Carboxymethyl Cellulose Substrate Research Articles

Purified cellulase-mediated simultaneous sugar utilization by Bacillus albus isolated from Similipal, Odisha, India.

Among 24 isolated cellulolytic bacteria from Similipal Biosphere Reserve, the most efficient isolate was recognized as a strain of Bacillus albus. This strain of B. albus was evaluated for cellulase production and the cellulase activity was measured in submerged fermentation using substrate carboxymethyl cellulose (CMC). Different nutritional (carbon, nitrogen, and metal-ion sources) and physical variables (pH, temperature, substrate concentration, and incubation time) during the growth of B. albus were optimized to obtain maximum cellulase activity. The highest cellulase activity of 5.79 U/mL for B. albus was observed at pH 6.75, temperature 37.5°C, CMC concentration 8.5 g/L, and 42 h incubation time. Further, supplementation of glucose as a subsidiary carbon source, yeast extract, peptone as nitrogen sources, and MgSO4 and MnSO4 as metal-ion sources enhance the cellulase activity of B. albus. The purified enzyme was reported to have a molecular weight of ∼54 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A zymogram analysis evidenced the cellulase activity of the purified enzyme fractions obtained from diethylaminoethyl cellulose chromatography. The purified cellulase was reported to have an optimum pH and temperature of 7.0°C and 50°C, respectively with a capacity of retaining its 60% residual activity within pH 6.0-8.0 and temperature 30-40°C, respectively. The metal ions, K+ and Na+ were the activators, while Pb2+ and Hg2+ were the inhibitors for the purified cellulase. The purified cellulase showed Km and Vmax values of 0.38 M and 8.19 U/mL, respectively, in presence of the substrate CMC and also simultaneous consumption of both hexose and pentose sugars.

High-performance homogeneous carboxymethylcellulose-stabilized Au@Ag NRs-CMC surface-enhanced Raman scattering chip for thiram detection in fruits

Cellulose material holds considerable promise for effective surface-enhanced Raman scattering (SERS) substrate construction due to its extensive availability, chemically modifying capacity, ease of manufacture, high flexibility and low optical activity. A large-area, high-sensitivity, stable and uniform Au@Ag nanorods (NRs)-CMC substrate was successfully developed via electrostatic repulsion by using negatively-charged core–shell Au@Ag NRs as SERS active plasmonic nanomaterial, combined with negatively-charged carboxymethylcellulose (CMC) hydrogel for nanoparticles stabilization, homodisperse and protection. The obtained Au@Ag NRs-CMC substrate showed excellent sensitivity for the detection of thiram residues in fruits containing low and abundant pigment interferents, such as apples and blueberries, with detection limits of 58 and 78 ppb, respectively. Additionally, it retained more than 80% SERS performance after storage for 9 months under ambient conditions, demonstrating its great potential in facilitating the commercialization of cellulose-based SERS technology for cost-effective detection of food contaminants with advantages of facile preparation procedure, uniformity, reproducibility and long-term stability.

The activity of BcsZ of Salmonella Typhimurium and its role in Salmonella-plants interactions.

Salmonella enterica is one of the most common human pathogens associated with fresh produce outbreaks. The present study suggests that expression of BcsZ, one of the proteins in the bcs complex, enhances the survival of Salmonella Typhimurium on parsley. BcsZ demonstrated glucanase activity with the substrates carboxymethylcellulose and crystalline cellulose, and was responsible for a major part of the S. Typhimurium CMCase activity. Moreover, there was constitutive expression of BcsZ, which was also manifested after exposure to plant polysaccharides and parsley-leaf extract. In an in-planta model, overexpression of BcsZ significantly improved the epiphytic and endophytic survival of S. Typhimurium on/in parsley leaves compared with the wild-type strain and bcsZ null mutant. Interestingly, necrotic lesions appeared on the parsley leaf after infiltration of Salmonella overexpressing BcsZ, while infiltration of the wild-type S. Typhimurium did not cause any visible symptoms. Infiltration of purified BcsZ enzyme, or its degradation products also caused symptoms on parsley leaves. We suggest that the BcsZ degradation products trigger the plant’s defense response, causing local necrotic symptoms. These results indicate that BcsZ plays an important role in the Salmonella-plant interactions, and imply that injured bacteria may take part in these interactions.

