CMCase Production Research Articles

Improved Production of Thermostable Cellulase from Thermoascus aurantiacus RCKK by Fermentation Bioprocessing and Its Application in the Hydrolysis of Office Waste Paper, Algal Pulp, and Biologically Treated Wheat Straw.

Thermostable cellulases have wide variety of applications and distinctive advantages, but their low titer becomes the hurdle in their commercialization. In the present work, an assessment of optimum levels of significant factors (temperature, moisture ratio, inoculum size, and ammonium sulfate) and the effect of their interactions on production of thermostable CMCase, FPase, and β-glucosidase by Thermoascus aurantiacus RCKK under solid-state fermentation (SSF) was carried out using central composite design (CCD) of response surface methodology (RSM). The study revealed 33, 13, and 8% improvement in FPase, CMCase, and β-glucosidase production, respectively. Moreover, crude cellulase from T. aurantiacus RCKK efficiently hydrolyzed office waste paper, algal pulp (Gracillaria verulosa), and biologically treated wheat straw at 60°C with sugar release of about 830mg/ml, 285mg/g, and 260mg/g of the substrate, respectively. The thermostable enzyme from T. aurantiacus RCKK holds potential to be used in biofuel industry.

Construction of a recombinant Escherichia coli JM109/A-68 for production of carboxymethylcellulase and comparison of its production with its wild type, Bacillus velezensis A-68 in a pilot-scale bioreactor

A gene encoding carboxymethylcellulase (CMCase) of Bacillus velezensis A-68 had been cloned in Escherichia coli JM109. Based on productivity and economic aspect, rice bran and ammonium chloride were chosen to be optimal carbon and nitrogen sources for production of CMCase by E. coli JM109/A-68. The optimal conditions for rice bran, ammonium chloride, and initial pH of medium for production of CMCase were established by the response surface methodology (RSM). The concentrations of four salts in the medium, K2HPO4, NaCl, MgSO4·7H2O, and (NH4)2SO4, for production of CMCase also were optimized. The optimal temperatures for cell growth and production of CMCase were 37°C. The maximal production of CMCase by E. coli JM109/A-68 was 880.2 U/mL, which was 10.5 time higher than its wild type, B. velezensis A-68. The production of CMCase by E. coli JM109/A-68 was compared with that by B. velezensis A-68 in a 100 L pilot-scale bioreactor under the optimized conditions. The production of CMCase by E. coli JM109/A-68 was found to be the mixed-growth associated unlike the growthassociated production of CMCase by B. velezensis A-68.

Novel Bacillus subtilis IND19 cell factory for the simultaneous production of carboxy methyl cellulase and protease using cow dung substrate in solid-substrate fermentation.

BackgroundHydrolytic enzymes, such as cellulases and proteases, have various applications, including bioethanol production, extraction of fruit and vegetable juice, detergent formulation, and leather processing. Solid-substrate fermentation has been an emerging method to utilize low-cost agricultural residues for the production of these enzymes. Although the production of carboxy methyl cellulase (CMCase) and protease in solid state fermentation (SSF) have been studied extensively, research investigating multienzyme production in a single fermentation process is limited. The production of multienzymes from a single fermentation system could reduce the overall production cost of enzymes. In order to achieve enhanced production of enzymes, the response surface methodology (RSM) was applied.ResultsBacillus subtilis IND19 utilized cow dung substrates for the production of CMCase and protease. A central composite design and a RSM were used to determine the optimal concentrations of peptone, NaH2PO4, and medium pH. Maximum productions of CMCase and protease were observed at 0.9 % peptone, 0.78 % NaH2PO4, and medium pH of 8.41, and 1 % peptone, 0.72 % NaH2PO4, and medium pH of 8.11, respectively. Under the optimized conditions, the experimental yield of CMCase and protease reached 473.01 and 4643 U/g, which were notably close to the predicted response (485.05 and 4710 U/g). These findings corresponded to an overall increase of 2.1- and 2.5-fold in CMCase and protease productions, respectively.ConclusionsUtilization of cow dung for the production of enzymes is critical to producing multienzymes in a single fermentation step. Cow dung is available in large quantity throughout the year. This report is the first to describe simultaneous production of CMCase and protease using cow dung. This substrate could be directly used as the culture medium without any pretreatment for the production of these enzymes at an industrial scale.

