Stable cellulases

The hydrogen bonding network analysis of softwood lignin is relevant to designing novel technologies to overcome the recalcitrance of plant biomass in the industrial deconstruction and manufacturing of ligninbased carbon fibers. In this work, we examine, by atomistic simulations, the hydrogen bonding network in guaiacyl-rich lignin and guaiacyl-type lignin over a wide range of temperatures. We determine the formation of stable water-bridged dimeric complexes by the interaction of phenolic and aliphatic hydroxyl groups and π−π stacking between phenol rings, causing a slow dynamic of lignin with temperature. The interaction strength between water oxygen and hydrogen of hydroxyl groups in these lignin complexes is established. The formation of the ice crystal structure at low temperatures in the complexes explains the anti-plasticizing action of water, increasing the Young's modulus (E) of lignin systems at high hydration. Glass-transition temperature and E were successfully predicted by CHARMM force field for lignin at different hydrations.

The effect of initial solid and moisture contents, temperature and time of incubation on the production of polygalacturonase, phytase, acid phosphatase, xylanase and β-glucanase by Aspergillus niger 377-4 during solid state fermentation was studied. Parameters of enzyme synthesis were optimized using statistical experimental designs. It was shown that the capacity of strain to synthesize the aforementioned enzymes could be modified within a wide range by culture parameters selection. The optimal polygalacturonase production efficiency was achieved with the initial medium mass of 19.9 g and humidity of 59.9%, after 77.7 h of incubation at 28.9 o C. The best combination of culture parameters for phytase synthesis was: initial medium mass 19.9 g, moistures 50%, temperature 33 o C and incubation time 83.9 h. The highest activity of acid phosphatase was obtained after 81.3 h of incubation at 27 o C, with initial substrate mass of 17.8 g and moistness content of 60%. The initial solid and...

The effects of glucose, crystalline cellulose and sawdust of Mitragyna cilata on the growth and cellulase production, inferred from cellulase (EC 3. 2. 1. 4) activity, of Penicillium chrysogenum PCL501 was determined. Glucose-containing media gave the highest mycelia weight of 1.78 mg mL -1 in 120 h of incubation. This is about 3.5 -4.5 times the maximum weights of 0.51 and 0.40 mg mL -1 respectively obtained from the cultures containing cellulose and sawdust. The cultures containing crystalline cellulose and sawdust produced extracellular protein with cellulase (EC 3. 2. 1. 4) activity whereas glucose-containing cultures yielded very low protein and no significant cellulase activity. Maximum protein content of 0.02, 0.13 and 0.46 mg mL -1 respectively were obtained from the cultures containing glucose, cellulose and sawdust. Peak cellulase activity values of 100.0 and 92.2 Units L -1 respectively were obtained for the cultures containing cellulose and sawdust. There is a correlation between the protein released and cellulase activity of the culture filtrates. P. chrysogenum PCL501 produces extracellular proteins with significant cellulase activity in media containing cellulose and sawdust but not in glucose-containing medium. Sawdust is indicated as a good inducer of cellulase activity in the organism. The waste cellulosic material can be used as low-cost carbon source for commercial cellulase production.

Cellulases can be applied as macerating and peeling enzymes in the orange juice processing industry. In this work, indigenous cellulose-degrading microorganisms were isolated from orange juice processing waste through successive enrichment procedures using carboxymethyl cellulose (CMC) as the sole carbon source. A total of 24 microbial isolates were screened for their ability to grow in CMC liquid medium, resulting in the selection of seven isolates. The latter were further assessed by determining their endo-1,4-β-d-glucanase, exo-1,4-β-d-glucanase, and β-1,4-d-glucosidase activities, of which their respective activities were as high as 3.89, 10.67, and 10.69 U/mg protein. All cellulose-degraders selected belonged to the genus Paenibacillus, although to distinct operational taxonomic units related to P. xylanexedens, P. tundrae, and P. pabuli (operational taxonomic unit—OTU#1) and to P. wynnii, P. odorifer, and P. donghaensis (OTU#2) spectrum. Regarding the cellulase activities of t...

Citric acid is one among the most adaptable organic acid and generally used in different industries including food, cosmetics, pharmacy, beverages and many others. Apart from its consumption as a food additive, citric acid is also considered as a vital component of various pharmaceuticals, synthetic detergents, cosmetics, and many other value-added products. It is predominantly produced by microbial fermentation of Aspergillus niger. The study was conduct to explore the potential of Aspergillus niger AsnO and AsnC (isolated from Orange and Carrot) for citric acid production. These two isolates were screened for citric acid production in Czapekdox agar incorporated with 1% Bromocerol green indicator and were subjected to citric acid production in Czapekdox broth. The effect of different pH, temperature, carbon source, nitrogen source, stimulators and incubation time on citric acid production by the isolate AsnO was analysed. The maximum amount of citric acid production was recorded i...

