Characterization Of Cellulase Research Articles

Partial Purification and Characterization of Cellulase from the Haemolymph of the African Giant Land Snail (Archachatina marginata)

The aim of this study is to partially purify and characterize the cellulase extracted from the snail haemolymph of the African giant land snail of the species, Archachatina marginata. The protein concentration of the haemolymph was found to be 168 mg mLG1 and the specific activity of the crude cellulase was found to be 0.007 U mgG1 protein using Carboxymethyl-cellulose (CMC) as the substrate. The partial purification on Sephadex G-200 produced two activity peaks. The molecular weights of the two cellulase fractions were estimated to be 112,202 and 67,000. The Michealis Menten constant (km) for cellulase was 4.3 and 2.8 mg mLG1 for the higher and lower molecular weight fractions, respectively. The multiplicity of cellulase complexes indicated that the haemolymph of A. marginata may convert derived cellulose in foodstuffs such as garlic, mushrooms and domestic wastes into sources of fuel. It is suggested that the enzyme can be used to produce value-added products, such as ethanol, citric acid, amino acids and vitamins including primary metabolites from cellulosic wastes.

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.

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.

Cellulase 생산균주의 Screening 및 Bacillus licheniformis CEL-II Cellulase 특성 규명

Cellulase 생산균주의 Screening 및 Bacillus licheniformis CEL-II Cellulase 특성 규명

Optimization and characterization of cellulases from thermophilic strain of Scytalidium thermophilum SKESMBKU01

Optimization and characterization of cellulases from thermophilic strain of Scytalidium thermophilum SKESMBKU01

Research on Extraction and Characterization of Cellulase from Commercial Enzyme Preparation

The extraction of cellulase from commercial enzyme preparation by ammonium sulfate precipitation and its enzymatic characterization were studied. The results were as follows: the conditions of precipitation was 60% saturation of ammonium sulfate, the recovery rate of carboxymethylcellulase (CMCase) and Filter Paper enzyme Activity (FPA) were 73.8 and 71.4%, respectively. The enzyme has optimal temperature of 55°C, optimal pH of 4.8, the ions of $Co^{2+}$, $Mn^{2+}$ in the buffer lowered the activity of CMCase, but the $Cu^{2+}$ in low concentration activated the CMCase. The ions of $Co^{2+}$, $Mn^{2+}$ and $Al^{2+}$ in the buffer lowered the activity of FPA, but the $Cu^{2+}$ and $K^+$ in low concentration activated the FPA; $K_m$ and $V_{max}$ of the enzyme were $2.22×10^{-3}g/mL$ and $1.11×10^4 U/h$ using Lineweaver-Burk graphic method, respectively.

Purification and characterization of cellulase from a marine Bacillus sp. H1666: A potential agent for single step saccharification of seaweed biomass

• Marine bacterium Bacillus sp. utilized without prior treated agricultural waste. • Cellulase production by Bacillus sp. in single step from wheat straw/rice bran. • Cellulase: molecular mass of 40 kDa, thermo-stable and active at wide pH 3–9. • High saccharification efficacy as tested on seaweed Ulva . In this study, 115 marine bacterial isolates were screened for cellulase enzymatic activity and enzyme with a molecular mass of 40 kDa was purified from culture supernatant of the marine bacterium Bacillus sp. H1666 using ion exchange and size exclusion chromatography method. Growth of bacterial strain H1666 with efficient cellulase enzyme production was observed on untreated wheat straw and rice bran. The biochemical properties of the extracted cellulase were studied and enzyme was found active over a range of pH 3–9. The optimum cellulase activity was observed at pH 7 and temperature 50 °C. The enzyme was also shown to be slightly thermo-stable with 40% residual activity at 60 °C for 4 h. The potential applicability of enzyme was tested on dried green seaweed ( Ulva lactuca ) and 450 mg/g increase in glucose yield was obtained after saccharification. MALDI TOF–TOF analysis of cellulase peptide fingerprint showed similarity to the sequence of the glycoside hydrolase family protein.