English

CMCase production by immobilized Aspergillus niger in submerged fermentation was studied .The isolated culture of Aspergillus niger was immob ilized in calcium alginate beads. Studies were carried out on different parameters li ke alginate concentration, incubation time, bead inoculum, CaCl2 concentration and curing time, which affect the pr oductivity and stability of the immobilized system. The maximum CM Case activity of 0.37 IU/ml was achieved with 4% alginate concentration, 48h of inc ubation time, 40 beads/flask of inoculum, 0.2M CaCl2 concentration and 60 min of curing time.

Optimization of cold-active CMCase production by psychrotrophic Sphingomonas sp. FLX-7 from the cold region of China

Organic solid waste consists of 70 % biodegradables, such as cellulose, which is unable to be degraded quickly and efficiently in the cold region of China. Psychrotrophic cellulose-degrading bacteria play an important role in cellulose degradation at low temperatures. In this study, a psychrotrophic cellulose-degrading FLX-7 strain with high carboxymethyl cellulase (CMCase) activity was isolated from the soil of a solid waste landfill under the environmental temperature of −10 °C. The FLX-7 strain was identified as Sphingobacterium sp. based on morphology and physiological and biochemical properties as well as 16S rRNA gene sequence analysis. To improve the cold-active CMCase production of FLX-7, fermentation conditions were optimized using response surface methodology based on a Box–Behnken design. The maximum CMCase activity (16.95 U/mL) was obtained at optimal fermentation conditions (inoculum size 2.1 %, temperature 10.4 °C, incubation time 73.5 h, and pH 7.2). The optimum pH and temperature for enzymatic reaction of cold-active CMCase were 5.0 and 25 °C, respectively. The cold-active CMCase was stable at a wide pH range of 5.0–9.0 and temperature range of 10–30 °C. The FLX-7 strain could be distributed via a psychrotrophic cellulose-degrading strain library and applied more broadly to organic solid waste composting in cold regions.

Determination of endoglucanase activity of paper decaying fungi from an old library at the ancient Medina of Fez

Paper from an ancient library of the cultural city of Fez (Morocco) is exposed to rapid deterioration by variety of microorganisms, especially cellulolytic fungi. For this, ten isolates fungi previously isolated from historical biodeteriorated paper were screened for their ability to produce endoglucanase (CMCase), amylase, polygalacturonase and ligninase enzymes. The CMCase activity of cellulolytic strains was essayed in liquid media at 25°C for 10 days. Influence of temperature and pH were assessed for the production of CMCase by all the fungus isolated from decaying paper. The research findings from the present study demonstrate that all the tested isolates had cellulase, amylase, pectinase and ligninase activities. It was found that Mucor racemosus PF15, Aspergillus niger, and Aspergillus oryzae exhibited the maximum endoglucanase activity in liquid medium (0.256, 0.236, and 0.216 UI/mL in descending order) for six days. Temperature profiling revealed optimum endoglucanase activity at 25 and 30°C. Maximum activity was observed at pH 5 and pH 6.

Enhanced alkaline cellulases production by the thermohalophilic Aspergillus terreus AUMC 10138 mutated by physical and chemical mutagens using corn stover as substrate.

A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and β-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0–11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for β-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and β-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.