Acetic acid (AC) hydrolysis has been reported to prepare xylooligosaccharides (XOS) from poplar. However, the influence of AC hydrolysis on the lignin structure changes is not clear, which is important for the following enzymatic hydrolysis of poplar. Herein, AC was used to produce XOS, and cellulase adsorption on cellulolytic enzyme lignin (CEL) from AC-hydrolyzed poplar and its inhibitory effect on two commercial cellulase preparations were investigated. AC hydrolysis gave a XOS yield of 39.8% from poplar. After AC hydrolysis at 170°C, the hydrophobicity and ζ-potential of CEL decreased to 2.3 L/g and 14.8 mV, respectively. The adsorption strength of CTec2 on CEL samples did not increase by AC hydrolysis, and the inhibitory effect of CEL on Celluclast 1.5L and β-glucosidase was observed, but not on CTec2. CEL samples improved the lytic polysaccharide monooxygenase (LPMO) activity of the enzymatic hydrolysis by CTec2. After CEL samples were added in enzymatic hydrolysis, the free filter paper activity of Celluclast 1.5L and β-G retained in the enzymatic hydrolysate decreased from 60.5 to 29.3−42.9%. The addition of CEL samples in enzymatic hydrolysis could not decrease the free filter paper activity of CTec2 retained in the enzymatic hydrolysate. In the enzymatic hydrolysis with CEL samples, higher glucose yields were obtained by CTec2 than those by Celluclast 1.5L and β-glucosidase. This work will help to understand the structure and inhibitory effects of AC-CELs and guide the development of AC hydrolysis for the production of XOS and monosaccharides from poplar.

Mango peel, a solid mango processing waste, comprises 15-20% of total fruit weight. This, being a rich source of lignocelluloses, was used as substrate for carboxymethyl cellulase (CMCase) production using Paenibacillus polymyxa. Maximum CMCase production (7.814 U mg-1) was observed in a medium containing 7% mango peel (w/v) with 1.5% ammonium sulphate (w/v) at 37 o C and pH 5.5. Purification to an extent of 28.24 fold was achieved by affinity column chromatography. Bands corresponding to 26.5 and 34.0 kDa molecular sizes were observed on 12% denaturing Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) while of 72 kDa on 10% non-denaturing Native-PAGE, proving its heteromeric multienzyme nature. The enzyme was stable over a range of 20-60 o C and pH of 4.0-7.5. Michaelis-Menten equation constant (K m and V max) values of purified CMCase were 8.73 mg ml-1 and 17.805 mM ml-1 min-1 , respectively.

A Bacillus coagulans strain isolated from palm fruit husk was tested for abilities to hydrolyse plant structural polysaccharides through the depolymerising activities of carboxymethylcellulase and polygalacturonase. The two enzymes were produced using solid substrate fermentation. Both had a working pH range of 4-9 with an optimum pH of 6.0 for the carboxymethylcellulase and 7.0 for the polygalacturonase. The respective enzymes remained active when allowed to stand at 27°C for 1 h over a wide pH and temperature range maintaining maximum activity at an optimum temperature of between 50°C and 60°C respectively. The Carboxymethylcellulase still retained full activity after being allowed to stand at 60°C for 10 min while the polygalacturonase retained full activity at 80°C for 5 min and had 50% activity at 70°C at 30 min. All enzymatic activities were fully inhibited by Mercury ions at 1.0 mM concentration. The carboxymethylcellulase had a molecular weight of between 116 and 205 KDa and...

Objective: The present study aimed to evaluate dried pomegranate peels as a substrate for citric acid production by Aspergillus niger. Methods: The morphological study of Aspergillus niger was carried out by wet mount with lactophenol cotton blue and slide culture method. A preliminary qualitative screening of citric acid-producing ability of this fungal strain was also performed by using the Czapek-Dox agar medium containing Bromocresol green. Dried and finely powdered Pomegranate peel was used as a principal substrate for the production of citric acid by submerged fermentation. Classical method of citric acid recovery involved a precipitation technique using calcium hydroxide followed by filtration and subsequent treatment with sulphuric acid. The citric acid produced was also chemically detected and titrimetrically estimated by 0.1 N NaOH. Results: The present experiment demonstrated that pomegranate peels may serve as an inexpensive medium for the production of citric acid with ...

The crystalline structure of cellulose makes it difficult to degrade and so most of the cellulosic waste in nature is disposed of by biomass burning. Cellulase enzyme system is potent enough to convert cellulose into glucose. Fungi are known to produce an array of hydrolytic enzymes. This study involves isolation of high potential cellulolytic fungal strains from the soil and optimizing pH and temperature conditions for enhanced cellulase production. The fungal strains were isolated from soil using serial dilution and pour plate techniques and screened using Congo red test and FPase method. Based on hydrolytic zones formation and cellulase enzyme production, Aspergillus fumigatus, Aspergillus terreus and Aspergillus flavus were found to show the highest potency for hydrolytic enzyme production at pH 5.8-6.0 and temperature range of 40°C-50°C.