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation.

Lignocellulosic biomass from plants has been used as a biofuel source and the potent acidic endoglucanase GtCel12A has been isolated from Gloeophyllum trabeum, a filamentous fungus. In this study, we established a plant-based platform for the production of active GtCel12A fused to family 3 cellulose-binding module (CBM3). We used the signal sequence of binding immunoglobulin protein (BiP) and the endoplasmic reticulum (ER) retention signal for the accumulation of the produced GtCel12A in the ER. To achieve enhanced enzyme expression, we incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C into the construct. In addition, to enable the removal of N-terminal domains that are not necessary after protein expression, we further incorporated the cleavage site of Brachypodium distachyon small ubiquitin-like modifier. The GtCel12A-CBM3 fusion protein produced in the leaves of Nicotiana benthamiana exhibited not only high solubility but also efficient endoglucanase activity on the carboxymethyl cellulose substrate as determined by 3,5-dinitrosalicylic acid assay. The endoglucanase activity of GtCel12A-CBM3 was maintained even when immobilized on microcrystalline cellulose beads. Taken together, these results indicate that GtCel12A endoglucanase produced in plants might be used to provide monomeric sugars from lignocellulosic biomass for bioethanol production.

A Novel Cold-Adaptive Endo-1,4-β-Glucanase From Burkholderia pyrrocinia JK-SH007: Gene Expression and Characterization of the Enzyme and Mode of Action

The efficient industrial conversion of plant-derived cellulose to simple sugars and other value-added chemicals requires various highly stable and reactive enzymes. Industrial processes especially synchronous saccharification and fermentation (SSF)-based production of cellulosic bio-ethanol require enzymes that are active at lower temperatures. In this study, we have identified, characterized, and expressed the cold-adaptive endo-1,4-β-glucanase (BpEG) isolated from the Burkholderia pyrrocinia JK-SH007. The analysis of the predicted amino acid sequence indicated that BpEG belongs to GH family 8. The BpEG without the signal peptide was cloned into the expression vector pET32a and significantly expressed in Escherichia coli BL21 (DE3) competent cells. The SDS-PAGE and Western blot analysis of BpEG revealed that the recombinant BpEG was approximately 60 kDa. Purified recombinant BpEG exhibited hydrolytic activity against carboxymethyl cellulose (CMC) and phosphoric acid swollen cellulose (PASC), but not crystalline cellulose and xylan substrates. High performance, anion exchange, chromatography-pulsed amperometric detector (HPAEC-PAD) analysis of the enzymatic products obtained from depolymerization of 1,4-β-linked biopolymers of different lengths revealed an interesting cutting mechanism employed by endoglucanases. The recombinant BpEG exhibited 6.0 of optimum pH and 35°C of optimum temperature, when cultured with CMC substrate. The BpEG enzyme exhibited stable activity between pH 5.0 and 9.0 at 35°C. Interestingly, BpEG retained about 42% of its enzymatic activity at 10°C compared to its optimal temperature. This new cold-adaptive cellulase could potentially achieve synchronous saccharification and fermentation (SSF) making BpEG a promising candidate in the fields of biofuel, biorefining, food and pharmaceutical industries.

Biotechnological potential of mangrove sediments: Identification and functional attributes of thermostable and salinity-tolerant β-glucanase

Microorganisms native to mangroves are expected to contain enzymes capable of hydrolyzing different carbon sources. However, most of these microorganisms aren't cultivable; hence, alternative techniques as metagenomics are tools for studying and obtaining some of the natural genomes, genes and enzymes of biotechnological interest. The β-glucanase was produced using a metagenomic clone of mangrove sediments and detected by functional screening on carboxymethylcellulose substrate. The enzyme was purified by cation exchange chromatography. The peptides detected by mass spectrometry showed 20% identity with the polypeptide deduced from the genomic fragment sequenced. The ORF identified as BglfosD9 possessed 729 bp and the encoded protein showed predicted MW and pI of 28kD and 6.8, respectively. The enzyme was active in a wide range of pH (5–10) with optimum pH at 8, had relative activity greater than 50% at all temperatures tested (5–90 °C), was stable at temperatures of 5, 50 and 90 °C and showed excellent relative activity at high NaCl concentrations. This β-glucanase also showed high relative activity in the presence of SDS and it could hydrolyze β-glucan, CMC and Avicel as substrates. These findings support the idea of a new thermostable and active enzyme at basic pH from metagenomic library of mangrove sediment.

Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆zface1 Strain

The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

Protein‐Polymer Bioconjugation Effects on Activity and Stability of Cellulase (FnCel5a) with Varying Molecular Weights of Polymer Chains

An alternative method for energy production which decreases dependency on nonrenewable resources and utilizes a greater amount of the available plant biomass is cellulosic ethanol production. However, there is a need for a more cost effective and energy efficient method to produce a stable form of cellulase that can withstand the harsh chemical and physical industrial processes. One method to enhance protein stability has been demonstrated through protein‐polymer bioconjugation. Thermophilic cellulase, FnCel5a, has been shown to have increased activity when conjugated to polymers via amidation reactions onto lysine residues of the enzyme. However, due to numerous lysine residues there was a lack of site‐specific binding of the polymers. Therefore, a novel site‐specific approach was taken in this study to conjugate polymers onto cellulase via a maleimide method using thiolene click chemistry onto a cysteine residue. In addition, polymer chain length was manipulated to investigate its effects on enzymatic stability and activity using the amidation method. Polymerization methods of RAFT and ATRP were utilized to synthesize N, N‐dimethylacrylamide (DMAm) polymers and OEOA polymers of varying degrees of polymerization. These polymers were conjugated using a “graft to” method onto lysine residues. SDS‐PAGE electrophoresis confirmed conjugation of the polymers. And an activity assay using carboxymethylcellulose substrate indicated the presence of a maximum percent enzymatic activity with increased polymer chain length. For site‐specific conjugation, a mutagenesis at C296S and C102S was done for selective polymer binding. SDS‐PAGE electrophoresis confirmed DMam and OEOA polymer binding at the C296 position of FnCel5a. Overall this study confirmed the effective use of click chemistry method as a technique for site‐specific polymer binding of RAFT and ATRP at a cysteine residue. This method can also be utilized to better investigate how polymer chain length can affect the stability and activity of FnCel5a with controlled site‐specific reactions.Support or Funding InformationREU Site: Summer Research in Chemistry and Biochemistry at Miami University at Miami University, Award No. 1460862This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Isolation and Characterization of Cellulase-producing Bacteria from Sugar Industry Waste

Cellulases are inducible enzymes that are synthesized by a large number of microorganisms during their growth on cellulosic materials. This study focuses on the isolation and screening of cellulase-producing bacteria from sugar industry waste (molasses) and characterization by morphological and biochemical analysis. Further, purification of cellulase was carried by ammonium sulfate precipitation and followed by column chromatography and molecular weight determined by SDS-PAGE. The isolated bacterial strains were grown on carboxymethyl cellulose (CMC) agar plate at various optimum conditions like pH, temperature, incubation period, carbon and nitrogen sources and substrate concentration. Three isolated strains showed clear hydrolyzing zone on agar plates containing CMC agar after Congo-red staining were identified as cellulase-producing bacteria. Based on cultural, morphological, and biochemical characteristics, the isolated strains were identified as <i>Paenibacillus sp., Aeromonas sp.</i>, and <i>Bacillus sp</i>. Among the isolated strains, <i>Paenibacillus sp</i>. showed the capability for highest cellulase production (0.89 µmol ml^-1 min^-1) at optimal pH 7.0 and 40°C temperature on 24 hour of the incubation period at 1% CMC substrate concentration and was selected for further cellulase purification. In the final step of cellulase purification, the specific activity, purification fold and recovery were 1720 U/mg, 9.74 and 35.6%, respectively. The molecular weight of the purified enzyme was determined 66.9 kDa and the enzyme showed a high specificity to CMC substrate. The bacterial strains present in molasses have the potential for cellulase production. Substrate specificity of the purified cellulase indicates it to be an endo-β-1, 4-glucanase. The cellulase produced from the selected strain may benefit for industrial application.