Characterization of cellulase, hemicellulase and lipase and its use in deinking of laser printed paper

Aims: It is recognized that laser printed paper are difficult to deink using conventional method. This had lead to the suggestion of enzymatic approach to overcome the problem encountered by commonly employed deinking techniques. The present study aimed to investigate 7 commercially available enzymes for their suitability use in deinking of laser printed paper. Methodology and results: 3 cellulases, hemicellulases, xylanase and 2 lipases were used in enzymatic deinking of laser-printed wastepaper. Cellulase A “Amano”3 (C), Hemicellulase (H) and lipase (L) were selected for used in deinking because they possess either highest activity or broad pH stability compared to others enzymes. Different combination of enzymes was carried out to evaluate their effectiveness in deinking process. CH enzymes sequence was determined to be the most effective sequence in toner removal with 1.90% of brightness increment. However, only 0.95% of brightness increment was gained by enzyme sequence L. Highest deinking efficiency obtained was not proportional to the highest total reducing sugar produced. Conclusion, significance and impact of study: Enzyme (cellulase and hemicellulase) can be used to de-ink laserprinted wastepaper, which are difficult to be deinked by conventional chemical deinking process. Thus, enzyme deinking has high possibility as alternative method to current chemical deinking process which is not environmental friendly.

Partial Purification and Characterization of Cellulase, Pectinase and Xylanase from Penicillium chrysogenum

Partial Purification and Characterization of Cellulase, Pectinase and Xylanase from Penicillium chrysogenum

Partial purification and characterization of cellulases from digestive tracts of the African giant snail (Achatina achatina)

Crude cellulase was extracted from the digestive tracts of 30 mature Achatina achatina and subjected to a 3-step purification process of ammonium sulfate precipitation, dialysis, and gel filtration. This purification procedure gave 3 prominent enzyme activity peaks that coincided with protein peaks and were designated A, B, and C, corresponding to high endoglucanase, \beta-glucosidase, and total cellulase activities, respectively. Temperature optima of 50 °C were recorded for \beta-glucosidase and total cellulase while 45 °C was recorded for endoglucanase. Total cellulase, glucosidase, and endoglucanase showed maximum activities at pH values of 5.5, 4.5, and 7.5, respectively. Kinetic studies show that total cellulase has a V_{max} and K_m of 2427.18 \mumol/min and 15.12 mg cellulose and endoglucanase has values of 955.11 \mumol/min and 2.39 mg Na-CMC, while the values of \beta-glucosidase are 946.97 \mumol/min and 4.3 mM cellobiose, respectively. This study shows that cellulases from digestive tracts of A. achatina could be utilized for degradation of cellulose-containing materials because of their high thermostability and acid/alkali stability, which reflect the potential commercial significance of the enzyme.

Purification and characterization of cellulase from North Pacific krill (Euphausia pacifica). Analysis of cleavage specificity of the enzyme

Krill are filter feeders that consume algae, plankton and detritus, indicating that krill possess an adequate cellulose digesting system. However, less is known about the enzymatic properties of crustacean cellulases compared to termite cellulases. In the present study, 48 kDa-cellulase was highly purified from krill (Euphausia pacifica) in an effort to determine the cleavage specificity of the enzyme. The most notable characteristic of the enzyme was its high activity against both lichenan and carboxymethyl cellulose. The enzyme hydrolyzed internal β-1,4 glycosidic bonds within lichenan as well as carboxymethyl cellulose to release oligosaccharides and glucose. The effects of pH and temperature on the activity and stability of both enzyme activities were almost identical. Cello-oligosaccharides with a degree of polymerization of 4-6 were hydrolyzed by the enzyme, and the same endo-products, cellotriose, cellobiose and glucose, were produced from these oligosaccharides. Neither cellotriose nor cellobiose was hydrolyzed by the enzyme. The enzyme digested filter paper and sea lettuce to produce cellobiose, cellotriose and glucose as major products. Although amino acid sequence homology of the enzyme with termite cellulases and the presence of oligosaccharides in the enzyme suggested that the enzyme is produced by krill itself, further analysis is necessary.

