s medium, the cellobiase productivity was significantly higher on wheat bran, followed by Leptochloa fusca (kallar grass) straw augmented with corn steep liquor. Maximum yield of cellobiase/g wheat bran was significantly higher than the values reported on other potent fungi, bacteria and recombinants, harboring heterologous gene for cellobiase. This enzyme in the presence and absence of Trichoderma reesei and celluloclast, saccharified the biomass and the percentage saccharification as well as glucose yield from lignocellulosic biomass was doubled in its presence. The partially purified enzyme was thermotolerant as evidenced by melting temperature, activation energy demand for active catalysis, enthalpy and entropy of activation for reversible or irreversible thermal inactivation.     

绿色木霉产生的葡萄糖苷酶类

由绿色木霉(Trichoderma viride)A10菌株的粗酶制剂中分离纯化得到 6个β-葡萄糖苷酶组分,对它们的物理、化学性质和对20种糖苷类化合物的水解作用进行了测试。其中5个组分都是既能水解β-构型的糖苷化合物,又能水解α-构型的糖苷化合物,表明它们对α-、β-异头专一性的要求并不象文献中报导的那样十分严格,对糖基和糖苷配基专一性的要求也并不十分严格。由此,对葡萄糖苷酶类的命名分类方法提出了讨论,并对在纤维素酶系研究中纤维二糖酶活力的检测方法进行了讨论。     

New isolate of Streptomyces sp. with novel thermoalkalotolerant cellulases

A Streptomyces sp. was isolated that produced novel thermoalkalotolerant cellulase activity after growth on crystalline cellulose at 50°C. Three major components of the cellulases (CMCase, Avicelase and cellobiase) were produced with maximal activities (11.8, 7.8 and 3.9 IU/ml) and maximum specific activities 357, 276 and 118 IU/mg protein, respectively, after 120 h growth. Maximum CMCase activity was between 50 and 60°C measured over 3 h. The enzyme also retained 88% of its maximum activity at 70°C and pH 5, and 80% of the activity at pH 10 and 50°C when assayed after 1 h. After incubation at 40°C for 1 h with commercial detergent (Tide) at pH 11, 95% activity was retained. The enzyme mixture produced glucose from crystalline cellulose.     

Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii

Industrial waste corn cob residue (from xylose manufacturing) without pretreatment was hydrolyzed by cellulase and cellobiase. The cellulosic hydrolysate contained 52.4 g l⁻¹ of glucose and was used as carbon source for lactic acid fermentation by cells of Lactobacillus delbrueckii ZU-S2 immobilized in calcium alginate gel beads. The final concentration of lactic acid and the yield of lactic acid from glucose were 48.7 g l⁻¹ and 95.2%, respectively, which were comparative to the results of pure glucose fermentation. The immobilized cells were quite stable and reusable, and the average yield of lactic acid from glucose in the hydrolysate was 95.0% in 12 repeated batches of fermentation. The suitable dilution rate of continuous fermentation process was 0.13 h⁻¹, and the yield of lactic acid from glucose and the productivity were 92.4% and 5.746 g l⁻¹ h⁻¹, respectively. The production of lactic acid by simultaneous saccharification and fermentation (SSF) process was carried out in a coupling bioreactor, the final concentration of lactic acid was 55.6 g l⁻¹, the conversion efficiency of lactic acid from cellulose was 91.3% and the productivity was 0.927 g l⁻¹ h⁻¹. By using fed-batch technique in the SSF process, the final concentration of lactic acid and the productivity increased to 107.6 g l⁻¹ and 1.345 g l⁻¹ h⁻¹, respectively, while the dosage of cellulase per gram substrate decreased greatly. This research work should advance the bioconversion of renewable cellulosic resources and reduce environmental pollution.     

Beta-D-glucosidase reaction kinetics from isothermal titration microcalorimetry

The cellobiase activities of nine thermal stable mutants of Thermobifida fusca BglC were assayed by isothermal titration microcalorimetry (ITC). The mutations were previously generated using random mutagenesis and identified by high-temperature screening as imparting improved thermal stability to the beta-D-glucosidase enzyme. Analysis of the substrate-saturation curves obtained by ITC for the wild-type enzyme and the nine thermally stabilized mutants revealed that the wild type and all the mutants were subject to binding of a second substrate molecule. Furthermore, the "inhibited" enzyme-substrate complexes were shown to retain catalytic activity. In the case of three of the BglC mutants (N178I, N317Y/L444F, and N317Y/L444F/A433V), binding of a second substrate molecule resulted in improved cellobiose turnover rates at lower substrate concentrations. No correlation between denaturation temperatures of the mutants and activity on cellobiose at 25 degrees C was evident. However, one particular mutant, BglC S319C, was significantly improved in both thermal tolerance and cellobiase activity with respect to those of the wild-type BglC. The triple mutant, N317Y/L444F/A433V, had a 5 degrees C increase in denaturation temperature while maintaining activity levels similar to that of the wild type at higher substrate concentrations. ITC provided a highly sensitive and nondestructive means to continuously monitor the reaction of BglC with cellobiose, resulting in abundant data sets that could be rigorously analyzed by fitting to known enzyme kinetics models. One distinct advantage of using data from the ITC was the empirical validation of the pseudo steady state assumption, a necessary condition for obtaining solutions to the proposed mechanisms.     

Increased enzyme secretion by 2-deoxy-d-glucose in presence of succinate by suppression of metabolic enzymes in Termitomyces clypeatus

Regulatory mode of secretion of proteins was detected for the industrial glycosidase, cellobiase, under secreting conditions (in presence of TCA cycle intermediates like succinate etc.) in the filamentous fungus Termitomyces clypeatus. The titers of key metabolic enzymes were investigated under secreting and non-secreting conditions of growth and compared to the corresponding production of intra and extracellular levels of cellobiase. Results were compared in presence of 2-deoxy-d-glucose, a potent glycosylation inhibitor in the secreting media. Inclusion of 2-deoxy-d-glucose in presence of succinate caused about 10 to 100 times decrease in titers of the metabolic enzymes hexokinase, fructose-1,6-bisphosphatase, isocitrate lyase and malate dehydrogenase leading to increased secretion of cellobiase by more than 100 times. The intracellular concentration of cAMP (86-fold decrease in presence of 2-deoxy-d-glucose under secreting conditions) and turnover rate of proteins also dropped significantly. In this suppressed metabolic state, a 10-fold increase in the titer of the secreted cellobiase was noticed. The results indicated elucidation of carbon catabolite repression like phenomenon in the fungus under secreting conditions which was more pronounced by 2-deoxy-d-glucose. The interdependence between secretion and regulation of metabolic enzymes will help in better understanding of the physiology of these highly adapted organisms for increasing their secretion potential of glycosidases like cellobiase with high industrial value.