Screening the capabilities of Indonesian indigenous mold in producing cellulase enzyme

This study aims to get the isolates of mold that can potentially produce cellulase enzyme. This research was conducted in December 2017 until January 2018 in Microbiology Laboratory of Universitas Negeri Jakarta. The screening test of potential isolates producing cellulase enzymes was performed on 31 isolates of UNJCC in CDA medium with addition of Carboxymethyl Cellulose (CMC) substrate and testing of clear zone formation using 0.1% congo red reagent. From the result of the research, there are 18 potential mold isolates which can produce cellulase enzyme. 3 isolates which have the highest Cellulolytic index are isolate A17, K4, and 1 which have cellulolytic index value of 1.03, 0.32, 0.29. From macroscopic and microscopic identification it can be assumed that the coded isolates of K4 and A17 belong to the genus Aspergillus, while, isolates code 1 belong to the genus Penicillium. Forming the clear zone on CMC subtrate with dropped congo red reagent it because congo red reagent can bind strongly with polysaccharides containing β (1-4) D-glucopyranosyl bonds. And the ability of clear zone formation on CMC substrates shows the presence of enzyme endo-β-1,4 glucanase (CMCase) that can breaks off the β -1,4-glycosidic bond linking D-glucose monomer to CMC.

Morphological, enzymatic screening, and phylogenetic analysis of thermophilic bacilli isolated from five hot springs of Myagdi, Nepal

The present study was conducted to identify and characterize the thermophilic bacteria isolated from five hot springs, viz., Sinkosh, Singha, Bhurung, Ratopani and Paudwar located in Myagdi district, Nepal by using phenotypic and genotypic methods. The hot spring has temperature 420-62 0C and pH 6.5-6.8. Isolation of thermophiles was done by using simple enriched nutrient broth media at 60 0C.Selected strains were screened for thermostable enzymes; cellulase, hemicellulase, amylase, protease, gelatinase, and lipase using substrates carboxymethyl cellulose (CMC), xylan, soluble starch, casein, gelatin and Tween (20,40,60 or 80) respectively. The bacteria were grouped in sixteen groups based on morphological and biochemical characteristics. 16S rRNA sequence analysis of sixteen isolates, one isolate representing one group and phylogenetic analysis showed the cluster of five distinct taxonomic groups. The groups were identified as for genus Anoxybacillus, Aeribacillus, Brevibacillus, Bacillus, and Geobacillus, based on ≥ 95% similarity with reference strains. This is the first study that reports Anoxybacillus sp. Brevibacillus sp. and Aeribaceillus sp. from the hot springs of Nepal.

Preliminary Characterization of a Cellulase Producing Bacterial Strain Isolated from a Romanian Hypersaline Lake

Cellulases are a group of enzymes that are used in many biotechnological processes. Since most of the enzymes synthesised by mesophilic microorganisms are unstable in industrial environments, it is necessary to direct research towards extremophile cellulolytic microorganisms because the enzymes synthesised by them are stable and active even in harsh physicochemical conditions. In the present investigation, our aim was to isolate and identify some microbial cellulolytic strains from a hypersaline lake located in Romania and to determine their optimal growth conditions. Of a total of 25 microbial strains isolated, only one extreme halotolerant bacterial strain was able to produce an endoglucanase. Based on molecular identification, we identified this cellulolytic strain as a species of Bacillus genus, most closely related to Bacillus zhangzhouensis. Optimal growth conditions were found to be at 15°C, pH 7.5 and 2 M NaCl. Endoglucanase activity of this bacterial strain is influenced by both salinity and temperature. The most significant endoglucanase activity was detected in the presence of 3 M NaCl, after 72 h of incubation at 15°C. In this situation, the amount of glucose released from a volume of 0.5 mL of 2% (w/v) carboxymethyl cellulose substrate is equivalent to 2.05 mg. In conclusion, this study represents the first preliminary characterization of a B. zhangzhouensis strain that has the ability to degrade cellulose and that demonstrates tolerance to high salt concentrations.

Polymer Conjugation to Enhance Cellulase Activity and Preserve Thermal and Functional Stability.