Quantitative Secretomic Analysis of Trichoderma reesei Strains Reveals Enzymatic Composition for Lignocellulosic Biomass Degradation

Trichoderma reesei is a mesophilic, filamentous fungus, and it is a major industrial source of cellulases, but its lignocellulolytic protein expressions on lignocellulosic biomass are poorly explored at present. The extracellular proteins secreted by T. reesei QM6a wild-type and hypercellulolytic mutant Rut C30 grown on natural lignocellulosic biomasses were explored using a quantitative proteomic approach with 8-plex high throughput isobaric tags for relative and absolute quantification (iTRAQ) and analyzed by liquid chromatography tandem mass spectrometry. We quantified 230 extracellular proteins, including cellulases, hemicellulases, lignin-degrading enzymes, proteases, protein-translocating transporter, and hypothetical proteins. Quantitative iTRAQ results suggested that the expressions and regulations of these lignocellulolytic proteins in the secretome of T. reesei wild-type and mutant Rut C30 were dependent on both nature and complexity of different lignocellulosic carbon sources. Therefore, we discuss here the essential lignocellulolytic proteins for designing an enzyme mixture for optimal lignocellulosic biomass hydrolysis.

Influence of mechanical agitation on the pH profile of total, soluble and insoluble filter paper activity of Hypocrea jecorina cellulase preparations

The characterization of cellulases is complex and has gained increasing importance due to the application of cellulases in finishing of textile fibres and the conversion of lignocellulose into second generation ethanol. Usually two or three different substrates such as filter paper Whatman No. 1, carboxymethylcellulose and avicel are used to characterize total, endo (EG) and exoglucanase (ExG) activity, respectively, and assays are carried out without any mechanical agitation. Unfortunately, the different activities obtained are not directly comparable and do not reflect cellulase behaviour in industrial applications, mainly due to physic-chemical differences between the substrates and the adopted procedures. In this study the pH profiles of three different cellulase preparations from Hypocrea jecorina (former Trichoderma reesei) have been characterized with modified filter paper assays which permitted the distinction between EG and ExG activities using a single insoluble substrate. Furthermore, the influence of mechanical agitation was studied. With the modified procedures, it could be shown that the contribution of EG and ExG activity to total cellulase activity and the endo/exo activity ratio depends on the cellulase preparation and varies with pH. When the assay was carried out under orbital agitation, EG activity increased significantly and the endo/exo-activity ratio increased.

