Hydrolysis Of Carboxymethyl Cellulose Research Articles

A Role of Cellulose Binding Module of the Thermophilic Endoglucanase TbCel12A

Endoglucanases are enzymes that play an important role in hydrolysis of lignocellulose by attacking glycosidic linkages in cellulose fibers and other glucans. The cellulose binding module (CBM) is responsible for binding the enzyme to the substrate. However, CBMs in certain enzymes interfere with substrate hydrolysis resulting in moderate or low activity. In a previous study, the processive endoglucanase TbCel12A including its CBM had low activity towards carboxymethyl cellulose (CMC). To assess the effect of the CBM, the catalytic domain of TbCel12A was produced without the CBM. The TbCel12A catalytic domain without the CBM hydrolyzed CMC 23 times more rapidly, while the pH and temperature optima and thermotolerance remained unchanged compared to full-length TbCel12A. Therefore, TbCel12A does not require the CBM for CMC hydrolysis and its application may be improved without it.

Functionalized iron oxide nanoparticles for covalent immobilization of cellic CTec2 cellulase: enabling enzyme reusability in cellulosic biomass conversion

A modified chemical co-precipitation method was employed to synthesize silicacoated-aminefunctionalized iron oxide nanoparticle (IONP@SiO2-NH2) which has been utilized as an anchoring surface for enzyme immobilization. Cellulase Cellic CTec2 cocktail was immobilized covalently onto IONP@SiO2-NH2 using glutaraldehyde as a linker. The morphological, structural, and compositional studies of nanoparticles were characterized by field emission scanning electron microscopy with energy dispersive X-ray (FESEM)-EDX, Fourier transform infrared (FTIR), X-ray diffraction (XRD), zeta potential, and Ultraviolet/visible (UV-Vis) spectroscopy. The FTIR, FESEM, and XRD spectra confirmed the successful immobilization of cellulase onto IONP@SiO2-NH2. The free enzymewith cellulase activity of 127 FPU/mL was optimized for the immobilization process which showed the best binding efficiency at 2% glutaraldehyde, protein loading 100 mg/g with carboxymethyl cellulase activity of 290 IU/g, and total immobilization yield of 70.8%. The immobilized cellulase exhibited enhanced stability for temperature and pH over a broader rangethan the free enzyme. The immobilized enzyme-mediated hydrolysis of 1% carboxymethyl cellulose (CMC) released 0.54 g glucose/g substrate at 24 h, showing potential for cellulosic biomass conversion. The immobilized cellulase which was investigated for reusability assay retained∼80% activity even after repeated 6 cycles of CMC hydrolysis. The results showed good reusability after magnetic separation, which is the main advantage of using this nanobiocatalyst system.

Manifestation of the Supramolecular Effect in the Enzymatic Hydrolysis of Sodium Carboxymethyl Cellulose

Manifestation of the Supramolecular Effect in the Enzymatic Hydrolysis of Sodium Carboxymethyl Cellulose

Purification of recombinant endoglucanase GH5-CBM72-CBM72 expressed in Escherichia coli

In a previous study, a gene GL0694641 coding for endoglucanase containing 3 domains GH5-CBM72-CBM72 was exploited from metagenomic DNA data of bacteria in Vietnamese goats’ rumen. The gene (eg5) encoding the mature enzyme (without signal peptide coding sequence) was optimized codons, artificially synthesized, and inserted into the pET22b(+) vector at NcoI and XhoI to generate expression vector pET22-eg5 for expression of the gene in Eschrichia coli. In this study, the gene eg5 was well expressed in E. coli BL21 and Rosetta 1 strains to produce recombinant endoglucanase of 77 kDa. The recombinant enzymes were expressed mainly in the soluble fractions of both strains. However, the enzyme expressed in E. coli BL21 was precipitated by imidazole at even a low concentration of 20 mM, whereas endoglucanase produced from E. coli Rosetta 1 strain was well soluble in buffers containing imidazole at concentrations of 20, 50, 200, and 250 mM. To our knowledge, this is the first study showing the negative effect of imidazole on recombinant protein. Endoglucanase expressed from strain E. coli Rosetta 1 was successfully purified by His-tag affinity chromatography using phosphate buffer saline (PBS). Protein contaminations were washed out by PBS buffer containing 20 mM and 75 mM imidazole then the target protein was harvested by the buffer containing 200 mM imidazole. After purification and desalting by the PD10 column, the recombinant endoglucanase had purity up to 97%. The pure enzyme exhibited endoglucanase activity hydrolyzing carboxymethyl cellulose in agar plate and by zymogram. The purified enzyme can be used as a material for its characterization.