Characterization of a thermostable endoglucanase produced by Isoptericola variabilis sp. IDAH9

This study aimed to isolate and evaluate the cellulase activity of cellulolytic bacteria in hot springs of Dehloran, Ilam province, Iran. Water and sludge samples were collected from the hot springs and the bacterial enrichment was performed in a medium containing rice barn and carboxymethyl cellulose (CMC). The cultures were incubated at 50 °C in aerobic conditions. The bacteria were isolated on CMC agar (1%) medium. Cellulase assay of the isolates was measured by the evaluation of endoglucanase enzyme activity, which is also called as carboxymethyl cellulase (CMCase). The isolated thermotolerant bacteria were then identified and optimized for the production of CMCase. Moreover, stabilizing elements of the enzyme were identified with in silico approach. The chosen isolate was identified as Isoptericola variabilis sp. IDAH9. The identified strain produced the most thermostable CMCase at a concentration of 5.6 g/L of ammonium sulfate, 9 g/L CMCase or 12 g/L rice bran, 0/6% Tween-80, and 0.2% sucrose. The produced enzyme showed 80% of the residual activity after 1 h of incubation at 65 °C. In silico data indicated that the remaining residual activity was due to the redundant stabilizing elements in the protein structure. Consequently, I. variabilis can be isolated from the extreme environment and has a thermostable endoglucanase which may be used for various applications after studying them.

Solid-substrate bioprocessing of cow dung for the production of carboxymethyl cellulase by Bacillus halodurans IND18

The production of carboxymethyl cellulase (CMCase) by Bacillus halodurans IND18 under solid substrate fermentation (SSF) using cow dung was optimized through two level full factorial design and second order response surface methodology (RSM). The central composite design (CCD) was employed to optimize the vital fermentation parameters, such as pH of the substrate, concentration of nitrogen source (peptone) and ion (sodium dihydrogen phosphate) sources in medium for achieving higher enzyme production. The optimum medium composition was found to be 1.46% (w/w) peptone, 0.095% (w/w) sodium dihydrogen phosphate and pH 8.0. The model prediction of 4210IU/g enzyme activity at optimum conditions was verified experimentally as 4140IU/g. The enzyme was active over a broad temperature range (40–60±1°C) and pH (7.0–9.0) with maximal activity at 60±1°C and pH 8.0. This study demonstrated the potential of cow dung as novel substrate for CMCase production.

A carboxymethyl cellulase from a marine yeast (Aureobasidium pullulans 98): Its purification, characterization, gene cloning and carboxymethyl cellulose digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase (CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U (mg protein)−1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40°C, much lower than that of the CMCases from other fungi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity (pH 5.0–6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. K m and V max values of the purified enzyme were 4.7 mg mL−1 and 0.57 µmol L−1 min−1 (mg protein)−1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose (CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp (EU978473). The protein deduced contained the conserved domain of cellulase superfamily (glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

Saccharification and hydrolytic enzyme production of alkali pre-treated wheat bran by Trichoderma virens under solid state fermentation.

BackgroundIn continuation of our previously interest in the saccharification of agriculture wastes by Bacillus megatherium in solid state fermentation (SSF), we wish to report an investigation and comparative evaluation among Trichoderma sp. for the saccharification of four alkali-pretreated agricultural residues and production of hydrolytic enzymes, carboxymethyl cellulase (CMCase), filter paperase (FPase), pectinase (PGase) and xylanase (Xylase) in SSF. The optimization of the physiological conditions of production of hydrolytic enzymes and saccharification content from Trichoderma virens using alkali-pretreated wheat bran was the last goal.MethodsThe physico-chemical parameters of SSF include incubation time, incubation temperature, moisture content of the substrate, incubation pH, supplementation with carbon and nitrogen sources were optimized.ResultsSaccharification of different solid state fermentation sources wheat bran, date's seeds, grass and palm leaves, were tested for the production of fermentable sugar by Trichoderma sp. The maximum production of hydrolytic enzymes CMCase, FPase, PGase and Xylase and saccharification content were obtained on wheat bran. Time course, moisture content, optimum temperature, optimum pH, supplementation with carbon and nitrogen sources were optimized to achieve the maximum production of the hydrolytic enzymes, protein and total carbohydrate of T. virens using alkali pre-treated wheat bran. The maximum production of CMCase, FPase, PGase, Xylase, protein and carbohydrate content was recorded at 72 h of incubation, 50-70 % moisture, temperature 25-35 °C and pH 5. The influence of supplementary carbon and nitrogen sources was studied. While lactose and sucrose enhanced the activity of PGase from 79.2 to 582.9 and 632.6 U/g, starch inhibited all other enzymes. This was confirmed by maximum saccharification content. Among the nitrogen sources, yeast extract and urea enhanced the saccharification content and CMCase, PGase and Xylase.ConclusionsThe results of this study indicated that alkali pre-treated wheat bran was a better substrate for saccharification and production of hydrolytic enzymes CMCase, FPase, PGase and xylase by T. virens compared to other alkali-pretreated agricultural residues tested.