Rice husk, a major waste product of rice milling factory in Nigeria, was hydrolyzed by cellulolytic enzymes of Aspergillus niger. Aspergillus niger synthesizes cellulase which biodegrade the cellulose content of rice husk. The cellulase activity was measured by the release of reducing sugar over the period of biodegradation. The reducing sugar content of the biodegraded rice husk showed a significant increase with an increase in days of biodegradation (0.560 -1.020mg/ml from day zero through day five, to day nine). The maximum yield of glucose concentration was observed on day five of biodegradation at pH of 5.4, while the minimum yield of glucose concentration was observed on day nine of biodegradation at pH 6.18. The study revealed that the growth of Aspergillus niger on rice husk increases its usefulness and that rice husk can through microbial degradation be converted to fermentable sugars and other useful product like alcohol. It also highlights the industrial potential of rice husks as possible substrates for cellulase enzyme production by Aspergillus niger which can eliminate agro-waste environmental pollution in Nigeria.

Abinash sharma (lovely professionl university student of 2010-2014 batch) Abstract Cellulose is most abundant biological component on terrestrial and aquatic ecosystem. Being the largest component of terrestrial and aquatic ecosystem conversion of this complex carbohydrate to a simpler sugar is having brighter future. The conversion of cellulose to one of the simplest form of sugar which is glucose is having bright future. Some and bacteria are capable of these conversion so in this experiment our main objective was to produce high yield of cellulase using Bacillus flexus strain. Various multiple factors was tested for cellulase productivity by Bacillus flexus strain using Taguchi methodology based on Design of experiments and dinitro salicylic acid (DNSA) technique was used for quantifying the cellulase productivity. The optimum pH and temperature for enzyme activity was 6 and 25 respectively and most prominent nitrogen source was peptone and whereas optimum cellulose and tween80 concentration was found to be 2% and 1% respectively. It shows that pH should be low, nitrogen should be high, cellulose should be low, tween80 should be low and temperature should be moderate. ANNOVA shows nitrogen has highest percentage contribution (38.07%) then pH has 24.784% contribution followed by tween80 which has 16.087% contribution then cellulose has 15.441% contribution and temperature has lowest % contribution (5.616). Maximum enzyme activity was found in trial condition 4 and is 0.25192 IU/ml.

Optimal enzyme activity occurs within a limited set of conditions such as temperature and pH which must thus be carefully controlled to promote high reaction rates. Therefore, watermelon peel was collected, processed, pretreated and subjected to hydrolysis in a series of batch reactors. Trichoderma reseei was isolated from decayed wood and the enzymes released by Trichoderma reseei were used for the hydrolysis for glucose production from watermelon dry peel. The effects of temperature at selected temperatures of 20 o C, 25 o C, 30 o C, 35 o C, 40 o C and 45 o C; as well as effects of pH on enzyme activity at selected pH ranges of 3.0 to 5.5 were studied. From the study, the results obtained within the limit of the experimental conditions show an optimal temperature of 30 o C, having a corresponding glucose concentration of 0.091728 g/l, optimal pH of 4.5 with a corresponding glucose concentration 0.6436 g/l, which implies that the enzymes from T. reseeigives high yield of glucose when controlled at these conditions. Also, the watermelon dry sample used in the study was highly deliginified, leading to high glucose yield.

Mango peel, a solid mango processing waste, comprises 15-20% of total fruit weight. This, being a rich source of lignocelluloses, was used as substrate for carboxymethyl cellulase (CMCase) production using Paenibacillus polymyxa. Maximum CMCase production (7.814 U mg -1 ) was observed in a medium containing 7% mango peel (w/v) with 1.5% ammonium sulphate (w/v) at 37 o C and pH 5.5. Purification to an extent of 28.24 fold was achieved by affinity column chromatography. Bands corresponding to 26.5 and 34.0 kDa molecular sizes were observed on 12% denaturing Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) while of 72 kDa on 10% non-denaturing Native-PAGE, proving its heteromeric multienzyme nature. The enzyme was stable over a range of 20-60 o C and pH of 4.0-7.5. Michaelis-Menten equation constant (K m and V max ) values of purified CMCase were 8.73 mg ml -1 and 17.805 mM ml -1 min -1 , respectively.

Production of endoglucanase, a good index for cellulase, by Penicillium atrovenetum using plantain peels as substrate was investigated. Cultural parameters varied include incubation time (1-15 days), pH (3-6), temperature (30-50oC) and nitrogen source. Enzyme production was analyzed by the dinitrosalicyclic acid (DNS) method. Highest production of endoglucanase was observed on the 7th day at pH 5 and at incubation temperature of 40oC. Ammonium oxalate, followed closely by ammonium persulfate, was observed as the best nitrogen source. The crude enzyme has optimum activities at 50oC and pH 5 while stable within 30oC to 50oC and pH 5 to 7. Addition of 5 mM CaCl2 enhanced the activities of the enzyme by 15.54%.