Novel, acidic, and cold-adapted glycoside hydrolase family 8 endo-β-1,4-glucanase from an Antarctic lichen-associated bacterium, Lichenicola cladoniae PAMC 26568.

Endo-β-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted β-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-β-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-β-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-β-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley β-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg–1) and kcat/Km value (6.35 mg–1 s–1mL) of rGluL toward barley β-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg–1) and kcat/Km value (2.83 mg–1 s–1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-β-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

Purification, Characterization, and Application of Endoglucanase from Rhizopus oryzae as Antibiofilm Agent.

Theenzyme endoglucanase is responsible for thedepolymerizationof cellulose. This study focuses on characterization and purification of endoglucanase fromRhizopus oryzaeMTCC 9642through a simple size exclusion method and its effective application as an antibiofilm agent. Extracellular ß-1,4-endoglucanase, an enzyme that catalyzes the hydrolysis of carboxymethyl cellulose, was found to be synthesized byRhizopus oryzaeMTCC 9642. The enzyme was purified up to homogeneity simply by size exclusion process through ultrafiltration and gel chromatography. The molecular weight ofpurified enzyme protein wasestimated to be 39.8kDa and it showed the highest substrate affinity towards carboxymethyl-cellulose with Kmand Vmaxvalues of 0.833mgml-1and of 0.33mmol glucose min-1mg-1protein, respectively. The purified enzyme exhibited optimal activity at pH 6 with a broad stability range of pH 3-8. The most preferred temperature was 35°C and 50% of activity could be retained after the thermal exposure at 40°C for 25min. The purified enzyme protein was inactivated by Cu2+, while the activity could be enhanced by the addition of exogenous thiols. Since biofilm is a challenge for health sector, with the aim of eradicating the biofilm, the purified endoglucanase was used to remove biofilm produced by two nosocomial bacteria. As predicted byin silicomolecular docking interaction, the purified enzyme could effectively degrade biofilm architecture of bacterial strains S. aureus and P. aeruginosa by 76.52 ± 6.52% and 61.67 ± 8.76%, respectively. The propertiesofpurified enzyme protein, as elucidated by in vitroandin silicocharacterization,may befavourable for its commercial applications as a potent antibiofilm agent.

Rheological properties and in vitro hydrolytic stability of the carboxymethylcellulose–dioxidine composition

The composition of carboxymethylcellulose–dioxydine was obtained and its structural, morphological and rheological characteristics in the processes of heat treatment and chemical hydrolysis were studied. Using IR spectroscopy, scanning electron microscopy, and viscometry, it has been established that the introduction of dioxidine into a solution of carboxymethylcellulose sodium salt with different molecular weights leads to partial disordering of hydrogen bonds between macromolecules, promotes thermal destruction of the polysaccharide and a decrease in the dynamic viscosity of solutions. Experimental kinetic curves of carboxymethylcellulose hydrolysis in the presence and absence of dioxidine were obtained, and it was shown that they could be described using a first-order equation. The hydrolysis rate coefficients were calculated, and it was shown that, as a result of heat treatment, as well as in the presence of an active substance, the rate of carboxymethylcellulose hydrolysis increased. The change in the rheological properties of carboxymethylcellulose sodium salt solutions with different degrees of polymerization in the presence of an antimicrobial substance should be taken into account when choosing the composition of viscous prolonged forms of biologically active substances.

