Screening of an Alkaline CMCase-Producing Strain and the Optimization of its Fermentation Condition.

OBJECTIVE: Alkaline Carboxymethyl Cellulase (CMCase) is an attractive enzyme for the textile, laundry, pulp, and paper industries; however, commercial preparations with sufficient activity at alkaline conditions are scarce. METHODS: High CMCase-producing bacterial isolate, SX9-4, was screened out from soil bacteria, which was identified as Flavobacterium sp. on the basis of 16S rDNA sequencing. RESULTS: The optimum pH and temperature for CMCase reaction were 8.0 and 55°C, respectively. Alkaline CMCase was stable over wide pH (3.0-10.6) and temperature (25-55°C) ranges. Enzyme activity was significantly inhibited by the bivalent cations Mn2+ and Cu2+, and was activated by Fe2+. To improve the alkaline CMCase production of SX9-4, fermentation parameters were selected through onefactor- at-a-time and further carried out by response surface methodologies based on a central composite design. CONCLUSION: High CMCase production (57.18 U/mL) was achieved under the optimal conditions: 10.53 g/L carboxymethylcellulose sodium, 7.74 g/L glucose, 13.71 g/L peptone, and 5.27 g/L ammonium oxalate.

Isolation of cellulose nanocrystals from grain straws and their use for the preparation of carboxymethyl cellulose-based nanocomposite films.

Cellulose nanocrystals (CNCs) were isolated from rice straw (RS), wheat straw (WS), and barley straw (BS) by using acid hydrolysis method. They were fibrous in shape with length (L) of 120-800nm and width (W) of 10-25nm, aspect ratio (L/W) of 18, 16 and 19, crystallinity index (CI) of 0.663, 0.710, and 0.634, and yield of 64, 75, and 69wt% for RS, WS, and BS respectively. Carboxymethyl cellulose (CMC)/CNC composite films were prepared with various concentration of the CNCs. SEM results showed that the CNCs were evenly distributed in the polymer to form homogeneous films. Mechanical and water vapor barrier properties were varied depending on the type of CNCs and their concentration. Tensile strength (TS) increased by 45.7%, 25.2%, and 42.6%, and the water vapor permeability (WVP) decreased by 26.3%, 19.1%, and 20.4% after forming composite with 5wt% of CNCs obtained from RS, WS, and BS, respectively.

Determination of the DS distribution of non-degraded sodium carboxymethyl cellulose by gradient chromatography.

Two series of sodium carboxymethyl celluloses (NaCMCs) derived from microcrystalline cellulose (Avicel samples, DP~160) and cotton linters (BWL samples, DP~1400) with average degrees of substitution in the range DS=0.45-1.55 were analyzed by gradient liquid adsorption chromatography (gradient LAC) in order to determine their chemical composition distributions (DS distributions or 1st order heterogeneities). Clear separations of samples having different average DS values were achieved for both sample series, allowing determination of the DS distributions of the samples. A slight molar mass influence on the eluent composition at elution was observed. From the DS distributions the DS standard deviations were calculated and taken as a measure for the extent of chemical heterogeneity of the single samples. While no noticeable dependence of the chemical heterogeneity on average DS was observed for Avicels, the heterogeneity decreases with increasing average DS for BWLs.

Purification and characterization of an alkaline cellulase produced by Bacillus subtilis (AS3).

An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and ß-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6-10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.

Physiological studies on carboxymethyl cellulase formation by Aspergillus terreus DSM 826

Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30ºC for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.

Pretreatment of wastewater containing a mixture of organic pollutants obtained from a CC2 plant by coagulation.

Coagulation is one of the most important physicochemical treatment steps in industrial wastewater to reduce the suspended and colloidal materials responsible for colour and turbidity of the wastewater. The manufacturing plant of N,N'-Dichloro bis (2,4,6-trichlorophenyl) urea (CC2) produces wastewater containing pyridine, acetic acid and diphenyl urea (DPU). The wastewater also contains lot of suspended solids like CC2 and various poly-aromatic compounds. In our present investigation, our basic aim was to find an effective coagulation process for the pretreatment of wastewater discharged from the CC2 plant. Studies were conducted to find out a suitable and effective coagulant for pretreatment of this wastewater. Various coagulating agents such as alum, ferric chloride, sodium carboxymethyl cellulose (Na-CMC) were used. Alum was found to be the most effective coagulant. Coagulation of the wastewater resulted in the total suspended solids (TSS) removal in the range of 92-94% and chemical oxygen demand (COD) removal in the range of 59 to 65% at a dose of 500 mg L(-1) of alum at a pH>or=7.0. After coagulation the concentration of pyridine in wastewater was found to be reduced by 10.0% and that of DPU 40-45% with a dosage of 500 mg L(-1) alum.

Carboxymethylcellulose from totally chlorine-free-bleached milox pulps.

High purity cellulose pulp was obtained from Eucalyptus globulus wood by using an environmentally friendly delignification technique (Milox pulping) and subsequent bleaching by totally chlorine free technology. The pulp obtained under optimised experimental conditions was used for the manufacture of carboxymethylcellulose in a heterogeneous medium. By means of an experimental design, the effects of selected operational variables on the composition and chemical properties of reaction products from the carboxymethylation reaction were assessed for optimisation purposes. The distribution of the different carboxymethylglucose mole fractions (un-, mono-, di- and tri-substituted) was determined and compared with theoretical predictions. The maximum average degree of substitution (1.26) was determined at a NaOH/cellulose mole ratio of 4.8 and at a MCA/cellulose mole ratio of 2.0.

Carboxymethylcellulase from Lenzites saepiaria, a brown-rotter.

Three wood-rotting fungi namely, Lenzites saepiaria, Polyporus xeranticus and Trametes gibbosa, were screened as cellulose-degraders from 20 different genera of both brown and white-rotters of Polyporaceae on the basis of their potential to degrade carboxymethylcellulose. The utilization of different carbon sources in the growth medium was studied with these fungi for identification of enzymes involved in saccharification. Carboxymethylcellulase and beta-glucosidase were identified as the two major enzymes involved in this process. Extracellular carboxymethylcellulase from L. saepiaria was purified to homogeneity and the enzyme partially characterized.

Purification and properties of a carboxymethylcellulase from phytopathogenic fungus Macrophomina phaseolina.

From the culture filtrate of Macrophomina phaseolina, two forms of carboxymethylcellulase were separated by ion-exchange chromatography and designated as CMCase-I and CMCase-II. CMCase-I was purified following a four-step procedure involving gel filtration on Sephadex G-75, Con-A Sepharose 4B affinity chromatography, fast protein liquid chromatography on mono Q anion-exchanger and on Superose 12 gel filtration. The final preparation was homogeneous by SDS-PAGE, isoelectric focussing in thin layers of polyacrylamide gels and immunoelectrophoresis. The enzyme showed optimum activity at pH 5.5 and 65 degrees C, was stable to heating at 65 degrees C for 10 min, and retained 31% of original activity after heating at 80 degrees C for 10 min. The molecular weight of the enzyme was 3.5 x 10(4) Da. A Km of 0.25 mg/ml was determined using carboxymethyl-cellulose as the substrate.

Purification of carboxymethylcellulose decreases toxicity of poly ICLC in mice.

The interferon inducer poly ICLC has toxic side effects; the carboxymethylcellulose (CMC) component is a possible source of this toxicity. We have purified CMC by ethanol extraction, and found that poly ICLC made with purified CMC is not significantly different in its effectiveness as an interferon inducer in mice than poly ICLC made with unpurified CMC, but is less toxic.