Purification and the effect of manganese ions on the activity of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea

Carboxymethylcellulases (CMCases) fromAspergillus niger andCellulomonas biazotea were purified by a combination of ammonium sulfate precipitation, anion-exchange and gel-filtration chromatography with a 12- and 9-fold increase in the purification factor. The native and subunit molar mass of CMCase fromA. niger were 40 and 25–57 kDa, respectively, while those fromC. biazotea were 23 and 20–30 kDa, respectively. Low concentrations of Mn²⁺ activated the enzymes from both organisms (mixed activation) with apparent activation constants of 0.80 and 0.45 mmol/L of CMCases fromA. niger andC. biazotea, respectively, while at higher CMC concentrations Mn²⁺ inhibited the enzymes (mixed and partial uncompetitive inhibition). The reason for this complex behavior is that more than one Mn²⁺ bind to the same enzyme form with the apparent average inhibition constants of 2.7 and 1.3 mmol/L for CMCases fromA. niger andC. biazotea, respectively.     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.     

Cloning of endoglucanase genes fromCellulomonas biazotea intoE. coli andS. cerevisiae using shuttle vector YEp24

We constructed aSmaI genomic library ofCellulomonas biazotea DNA inE. coli and in theS. cerevisiae shuttle vector, YEP 24. Three clone were identified that conferred the ability forE. coli orS. cerevisiae transformants to produce carboxymethylcellulase (CMCase). Cells transformed with these clones were compared with one another and with nontransformed cells for hyper-production of CMCase.In vivo andin vitro studies indicated that the CMCase genes were fully expressed and the enzyme activity was located extracellularly. The optimum pH and temperature for the CMCase thus cloned were pH 7 and 50°C, respectively, as was the case for the donor.     

Enhanced production of cellulases byCellulomonas strains grown on different cellulosic residues

Cellulomonas strains consumed commercial cellulose, cellulosic residues, xylan, cellobiose and carboxymethyl cellulose (CMC) as carbon sources in liquid culture, the growth being the most on cellobiose medium. All three components of the cellulase complex ofCellulomonas were produced when the organisms utilized all substrates as sole carbon and energy sources. The filter-paper cellulase (FPase) and endo-glucanase (CMCase) activities were higher in media containing α-cellulose and cellulosic residues than in media containing CMC, cellobiose, and xylan. Cell-free supernatants of all organisms exhibited greater CMC hydrolyzing activity than filter paper and β-glucoside hydrolyzing activities. All strains synthesized β-glucosidase maximally on cellobiose followed by commercial cellulose and cellulosic residues.C. biazotea produced the highest FPase and CMCase activity during growth on α-cellulose. It was followed byC. flavigena, C. cellasea, andC. fimi. Endo-glucanase and FPase from all organisms were secreted into the medium; 10–13 % became adsorbed on the surface of the insoluble substrates and could be successfully eluted using Tween 80. β-Glucosidase was located in cell extracts from all organisms.C. biazotea produced FPase and β-glucosidase activities several-fold greater than those produced by many other strains ofCellulomonas and some other cellulolytic bacteria and fungi. These studies were supported byPakistan Atomic Energy Commission. Some chemicals were purchased from funds allocated byUnited States Agency for International Development, Washington (DC, USA), under PSTC proposal 6.163.     

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 β-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.     

Activity and thermostability of carboxymethylcellulase from Aspergillus niger is strongly influenced by noncovalently attached polysaccharides

Removal of non-covalently attached polysaccharides from carboxymethylcellulase (CMCase) of Aspergillus niger improved its activity but decreased its thermostability and protease resistance. The activation energy profile of the hydrolysis of carboxymethylcellulose (CMC) was triphasic with increasing values of 17,-55 and-562 kJ/mol for polysaccharide-free and 19, -21 and -207 kJ/mol for polysaccharide-complexed CMCase. The specificity constant (V_max/K_m) of polysaccharide-free CMCase was 1.41 compared to polysaccharide-complexed CMCase which was only 0.68. The polysaccharide free CMCase had lower thermostability (melting point = 82°C) and higher protease susceptibility compared to polysaccharide-complexed CMCase (melting point>100°C).The authors are with the National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan;     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

Production and characterization of extracellular protease of mutant Aspergillus niger AB100 grown on fish scale

Fish scale, the chief waste material of fish processing industries was processed and tested for production of extracellular protease by mutant Aspergillus niger AB₁₀₀. Protease production by A. niger AB₁₀₀ was greatly enhanced in presence of processed fish scale powder. Where as among the three complex nutrients tested, soya bean meal shows maximum stimulatory effect over protease production (2,776 μmol/ml/min) when used in combination with glucose (5% w/v) and urea (2.5% w/v). The protease was optimally active at pH 7.0, retaining more than 60% of its activity in the pH range of 5–9. The enzyme was found to be most active at 50°C and stable at 30°C for 1 h. Purification of enzyme by CM-Cellulose and SDS-PAGE resulted in about 26-fold increase in the specific activity of the enzyme with a molecular weight of 30.9 kDa. HPLC study shows the purity of the enzyme as 75.92%. By the activating effect of divalent cations (Fe²⁺, Zn²⁺, Mn²⁺, Ca²⁺and Mg²⁺) and inhibiting effect of chelating agent (EDTA) and Hg²⁺, the enzyme was found to be a metalloprotease.     

Enzymatic hydrolysis of cotton fibers in supercritical CO2

A study was carried out on the application of supercritical fluid to the hydrolysis of boll fibers of cotton (cultivar Tashkent-6 ofGossypium hirsutum L.) by cellulase enzymes fromTrichoderma viride, Trichoderma reesei andAspergillus niger. Conditions of the enzymatic process were optimized. The stabilities of cellulase enzymes were sustained, at the pressure of up to 160 atm for 48 hours at 50°C in supercritical carbon dioxide.     