Facile method for preparation of oligo-carboxymethyl cellulose and other oligosaccharides: Physicochemical properties and bioactivity

Widely available enzymes, α-amylase from Aspergillus oryzae and pectinase from Aspergillus aculeatus, have been examined for their ability to perform nonspecific hydrolysis of polysaccharides chitosan, pectin, alginate, carboxymethyl cellulose (CMC), hydroxypropyl methylcellulose (HPMC) and methyl cellulose (MC) to form the corresponding oligosaccharides. The good yields of oligo-CMC (50–60%), oligo-pectin (40–60%) and oligo-alginate (25–54%) have been achieved. The hydrolysis of chitosan yielded 26-10% oligosaccharides, while HPMC and MC did not undergo significant hydrolysis process, pointing out the importance of polar groups, preferably carboxyl, for this reaction. Among the received oligomers, oligo-CMC is the least explored. Therefore, its bioactivity was examined, demonstrating a notable elicitation potential increasing the content of phenolic compounds and glucosinolates in broccoli sprouts. Furthermore, oligo-CMC was combined with H2O2 and found to diminish drawbacks and enhance advantages of this elicitor.As oligosaccharides find numerous applications in food, health, beauty and agriculture fields, sustainable and cost-effective method for their mass production is of importance. Moreover, the eliciting potential of oligo-CMC may allow its use in “super treatment” formulations that prevent damages, accelerate growth and increase content of beneficial nutrients in plants.

Differential Influence of Imidazolium Ionic Liquid on Cellulase Kinetics in Saccharification of Cellulose and Lignocellulosic Biomass Substrate

The effect of [Emim][OAc] on Celluclast 1.5 L, Accellerase 1500, and IL-tolerant (MSL2) cellulase during the saccharification of carboxymethylcellulose (CMC), Avicel (AV), rice straw (RS) was studied in one pot process (pretreatment and saccharification). The inhibition caused by [Emim][OAc] (0.5–2.0 M) with substrate loading (20–50 mg/mL) were also evaluated. In most cases, the inhibition mode of saccharification for CMC and AV was identified to be uncompetitive inhibition when the concentration of [Emim][OAc] was higher than 0.5 M. Under the influence of 0.5 M of [Emim][OAc], the Critical Concentration of Substrate (CCS) values of the Celluclast 1.5 L and Accellerase 1500 on CMC hydrolysis were determined to be at 26.59 and 33.65 mg/mL, respectively. Also, increasing in [Emim][OAc] concentration could increase in CCS values, suggesting the positive effect of [Emim][OAc] on the improvement of enzymatic saccharification. This study provides insight into the process optimization for integration of [Emim][OAc] in one pot process of biorefinery.

Glucomannan and beta-glucan degradation by Mytilus edulis Cel45A: Crystal structure and activity comparison with GH45 subfamily A, B and C

The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a β-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis, which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported.Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 °C relative to 30 °C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose.

CalkGH9T: A Glycoside Hydrolase Family 9 Enzyme from Clostridium alkalicellulosi

Glycoside hydrolase family 9 (GH9) endoglucanases are important enzymes for cellulose degradation. However, their activity on cellulose is diverse. Here, we cloned and expressed one GH9 enzyme (CalkGH9T) from Clostridium alkalicellulosi in Escherichia coli. CalkGH9T has a modular structure, containing one GH9 catalytic module, two family 3 carbohydrate binding modules, and one type I dockerin domain. CalkGH9T exhibited maximal activity at pH 7.0–8.0 and 55 °C and was resistant to urea and NaCl. It efficiently hydrolyzed carboxymethyl cellulose (CMC) but poorly degraded regenerated amorphous cellulose (RAC). Despite strongly binding to Avicel, CalkGH9T lacked the ability to hydrolyze this substrate. The hydrolysis of CMC by CalkGH9T produced a series of cello-oligomers, with cellotetraose being preferentially released. Similar proportions of soluble and insoluble reducing ends generated by hydrolysis of RAC indicated non-processive activity. Our study extends our knowledge of the molecular mechanism of cellulose hydrolysis by GH9 family endoglucanases with industrial relevance.

Differential effects of inorganic salts on cellulase kinetics in enzymatic saccharification of cellulose and lignocellulosic biomass.