PRODUCTION AND CHARACTERIZATION OF THERMOPHILIC CARBOXYMETHYL CELLULASE SYNTHESIZED BY Bacillus sp. GROWING ON SUGARCANE BAGASSE IN SUBMERGED FERMENTATION

The production and characterization of cellulase from thermophilic strain Bacillus sp. C1AC5507 was studied. For enzyme production, sugarcane bagasse was used as carbon source. The produced carboxymethyl cellulase (CMCase) had a molecular weight around 55 kDa and its activity varied between 0.14 and 0.37 IU mL-1 in conditions predicted by Response Surface Methodology. The optimum temperature and pH for the CMCase production were 70 °C and 7.0, respectively. The enzyme activity was inhibited mostly by Cu+2 and activated mostly by Co+2, Mn2+, Ca+2 and Fe+3. Our findings provide a contribution to the use of natural wastes such as sugarcane bagasse as substrate for growth and production of thermophilic CMCase. Further optimization to increase the production of cellulase enables the use in industrial applications.

Application of Statistical Approach for Optimizing CMCase Production by Bacillus tequilensis S28 Strain via Submerged Fermentation Using Wheat Bran as Carbon Source

Application of Statistical Approach for Optimizing CMCase Production by Bacillus tequilensis S28 Strain via Submerged Fermentation Using Wheat Bran as Carbon Source

Optimization of pretreatment and fermentation conditions for production of extracellular cellulase complex using sugarcane bagasse.

Sugarcane bagasse (SCB), a lignocellulosic byproduct of juice extraction from sugarcane, is rich in cellulose (40-42%). This could be used as a substrate for the production of cellulase complex. Fermentation conditions were optimized for production of cellulase complex (CMCase, Cellulobiase and FPase) by wild type Trichoderma sp. using sugarcane bagasse as sole carbon source. Alkaline treatment (2% NaOH) of bagasse (AlSCB) was found suitable for the production of reducing sugar over the acidic pretreatment method. After 5 days of incubation period, 5% substrate concentration at pH 5.0 and 400C resulted in maximum production of CMCase (0.622 U), while maximum (3.388 U) production of cellulobiase was obtained at 300C. The CMCase was precipitated and purified to the extent of 59.06 fold by affinity chromatography with 49.09% recovery. On 12% SDS-PAGE, a single band corresponding to 33 kDa was observed. The Km and Vmax for CMCase from Trichoderma was found 507.04 mg/ml and 65.32 mM/min, respectively. The enzyme exhibited maximum activity at 300C at pH-5.0 (0.363 U) and was stable over range of 20-60°C and pH 5.0-7.5.

Optimization of medium composition for enhanced cellulase production by mutant Penicillium brasilianum KUEB15 using statistical method

Penicillium brasilianum KUEB15 was utilized for cellulase production and medium optimization was investigated. The optimal compositions of the variables for maximum CMCase production by central composite rotatable design were 12.39g/L of corn stover, 0.48g/L of urea, and 6.12g/L of ammonium sulfate. The predicted CMCase activity by the model was 1.207Unit/ml, and the observed CMCase activity which was produced in optimal media was 1.180Unit/ml. The activity of CMCase from P. brasilianum KUEB15, which was fermented in optimal media by RSM, was found to be 1.61-fold increased than that in the Mandel's medium (0.731Unit/ml).