A thermophilic cellulase, FnCel5a, from Fervidobacterium nodosum was conjugated with various functional polymers including cationic, anionic, and strongly and weakly hydrogen bonding polymers. The activity of FnCel5a toward a high-molecular-weight carboxymethyl cellulose substrate was enhanced by polymer conjugation. Activity enhancements of 50% or greater observed for acrylamide and mixed N,N-dimethyl acrylamide-2-(N,N-dimethylamino)ethyl methacrylate polymers, suggesting that the greatest enhancements were caused by polymers capable of noncovalent interactions with the substrate. The conjugates were found to have nearly identical thermodynamic stability to the native enzyme, as assessed by free energy (ΔG), enthalpy (ΔH), and entropy (TΔS) parameters extracted from differential scanning fluorimetry. Polymers tended to confer comparable tolerance to high concentrations of dimethylformamide, with longer polymers typically enabling higher activity relative to shorter polymers. The new FnCel5a conjugates represent an advance in the production of cellulases that maintain activity at high temperatures or in the presence of denaturing organic solvents.

Purification and characterization of cellulase from a novel isolate of Trichoderma longibrachiatum

ABSTRACTIn the present study, the isolation, purification and characterization of a complex enzyme, cellulase, was carried out using a new local isolate of the fungus Trichoderma longibrachiatum (KM274866) from wood chips, with a view to explore its utility in the biofuel industry. The fungus was grown on a selected natural substrate, sugarcane bagasse, based on cost considerations for enzyme production. The enzyme was purified 14.82 fold with a yield of 25.8% and specific activity of 30 U/mg of protein. The molecular mass of the enzyme was found to be 67 ± 1 KDa. The optimum pH was 4.8, but the enzyme was stable at a pH range of 3–6. Optimum temperature was 45 °C, but the stability range of the enzyme was 30–55 °C. Metal ions such as Ca2+, Na+, Mg2+, Zn2+ and Fe2+ enhanced enzyme activity. Triton X100 in the medium resulted in a significant increase of enzyme activity compared to other group specific reagents. KM and Vmax for the enzyme were found to be 0.121 mg/ml and 0.421 μmol/min, respectively, against the substrate carboxy methyl cellulose. As the enzyme is from an inexpensive source, it will be most useful in the preparation of bioethanol for the biofuel industry.

Characterization of a novel thermostable GH45 endoglucanase from Chaetomium thermophilum and its biodegradation of pectin

A novel thermostable endoglucanase (CTendo45) encoding gene was cloned from Chaetomium thermophilum and heterologously expressed in Pichia pastoris. Sequence alignment indicated that the CTendo45 enzyme belonged to glycoside hydrolase family 45. The recombinant enzyme was purified by Ni2+ affinity chromatography, and its apparent molecular mass was estimated to be 32kDa by SDS-PAGE. The purified enzyme displayed maximum activity at 70°C and pH 4. CTendo45 was stable at 60°C for 1h, and residual activities of 78.9% and 65.6% were estimated after 1h at 70°C and 80°C, respectively. Ca2+, Zn2+, Mg2+, Cu2+ and Mn2+ were found to have beneficial effects on the enzyme activity to different degrees. The specific activity of purified CTendo45 was 1.52IUmg-1 and the Km value was 59.6μgml-1 with a sodium carboxymethyl cellulose substrate. Moreover, CTendo45 exhibited high hydrolysis activity towards pectin, and the hydrolysis products were mainly galacturonic acid oligosaccharides. CTendo45 is the first reported bifunctional enzyme in glycoside hydrolase family 45 from C. thermophilum that is able to hydrolyze both cellulose and pectin. The biochemical properties of this recombinant CTendo45 make it a potentially effective glycoside hydrolase for industrial applications.

Characterization and kinetic parameters of endo-1,4-β-d-glucanase of ultraviolet mutated Aspergillus niger and Penicillium citrinum