Inorganic salt pretreatment of lignocellulosic biomass has proven to be an efficient way to increase the efficiency of enzymatic saccharification. However, it is not clear that this improvement is the result of modification of the lignocellulosic substrate after pretreatment, or removal of inhibitor, or enhancement of cellulase or a combination of these events. Therefore, this study aimed to analyze the effects of inorganic salts on kinetics of cellulase enzymes (celluclast 1.5L and accellerase 1500). Two substrates rich in cellulose content [carboxymethylcellulose (CMC), avicel (AV)] and lignocellulose substrate [sugarcane bagasse (SB)] were considered. The enzymatic saccharification was carried with and without the addition of inorganic salts (NaCl and KCl) at 0.5M and 1.0M concentration. The kinetic parameters, Km and Vm, were determined to mechanically understand the pattern of inhibition and enhancement of inorganic salts on enzymatic saccharification. The kinetics parameters of celluclast 1.5L and accellerase 1500 for hydrolysis of CMC and AV with NaCl showed uncompetitive inhibition. Whereas, influences of KCl on both cellulase were differentiated to function in inhibition or enhancement modes when challenged with different substrates. On the other hand, enzymatic hydrolysis efficiencies of SB using both cellulases were enhanced under addition of NaCl and KCl, by increasing Vm of celluclast 1.5L from 0.303 to 0.635mg/mLmin (0.5M KCl) and accellerase 1500 from 0.383 to 0.719mg/mLmin (1.0M NaCl). The details of kinetic analysis in this work revealed the mechanism of inorganic salts on cellulase kinetics to be involved in substrate modification and removal of inhibitor.

Characterization and Cellulase Production Activity of different Aspergillus Sp. Isolated from “Puri, Odisha” Marine Fungal Strains

Cellulose undergoes hydrolysis utilizing chemicals to deliver glucose, which might be utilized for the production of ethanol, organic acids, and various chemicals. Cellulases are a collection of hydrolytic catalysts that can hydrolyze the most plentiful natural polymer for example cellulose into smaller sugar components including glucose subunits. Cellulase is overpriced and contributes simply half to the general expense of hydrolysis because of the low explicit activity. This enzyme has enormous potential in industries and its use in food, beverages, textile, laundry, paper, and mash industries, and so forth. Consequently, there has been a lot of examination focused on new microorganisms producing cellulose enzymes with higher specific activities and greater efficiency. Currently, work is pointed toward screening and disconnecting cellulolytic growths from the marine samples gathered from the Bay of Bengal, Puri coast, Odisha. All out 7 fungi were isolated from these dirt examples, out of which 2 fungi were portraying the extensive cellulase activity. The fungal isolate, for example, MWF-1 andMSF-6 isolated from water silt respectively were recognized to show the most extreme zone of hydrolysis of carboxy-methyl cellulose. The cellulase activity was assayed by Carboxymethylcellulose "CMCase" (endoglucanase) measure.

Purification and characterization of novel, thermostable and non-processive GH5 family endoglucanase from Fomitopsis meliae CFA 2

Endoglucanases from glycoside hydrolase family 5 (GH5) are the key enzymes in degradation of diverse plant polysaccharides. Present study reports purification, characterization and partial sequencing of novel thermostable GH5 family endoglucanase from a newly isolated brown rot fungi Fomitopsis meliae CFA 2. Endoglucanase was purified 34.18 fold with a specific activity of 302.90 U/mg. The molecular weight of the endoglucanase was 37.87 kDa as determined by SDS PAGE. LC MS/MS analysis identified the protein to be a member of GH5_5 family. The temperature and pH optima for endoglucanase activity were 70 °C and 4.8, respectively. The enzyme catalyzed the hydrolysis of carboxymethyl-cellulose with a Km of 12.0 mg/ml, Vmax of 556.58 μmol/min/mg and Kcat of 129.41/sec. The enzyme was stimulated by Zn+2 and K+ metal ions and DTT. Half-life (t1/2) for endoglucanase was found to be 11.36 h with decimal reduction time (D) of 37.75 h at 70 °C. The activation energy for endoglucanase was found to be 30.76 kJ/mol (50 °C–70 °C). Looking at the results, the endoglucanase from Fomitopsis meliae CFA 2 seems to be a promising thermostable enzyme which may be applicable in applications like biomass hydrolysis.