Effect of salinity on biology of Saprolegnia ferax and Pythium ultimum

<i>Saprolegnia ferax</i> and <i>Pythium ultimum</i> were cultivated on the leaves of <i>Bromus catharicus</i>. Different concentrations of NaCl were used in the medium. <i>S. ferax</i> appears to be more tolerant to salinity than <i>P. ultimum</i> in CMC-ase production and the ability for growth.

Enhanced production of carboxymethylcellulase of Bacillus subtilis subsp. subtilis A-53 by a recombinant Escherichia coli JM109/A-53 with pH and temperature shifts

Effect of shifts in pH of the medium and temperature on cell growth and production of carboxymethylcellulase (CMCase) by a recombinant Escherichia coli JM109/A-53 was investigated. Lower initial pH (pH 7.0) of the medium and higher temperature (40 °C) stimulated cell growth of E. coli JM109/A-53, whereas higher initial pH (pH 8.0) and lower temperature (35 °C) enhanced production of CMCase. The maximal production of CMCase was obtained when initial pH of 7.0 and temperature of 40 °C were simultaneously changed to 8.0 and 35 °C after 36 h. The production of CMCase with simple shifts in pH and temperature was 783.1 U/mL, which was 2.13 times higher than that under optimal conditions for cell growth. This indicated that a simple process with shifts in pH and temperature could be economically applied for enhanced production of CMCase by E. coli JM109/A-53 with industrial scales.

Enhanced Production of carboxymethylcellulase by a marine bacterium, Bacillus velezensis A-68, by using rice hulls in pilot-scale bioreactor under optimized conditions for dissolved oxygen.

The optimal conditions for the production of carboxymethylcellulase (CMCase) by Bacillus velezensis A-68 at a flask scale have been previously reported. In this study, the parameters involved in dissolved oxygen in 7 and 100 L bioreactors were optimized for the pilot-scale production of CMCase. The optimal agitation speed and aeration rate for cell growth of B. velezensis A-68 were 323 rpm and 1.46 vvm in a 7 L bioreactor, whereas those for the production of CMCase were 380 rpm and 0.54 vvm, respectively. The analysis of variance (ANOVA) implied that the highly significant factor for cell growth was the aeration rate, whereas that for the production of CMCase was the agitation speed. The optimal inner pressures for cell growth and the production of CMCase by B. velezensis A-68 in a 100 L bioreactor were 0.00 and 0.04 MPa, respectively. The maximal production of CMCase in a 100 L bioreactor under optimized conditions using rice hulls was 108.1 U/ml, which was 1.8 times higher than that at a flask scale under previously optimized conditions.

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.

Simplification and optimization of media ingredients for enhanced production of CMCase by newly isolatedBacillussubtilisNA15

Cellulose degrading bacterium was isolated from composting biomass and identified as Bacillus subtilis on the basis of phenotypic fingerprint using GEN III MicroPlateTM and 16S ribosomal RNA (rRNA) gene sequence analysis. 16S rRNA gene sequence similarity analysis showed 99% similarity with Bacillus subtilis. The screening of 11 nutrients was done using Plackett-Burman design to explicate the parameters that significantly influence the carboxy methyl cellulase (CMCase) production. Maximum CMCase production of 0.47 U/mL was obtained using response surface methodology with carboxy methyl cellulose (CMC): 18 g/L, peptone: 5 g/L, yeast extract: 5 g/L and MnCl2: 0.5 g/L. The model predicted the maximum CMCase activity (0.45 U/mL), which was in good agreement with the experimental value of 0.47 U/mL showing sevenfold increase as compared to unoptimized medium. Presence of MnCl2 in the medium significantly enhanced the CMCase production. © 2014 American Institute of Chemical Engineers Environ Prog, 34: 533–541, 2015