Schizophyllum commune에 의한 Cellulase 생산 및 섬유소계 바이오매스의 당화를 위한 효소적 특성

본 연구에서는 Schizophyllum commune외 당 분해효소 생산을 위한 최씩 배양 조건과 목질바이오매스에 대한 당화 특성에 대하여 연구하였다. S. commune 균체 외 효소에는 endo-β-1.4-g1ucanase (EG). cellobiohydrolase (CBH) β-glucosidase (BGL)와 같은 cellulase와 β-l,4-xylosidase (EXL)이 함유되어 있고 그 중에서 EG 및 BGL활성이 비교적 높은 활성을 나타낸 것으로 밝혀졌다 S. commune에서 생산된 EG. BOL. 및 CBH의 최적 온도는 50°C이었으나. 열안정성을 기지는 온도범위는 30~40°C였다. 그리고 최적 pH는 5.5이었으며 열안정성을 나타내는 온도범위에서의 적정 pH는 동일한 pH 5.5이었다. Cellulase 생산을 위한 S. commune9.1 최적 배양 조건은. 탄소원으로 천연 cellulose. 질소원으로는 corn steep. 또는 peptone/yeast extract 혼합을 비타민 은 첨가하지 않는 것이 cellulase 효소활성 증가에 적절한 것으로 밝혀졌던. 또한 탄소원의 최적 첨가 농도는 2% (w/w). 적정 배양 pH 및 온도는 5.5~6.0과 25~30°C로 밝혀졌다 본 연구에서 도출된 최적 배양 조건으로 S. commune를 배양시키고 40배로 농축한 절과. EG가 3670.5 U/㎖. BOL과 CBH가 각각 631.9 u/㎖. 398.5 u/me. BXL이 15.2 U/Me로 매우 높은 효소 활성을 나타냈다 동일한 효소의 Filter Paper Unit도 11 FPU/㎖로 상당히 높았다. 최적배양조건에서 얻어진 S. commune 효소로 다양한 기질에 대해 당화 시험을 실행한 결과, 전처리를 하지 않은 공시 활엽수에 대하여 낮은 당화율을 나타냈으나 천연 cellulose (Aldrich. ~20 micron) 및 볏짚의 경우에는 각각 50.5% 및 33.1%의 높은 당화성능을 나타냈다. 이 같은 당화 수준은 동일 효소농도 (30 FPU/g.glucan)로 비교했을 때 Trichoderma reesei 유래 상용화 효소인 Celluclaset 1.5L의 약 110% 수준을 나타냄으로써. 대량생산기술 개발과정을 통해 목질계 당화 효소로의 사용화 가능성이 높은 균주로 평가되었다.

Production and characterization of cellulases and hemicellulases by Acremonium cellulolyticus using rice straw subjected to various pretreatments as the carbon source

Cellulases and hemicellulases are key enzymes in the production of alternative fuels and chemicals from lignocellulosic biomass—an abundant renewable resource. Carbon source selection is an important factor in the production of cellulases and hemicellulases. Rice straw – a potential ethanol source – has recently gained considerable interest in Asian countries. Here, we investigated the production of cellulases by using rice straw subjected to various pretreatments as substrates in order to produce cellulases at low costs; we also identified the enzymes’ characteristics. Rice straw cutter milled to <3 mm was pretreated by wet disk milling, dry ball milling, or hot-compressed water treatment (HCWT). Pretreated rice straw and commercial cellulose, Solka Floc (SF), were used as carbon sources for cellulase production by the fungus Acremonium cellulolyticus. Filter paper cellulase, β-xylanase, and β-xylosidase production from ball- and disk-milled samples were higher than those from SF. Enzymatic activity was absent in cultures where HCWT rice straw was used as carbon source. Wet disk-milled rice straw cultures were more suitable for enzymatic hydrolysis of pretreated rice straw than SF cultures. Thus, wet disk milling may be a suitable pretreatment for producing substrates for enzymatic hydrolysis and generating inexpensive carbon sources for cellulase production.

Production and characterization of cellulases and xylanases of Cellulosimicrobium cellulans grown in pretreated and extracted bagasse and minimal nutrient medium M9

Bagasse, the main coproduct in the sugar industry, has been shown to be the sole carbon source for liquid-state fermentation to produce hydrolytic enzymes (cellulases and xylanases) using Cellulosimicrobium cellulans in flask during 3 days and the enzymatic extracts obtained at different time intervals were analysed. The degradation of bagasse indicate the lignocellulolytic capacity of C. cellulans, 26% decrease in hemicellulose content at 96 h, while 10% decrease in cellulose. This accord to that xylanases fairly predominated over cellulases. A rapid rise in the activity of cellulases and xylanases was observed in the early stages of incubation (within the first 24 h). The optimum pH for these enzymes by C. cellulans in liquid cultures was between 5.0 and 5.6 and the optimum temperature was 50 °C. Thermal stability of the enzymes was appreciable up to 45 °C, pH stability at pH 5.0.