Production of cellulase enzyme by Aspergillus niger, Aspergillus terreus and Penicillium sp. isolated from soil

This research was aimed to isolate cellulolytic fungi showing hyper-cellulase activity. Potential cellulase-producing fungi were isolated from different soil samples, particularly near agro-wastes dumping sites. Among the various isolates obtained from different sampling sites, three different fungi were selected depending upon the clear zone diameter produced in Carboxymethyl Cellulose (CMC) agar. CMC hydrolysis by all three fungal strains exhibited their activities at pH 5.5-7.5, whereas maximum activity occurred at pH 5.5. Enzymes were also pH stable. Based on K m and V max values, the endoglucanase and β-glucosidase enzymes of Aspergillus niger were most efficient. The characterization of these fungi may also provide an opportunity to screen the cellulase enzymes for their further utilization in bio-ethanol production using lignocellulosic agro-wastes.

Characterization of FsXEG12A from the cellulose-degrading ectosymbiotic fungus Fusarium spp. strain EI cultured by the ambrosia beetle

Despite the threat of Fusarium dieback posed due to ambrosia fungi cultured by ambrosia beetles such as Euwallacea spp., the wood-degradation mechanisms utilized by ambrosia fungi are not fully understood. In this study, we analyzed the 16S rRNA and 18S rRNA genes of the microbial community from the Ficus tree tunnel excavated by Euwallacea interjectus and isolated the cellulose-degrading fungus, Fusarium spp. strain EI, by enrichment culture with carboxymethyl cellulose as the sole carbon source. The cellulolytic enzyme secreted by the fungus was identified and expressed in Pichia pastoris, and its enzymatic properties were characterized. The cellulolytic enzyme, termed FsXEG12A, could hydrolyze carboxymethyl cellulose, microcrystalline cellulose, xyloglucan, lichenan, and glucomannan, indicating that the broad substrate specificity of FsXEG12A could be beneficial for degrading complex wood components such as cellulose, xyloglucan, and galactoglucomannan in angiosperms. Inhibition of FsXEG12A function is, thus, an effective target for Fusarium dieback caused by Euwallacea spp.

Enhanced cellulase recovery in SSF from Rhizopus oryzae SN5 and immobilization for multi-batch saccharification of carboxymethylcellulose

In this study enhanced the cellulase recovery was achieved from wheat bran solid state fermentate of a locally isolated fungus, Rhizopus oryzae SN5 by using “one factor at a time” technique. Interestingly, 18.9% enhanced cellulase production (452.16 ± 2.38 U/gds) was observed with extraction optima of Na-citrate buffer (50 mM, pH 4.8), 1:10 solid: solvent ratio, 90 min soaking time at 40 °C. Further, cellulase was partially purified, where in 5.73-fold enhanced specific activity was achieved with product recovery of 7.1%. Moreover, the molecular weight of the purified cellulase was determined as~80 kDa using SDS-PAGE and zymogram analysis. Furthermore, Ca-alginate immobilized cellulase (IMC) exhibited increased Km (2.2 ± 0.07 to 2.78 ± 0.09 mg/ml) and decreased Vmax (31.36 ± 1.37 to 27.12 ± 1.21 μmol/min/ml) than free enzyme. In addition, the turn over number (Kcat) was found to be increased (542.4 ± 14.38 to 625.2 ± 12.11 Min−1), whereas catalytic efficiency (Kcat/Km) was decreased (246.54 ± 8.34 to 224.89 ± 9.07 Min−1.mg−1.ml−1) for IMC. The pH and thermal stability of IMC was found to be 30–40% higher in comparison to the free cellulase. Notably, the IMC displayed, easy recovery and high reusability for Carboxymethylcellulose (CMC) hydrolysis, with 70.6% cellulase activity retention after five successive cycles of their reuse. Taken together, the optimized extraction process and developed IMC could be used in cellulose based industries to cut down the saccharification cost.