An efficient method for the preparation of protoplasts fromTrichoderma viride

Conditions for an efficient high-yield procedure for the preparation of protoplasts fromTrichoderma viride have been determined. The optimum yield of protoplasts was obtained using 15–18-h-old unbranched mycelia, 0.7 mol/L KCl in phosphate buffer (pH 6), and 5 % (W/V) of lyophilized snail gut-juice enzyme. The conversion of mycelia to protoplasts was complete within 40–60 min incubation at 30 °C.     

Purification and characterization of cellulase fromBacillus thermoalcaliphilus isolated from a termite mound

An extracellular carboxymethyl cellulase (CMCase) was purified to homogeneity fromBacillus thermoalcaliphilus sp. nov. The cellulase was composed of a single subunit with molar mass of 46 kDa. The apparentK _m was at 3.5 mg cellulose per mL. Its optimum pH was 8.5, it was most stable at pH 8.5–9.5 but 50% of enzyme activity was present after 30 min at pH 11.0. The activity was highest at 70°C.     

Isolation of fluconazole-tolerant micromycetes onto different cultivation media

On studying the dependence of quality and/or quantity of environmental fluconazole-tolerant micromycetes on culture mediumin vitro, the highest number of isolates was obtained in Sabouraud agar (SA) without fluconazole (59 CFU/mg of soil sample at 25°C and 31 CFU/mg at 37°C). In the case of SA, fluconazole inhibited the growth of micromycetes to 83%. Significant differences were found between the average concentration of CFU/mg for six cultivation media without and with fluconazole (70 mg/L) at 25 and 37°C. The genusAspergillus (A. flavus, A. fumigatus, aspergilli fromA. glaucus andA. niger group,A. nidulans, A. versicolor, A. ustus), Scopulariopsis brevicaulis andPenicillium sp. were the most common isolates. Sabouraud agar combined with malt extract agar, dichloran-18% glycerol agar and potato-dextrose agar were the most suitable for isolation of fluconazole-tolerant fungi.     

Production and Purification of Thermostable Cellulases from Humicola insolens YH-8

Humicola insolens YH-8, a thermophilic fungus isolated from manure and compost heaps, produced a significant amount of thermostable cellulases in cultures on wheat bran medium (50°C, 4 days). The mold bran extract hydrolyzed Avicel, CMC and newsprint at 90%, 45% and 35%, respectively, to glucose. Then, Avicelase and CMCase were purified from the culture extract by adsorption onto Avicel, heat and acid treatment and consecutive column chromatographies to a homogeneous state on polyacrylamide gel disc electrophoresis. The purified cellulases, especially CMCase, was found highly thermostable. The optimal temperature of both enzymes was 50°C. Avicelase was stable after heating at 65°C for 5 mm and CMCase retained 45% of the original activity after heating at 95°C for 5 min.     

Comparative studies on glucoamylases from three fungal sources

Five commercial preparations of glucoamylases (three fromAspergillus niger, one each fromAspergillus foetidus andAspergillus candidus) were purified by ultrafiltration, Sepharose-gel filtration and DEAE-sephadex chromatography. Two forms of the enzyme, namely glucoamylase I and glucoamylase II were obtained from the fungi except from one strain ofA. Niger. All the enzymes appeared homogeneous by electrophoresis and ultracentrifugation. The specific activities varied between 85 and 142 units. The pH and temperature optima were between 4 and 5, and 60‡C respectively. The molecular weight as determined by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis ranged from 75,000 to 79,000 for glucoamylase I and 60,000 to 72,000 for glucoamylase II. OnlyA. niger glucoamylases contained phenylalanine at the N-terminal end. The amino acid composition of the enzymes was generally similar. However,A. niger andA. foetidus glucoamylases, in contrast toA. candidus enzymes, contained greater percentage of acidic than of basic amino acids. The enzymes contained 15 to 30% carbohydrate and 49 to 57 residues of monosaccharides per mol.A. niger enzymes contained mannose, glucose, galactose, xylose and glucosamine but theA. candidus enzyme lacked xylose and glucose and only xylose was absent inA, foetidus enzymes. Majority of the carbohydrate moieties were O-glycosidically linked through mannose to the hydroxyl groups of seline and threonine of the polypeptide chain.     

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn²⁺, Hg²⁺, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K _m and V _max of the enzyme for CMC were 3.4 mg ml^–1 and 44.7 mol min^–1 (mg protein)^–1, respectively.     

Kinetic analysis of the active site of an intracellular β-glucosidase fromCellulomonas biazotea

Purified β-glucosidase fromCellulomonas biazotea had an apparentK _m andV for 2-nitrophenyl β-d-glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of β-glucosidase was 65 kJ/mol. The inhibition by Mn²⁺ vs. oNPG of parental β-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 μmol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK _a’s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of β-glucosidase decreased in the presence of 10 % ethanol. The half-life of β-glucosidase in 1.75 mol/L urea at 35 °C was 145 min, as determined by 0–9 mol/L transverse urea gradient-PAGE. This work was financed in part by a grant made by theUS Agency for International Development under PSTC proposal 6-163,USAID grant no. 9365542-G-00-89-42-00, and PAEC.     

Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water-miscible organic mixtures

We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.     

Pre and post cloning characterization of a β-1,4-endoglucanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Consistent with its precloning characterization from the cellulolytic Bacillus sp., β-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60°C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70°C and over a pH range of 6.0–8.0. The K _m and V _max values for CMCase activity of the enzyme were 4.1 mg/ml and 25 μmole/ml min⁻¹, respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the actual one.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.