Mutagenic agents had been used to enhance production of enzymes in microorganisms, but the effects of the agents on the kinetic parameters and catalytic efficiency of the produced enzymes had not been critically evaluated. A study was conducted to assess the effects of ultraviolet (UV) mutagenesis on the kinetic parameters and catalytic efficiency of endo-1,4-β-D-glucanase enzyme obtained from wild and mutated strains of Aspergillus niger and Penicillium citrinum obtained from decaying wood wastes modified by ultraviolet radiation. The endo-1, 4-β-Dglucanase from both wild and mutant strains of the fungi were purified by centrifugation, ammonium sulfa-te precipitation and anion exchange chromatography using Sephadex A25-120 and Whatman DE-52 resins. The kinetic parameters determined from Lineweaver-Burk plot, using the velocity or reaction rates at varying concentrations of carboxymethyl cellulose substrate ranging from 0 - 20 mg/mL were validated by Hanes-Woolf plots for convergence, as the Michaelis-Menten kinetic equation is non-linear. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for the wild A. niger were 15.31 g/L and 4.67 g/L respectively, while values for corresponding mutated strain were 7.9 g/L and 3.39 g/L respectively. The Km and Vmax for wild P. citrinum were 7.94g/L and 6.81g/L respectively, while the values for the mutated strain were 6.60 g/L and 6.84 g/L respectively. Catalytic efficiency of endoglucanase of wild and mutated strains of A. niger were 0.305 and 0.42 M-1s-1 respectively, while the values for endoglucanase of wild and mutated strains of P. citrinum were 0.858 and 1.036 M-1s-1 respectively. Mutagenesis using ultraviolet radiation therefore enhanced the catalytic efficiency of endoglucanase enzyme.Keywords: Endo-1,4-β-D-glucanase, Aspergillus niger, Penicillium citrinum, UV Mutagenesis, Michaelis-Menten Kinetic Equation, Lineweaver-Burk Plot, Hanes Woolf Plot, Catalytic Efficiency

A novel pH-stable, endoglucanase (JqCel5A) isolated from a salt-lake microorganism, Jonesia quinghaiensis

Background: Endoglucanase, one of three type cellulases, can randomly cleave internal β-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene ( JqCel5A ) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance. Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Times New Roman,serif;} Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Times New Roman,serif;}

Thermodynamic and saccharification analysis of cloned GH12 endo-1,4-β-glucanase from Thermotoga petrophila in a mesophilic host.

The thermotolerant endo-1,4-β-glucanase gene, of Thermotoga petrophila RKU-1, was cloned and over-expressed in E. coli strain BL21 CodonPlus. Enzyme was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 38 kDa, by purification steps of heat treatment combined with ion-exchange column chromatography. The purified enzyme was optimally active, with specific activity of 530 Umg(-1) against carboxymethyl cellulose (CMC), at pH 6.0 and 95°C and was also stable upto 8 h at 80°C. The enzyme also showed activity against β-glucan barley: 303 %, laminarin: 13.7 %, Whatman filter paper: 0.017 % with no activity against starch and Avicel. The recombinant enzyme exhibited Km, Vmax and Kcat of 12.5 mM, 735 µmol mg-1min-1 and 2351.23 s-1, respectively against CMC as a substrate. The stable recombinant enzyme manifested half life (t1/2) of 6.6 min even at temperature as high as 97°C, with free energy of denaturation (ΔG*D), enthalpy of denaturation (ΔH*D), and entropy of denaturation (ΔS*D) of 98.2 kJ mol(-1), 528.9 kJ mol(-1), and 1.17 kJ mol(-1)K(-1), respectively at 97°C. In addition, the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for hydrolysis of CMC substrate by endo-1,4-β-glucanase were calculated at 95°C as 48.2 kJ mol(-1), 54.6 kJ mol(-1) and -17.4 J mol(-1) K(-1), respectively. The recombinant enzyme saccharified pre-treated wheat straw and bagasse to 3.32 % and 3.2 %, respectively after 6 h incubation at 85°C. Its thermostability, resistance to heavy metal ions and specific activity make this enzyme an interesting candidate for industrial applications.

Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon.

Extreme habitats serve as a source of enzymes that are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8–70°C). Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70°C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12) endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45% sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92°C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential.

키토산 비드에 고정화된 셀룰라아제의 특성

Recently, there is a growing interest in efficient biomass pretreatment and saccharification processes to produce biofuels and biochemicals from renewable non-food biomass resources. In this study, glucose was produced from cellulose by immobilizing cellulase enzyme on chitosan beads which was reported to have high pH and temperature stability. The immobilized amounts of cellulase on chitosan beads linearly increased with increasing the concentrations of cellulase solution. The glucose production increased to 7.2 g/L from 1% carboxymethyl cellulose (CMC) substrate when immobilized at 20% cellulase solution. The maximum specific activity was 0.37 unit/mg protein when immobilized at 8% cellulase solution. At pH 7 and <TEX>$37^{\circ}C$</TEX>, the optimum reaction composition was 0.5 g beads/L from 1% CMC substrate. At this condition, the conversion to glucose completed at ca. 20 min.