Impact of ultrasonic irradiation pretreatment of water-soluble cellulose on enzymatic hydrolysis

This study investigated the effects of ultrasonic irradiation on the enzymatic hydrolysis of water-soluble cellulose. Two types of cellulases (from Aspergillus niger and Trichoderma reesei) were employed for the enzymatic hydrolysis of carboxymethyl cellulose (CMC). An assay of the enzymatic hydrolysis of water-soluble cellulose was evaluated based on the production of glucose. The optimum set of operating conditions for ultrasonic irradiation involved an ultrasonic irradiation power and time of 116 W and 30 min, respectively. The initial reaction rate of enzymatic hydrolysis of CMC was increased with ultrasonic pretreatment (116 W) compared to that of the ultrasonication-free system. The kinetic parameters (Michaelis–Menten constant; Km and maximum reaction rate; Vmax) were examined to evaluate the reactivity. As a result of ultrasonic irradiation, the value of Km was decreased to 0.2 times its original value in the system employing A. niger, and to 0.6 times its original value in the system employing T. reesei. Ultrasonication increased the value of Vmax to 1.3 times its original value for both cellulases. Moreover, the viscosity of the CMC solution was decreased by ultrasonic irradiation, decreasing the activation energy of the reaction and increasing its frequency factor. Ultrasonic irradiation can therefore be employed in process design to control viscosity and achieve high-performance enzymatic hydrolysis of water-soluble cellulose. Ultrasonication is an effective application for accelerating enzymatic hydrolysis of cellulose and design lower energy industrial processes for cellulose degradation.

Purification and Biochemical Characterization of Alkalophilic Cellulase from the Symbiotic Bacillus subtilis BC1 of the Leopard Moth, Zeuzera pyrina (L.) (Lepidoptera: Cossidae)

In the current study,an extracellular cellulase belonging to symbiotic Bacillus subtilis Bc1 of the leopard moth is purified and characterized. The molecular mass of enzyme was 47.8kDa using SDS-PAGE. The purified enzyme had optimum activity in temperature and pH around 60°C and 8, respectively. The purified cellulase was introduced as a stable enzyme in a wide variety of temperature (20-80°C) and pH (4-10) and remained active to more than 74% at 80°C for 1h. Moreover, the cellulase extremely was stabled in the presence of metal ions and organic solvents and its activity was increased by acetone (20% v/v), CaCl2 and CoCl2 and inhibited by MnCl2 and NiCl2. The values of enzyme's Km and Vmax were found to be 1.243mg/mL and 271.3µg/mL/min, respectively. The purified cellulase hydrolyzed cellulose, avicel and carboxymethyl cellulose (CMC) and the final product of CMC hydrolysis was cellobiose using thin-layer chromatography analysis. Consequently, owing to exo/endoglucanase activity and organic solvent, temperature and pH stability of the purified cellulase belong to B. subtilis BC1, it can be properly employed for various industrial purposes.

Discovery and Expression of Thermostable LPMOs from Thermophilic Fungi for Producing Efficient Lignocellulolytic Enzyme Cocktails.

In this study, two novel thermostable lytic polysaccharide monooxygenases (LPMOs) were cloned from thermophilic fungus Scytalidium thermophilum (PMO9D_SCYTH) and Malbranchea cinnamomea (PMO9D_MALCI) and expressed in the methylotrophic yeast Pichia pastoris X33. The purified PMO9D_SCYTH was active at 60 °C (t1/2 = 60.58 h, pH 7.0), whereas, PMO9D_MALCI was optimally active at 50 °C (t1/2 = 144 h, pH 7.0). The respective catalytic efficiency (kcat/Km) of PMO9D_SCYTH and PMO9D_MALCI determined against avicel in presence of H2O2 was (6.58 × 10-3 and 1.79 × 10-3 mg-1 ml min-1) and carboxy-methylcellulose (CMC) (1.52 × 10-1 and 2.62 × 10-2 mg-1 ml min-1). The HRMS analysis of products obtained after hydrolysis of avicel and CMC showed the presence of both C1 and C4 oxidized oligosaccharides, in addition to phylogenetic tree constructed with other characterized type 1 and 3 LPMOs demonstrated that both LPMOs belongs to type-3 family of AA9s. The release of sugars during saccharification of acid/alkali pretreated sugarcane bagasse and rice straw was enhanced upon replacing one part of commercial enzyme Cellic CTec2 with these LPMOs.