Characterization of cellulase under various intensities of static magnetic fields

Abstract The activity and secondary conformation of cellulase were investigated under static magnetic fields (0.15, 0.30, and 0.45 T). Compared to control group, activity and conformational change of cellulase were observed by means of UV spectrophotometer and far-UV circular dichroism detection after exposed in various static magnetic fields. The cellulase exhibited its maximal specific activity in the pH range of 4.0–4.6, and the optimal temperature was 60 °C. In addition, the circular dichroism spectra and kinetic parameters of cellulase have also been changed by magnetic fields.

Multiplex PCR for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolated from Bandung, Indonesia

Alarge number of microorganism especially filamentous fungi has ability to degrade cellulose. The purpose of this study was to conduct screening, purification, and characterization of cellulase from fungi which was isolated from used paddy straw mushroom substrate. Screening of cellulytic activity using CMC medium shown that 11 out of 20 isolates of fungi produced a clearing zone surrounding fungal colony. Among them isolate number JMF 12 showed the highest cellulase activity and was further used for purification and characterization. The cellulase was purified to electrophoretical homogeneity by ammonium sulfate precipitation, dialyzed by Novagen D-Maxi TubeTM Dialyzer, and Sephadex G-100 gel filtration chromatography. The recovery and purification fold was 3.82 % and 1.98 respectively, after Sephadex G-100 gel filtration chromatograph. The o purified cellulase had an optimal pH and temperature at 6 and 45 C. The Km and Vmax of cellulase was 11.43 mM and 0.006mmol/min respectively. The purified cellulase was activated by Na+ and Zn++ but inhibited by Ca++, Co++, Fe++, and Hg++ .

Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens

A total of six lactic acid bacteria (LAB) isolates were selected from five indigenously fermented cereal gruels and identified as Lactobacillus fermentum OYB, Lb. fermentum RS2, Lb. plantarum MW, Lb. plantarum YO, Lb. brevis WS3, and Lactococcus spp. RS3. Six aflatoxin-producing aspergilli were also selected from the various food sources. Two of the isolates, identified as Aspergillus parasiticus C2 and A. parasiticus AF7, produced both aflatoxin B1 and G while the other four identified as A. flavus M1, A. flavus B4, A flavus B5 and A. flavus C6, produced only aflatoxin B1. Each of the LAB isolates inhibited the growth of at least a toxin-producing Aspergillus. The maximum inhibitions were shown by Lb. plantarum YO, which was able to inhibit the vegetative and sporulative growth of all the aflatoxinproducing aspergilli. Lactococcus spp. RS3 and Lb. brevis WS3 were only able to inhibit A. parasiticus C7 reasonably and A. flavus B5 and C6 mildly.

Characterization of cellulases and hemicellulases produced by Trichodermareesei on various carbon sources

Cellulase and hemicellulase enzymes were produced by Trichoderma reesei RUT C30 on steam pretreated spruce, willow, corn stover and delignified lignocellulose (Solka Floc), as a reference. The enzymes produced were characterized by protein and various enzyme activity measurements. On steam pretreated corn stover higher cellulolytic enzyme activities were reached than on Solka Floc, while the activities obtained on steam pretreated spruce and willow were considerably lower. The produced and two commercial cellulases (Celluclast 1.5 L and Econase CE) were compared by determining specific activities. There were minor differences among the enzymes corresponding to their specific cellulase activities. In contrast, within hemicellulase and β-glucosidase activities, the differences were found to be more significant. It should be also noted that commercial cellulases had considerably higher specific acetyl xylan esterase activities than the produced enzymes. According to subsequent hydrolysis experiments, performed to characterize the produced enzyme complexes to evaluate their applicability for hydrolysis and enzyme production, it seems that the application of the enzyme that was produced on the same substrate as was used for hydrolysis can be advantageous in the case of some substrates. As a result, these experiments demonstrated that pretreated corn stover is a good substrate both for enzyme production and hydrolysis, since high cellulolytic activities could be reached using it as carbon source. Moreover, high sugar yields could be obtained in the hydrolysis by the enzyme produced on steam pretreated corn stover.