Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts

β-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of β-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or β-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for β-1,3-1,4 glucans, showing maximum activity on barley β-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley β-glucan and lichean were 3-O-β-cellotriosyl-D-glucose and 3-O-β-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for β-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase.     

Degradation of cellulose by the major endoglucanase produced from the brown-rot fungus Fomitopsis pinicola

An endoglucanase that is able to degrade both crystalline and amorphous cellulose was purified from the culture filtrates of the brown-rot fungus Fomitopsis pinicola grown on cellulose. An apparent molecular weight of the purified enzyme was approximately 32 kDa by SDS-PAGE analysis. The enzyme was purified 11-fold with a specific activity of 944 U/mg protein against CMC. The partial amino acid sequences of the purified endoglucanase had high homology with endo-beta-1,4-glucanase of glycosyl hydrolase family 5 from other fungi. The K(m) and K(cat)values for CMC were 12 mg CMC/ml and 670/s, respectively. The purified EG hydrolyzed both cellotetraose (G4) and cellopentaose (G5), but did not degrade either cellobiose (G2) or cellotriose (G3).     

Molecular cloning,expression, and characterization of an endo-beta-1,4-glucanase cDNA from Epidinium caudatum1

An endo-beta-1,4-glucanase gene (epi3) from the rumen ciliated protozoan Epidinium caudatum was cloned from a cDNA library constructed by using the lambda ZAP II vector. The enzymatic activity of the gene product was detected by the Congo red assay, using carboxymethyl cellulose (CMC) as substrate. The nucleotide sequence of epi3 revealed 1,253 nucleotides with an open reading frame for a protein (Epi3) of 356 amino acids (Mr -41,014). Epi3 shows high homology with family 5 endoglucanase genes and with genes from protozoa isolated from sources other than the rumen. The specific activity of Epi3 produced in Escherichia coli was 5.544, 2.754, and 0.295 mmol of glucose min(-1) mg(-1) protein when the substrates used were CMC, beta-glucan, and xylan, respectively. A beta-1,4-linked trisaccharide of glucose was the preferred substrate of Epi3, as determined by analysis with the p-nitrophenyl form of the substrate. To our knowledge, this is the first report of the isolation of an endoglucanase gene from a rumen protozoan.     

Production and partial purification of cellulase from a novel fungus,Aspergillus flavus BS1

This study describes the isolation and characterization of a novel fungus, Aspergillus flavus BS1 and its cellulolytic activities with special emphasis on endoglucanase production. Preliminary screening studies showed that A. flavus BS1 was a potent strain for the production of cellulase. To study the cellulolytic activities in detail by submerged fermentation (SmF), productions of endoglucanase, exoglucanase, and β-glucosidase were estimated from the basal salt medium (BSM) supplemented with 1 % carboxy methyl cellulose (CMC). CMC medium supported the maximum yield of endoglucanase (2,793 U/ml) on day 5 of incubation at 28 °C and 150 rpm, which was higher than that obtained with naturally available supplements (flour) from banana, tapioca, potato, or banana peel. During cellulase production by solid-state fermentation, 10 % (w/w) tapioca flour in sawdust (teak wood) moisturized with BSM (1:2, w/v) supported maximum cellulase yield (5,408 U/g dry substrate) on day 3 at 28 °C, which was 2-fold higher than that obtained during SmF. The active cellulase was qualitatively estimated by polyacrylamide gel electrophoresis (PAGE). Native-PAGE (0.25 % CMC impregnated on the 10 % gel) activity staining with congo-red showed a clear zone for CMCase activity, whereas SDS-PAGE showed a distinct band. In conclusion, this study showed that A. flavus strain BS1 is a potent strain for the production of cellulase on lignocellulosic media, the hot enzyme for bioethanol production from the lignocellulosic biomass by SSF.     

Sequence of celQ and properties of CelQ, a component of the Clostridium thermocellum cellulosome

The nucleotide sequence of the Clostridium thermocellum F1 celQ gene, which codes for the endoglucanase CelQ, consists of 2,130 bp encoding 710 amino acids. The precursor form of CelQ has a molecular weight of 79,809 and is composed of a signal peptide, a family 9 cellulase domain, a family IIIc carbohydrate-binding module (CBM), and a dockerin domain. Truncated derivatives of CelQ were constructed: CelQdeltadoc consisted of the catalytic domain and the CBM; CelQcat consisted of the catalytic domain only. CelQdeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan and low activity toward Avicel, acid-swollen cellulose, lichenan, and xylan. The Vmax and Km values were 235 micromol/min/mg and 3.3 mg/ml, respectively, for CMC. By contrast, CelQcat, which was devoid of the CBM, showed negligible activity toward CMC, i.e., about 1/1,000 of the activity of CelQdeltadoc, supporting the previously proposed idea that family IIIc CBMs participate in the catalytic function of the enzyme. Immunological analysis using an antiserum raised against CelQdeltadoc confirmed that CelQ is a component of the C. thermocellum cellulosome.     

Endo-1,4-beta-xylanase B from Aspergillus cf. niger BCC14405 isolated in Thailand: purification, characterization and gene isolation

During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of 55 degrees C. When tested using xylan from birchwood, it showed K(m) and V(max) values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by CuSO(4) EDTA, and by FeSO(4) The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-beta-xylanase. The full-length gene encoding endo-1,4-beta-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-a-xylanase B previously identified in A. niger.     

Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan

The mature peptide of Aspergillus niger xylanase A (AnxA) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant AnxA (reAnxA) was secreted into culture medium. After 96-h 0.25% methanol induction, the activity of reAnxA in the culture supernatant reached the peak, 175 U/mg, which was 1.9 times as high as that of the native AnxA (92 U/mg). Studies on enzymatic properties showed that the optimum temperature and optimum pH of reAnxA were 50 degrees C and 5.0, respectively. The reAnxA was very stable in a wide pH range of 3.0-8.0. After incubation at the pH 3.0-8.0, 25 degrees C for 1h, all the residual activities of reAnxA were over 80%. The K(m) and k(cat) values for reAnxA were 4.8 mg/ml and 123.2s(-1), respectively. HPLC analysis showed that xylotriose was the main hydrolysis product of birchwood xylan and bran insoluble xylan by reAnxA.     

Kinetic and thermodynamic study of cloned thermostable endo-1,4-β-xylanase from Thermotoga petrophila in mesophilic host

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(?)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.     

Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal-0.77% (w/v); (NH(4))(2)SO(4)-0.1m; KH(2)PO(4)-0.05 m; rice bran oil-2% (v/v); CaCl(2)-0.05 m; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35°C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6-fold increase in lipase production was achieved. Partial purification by (NH(4))(2)SO(4) precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40°C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.     

Molecular cloning and expression of a novel family A endoglucanase gene from Fibrobacter succinogenes S85 in Escherichia coli

A Fibrobacter succinogenes S85 gene that encodes endoglucanase hydrolysing CMC and xylan was cloned and expressed in Escherichia coli DH5 by using pUC19 vector. Recombinant plasmid DNA from a positive clone hydrolysing CMC and xylan was designated as pCMX1, harboring 2,043 bp insert. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The nucleotide sequence accession number of the cloned gene sequence in Genbank is U94826. The endoglucanase gene cloned in this study does not have amino sequence homology to the other endoglucanase genes from F. succinogenes S85, but does show sequence homology to family 5 (family A) of glycosyl hydrolases from several species. The ORF encodes a polypeptide of 654 amino acids with a measured molecular weight of 81.3 kDa on SDS-PAGE. Putative signal sequences, Shine-Dalgarno-type ribosomal binding site and promoter sequences (-10) related to the consensus promoter sequences were deduced. The recombinant endoglucanase by E. coli harboring pCMX1 was partially purified and characterized. N-terminal sequences of endoglucanase were Ala-Gln-Pro-Ala-Ala, matched with deduced amino sequences. The temperature range and pH for optimal activity of the purified enzyme were 55 approximately 65 degrees C and 5.5, respectively. The enzyme was most stable at pH 6 but unstable under pH 4 with a K(m) value of 0.49% CMC and a V(max) value of 152 U/mg.     

Overproduction and characterization of xylanase B from Aspergillus niger

The xynB gene, which encodes endo-beta-1,4-xylanase XynB, in Aspergillus niger BRFM281 was amplified by RT-PCR using mRNA isolated from a culture containing sugar beet pulp as an inducer. The cDNA was cloned into an expression cassette under the control of the strong and constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme XynB with a six-histidine peptide fused to the carboxy end of the protein. Homologous overproduction of XynB was successfully achieved in shake flask cultures, and the secretion yield was estimated to be 900 mg x L(-1). The recombinant XynB was purified 1.5-fold by immobilized metal affinity chromatography to homogeneity using a one-step purification protocol with 71% recovery. The purified recombinant enzyme was fully characterized and has a molecular mass of 23 kDa and an optimal activity at pH 5.5 and 50 degrees C with stability in the pH range 4.0-7.0 and temperature up to 50 degrees C. Using soluble oat spelts xylan, the determined Km and Vmax values were 7.1 mg x mL(-1) and 3881 U x mg(-1), respectively.     

Isolation and Characterization of a Novel Endoglucanase from a Bursaphelenchus xylophilus Metagenomic Library

A novel gene (designated as cen219) encoding endoglucanase was isolated from a Bursaphelenchus xylophilus metagenomic library by functional screening. Sequence analysis revealed that cen219 encoded a protein of 367 amino acids. SDS-PAGE analysis of purified endoglucanase suggested that Cen219 was a monomeric enzyme with a molecular mass of 40 kDa. The optimum temperature and pH for endoglucanase activity of Cen219 was separately 50°C and 6.0. It was stable from 30 to 50°C, and from pH 4.0 to 7.0. The activity was significantly enhanced by Mn²⁺ and dramatically reduced by detergent SDS and metals Fe³⁺, Cu²⁺ or Hg²⁺. The enzyme hydrolyzed a wide range of β-1, 3-, and β-1, 4-linked polysaccharides, with varying activities. Activities towards microcrystalline cellulose and filter paper were relatively high, while the highest activity was towards oat gum. The K_m and V_max of Cen219 towards CMC was 17.37 mg/ml and 333.33 U/mg, respectively. The findings have an insight into understanding the molecular basis of host–parasite interactions in B. xylophilus species. The properties also make Cen219 an interesting enzyme for biotechnological application.     

Cloning,heterologous expression,and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii

The gene encoding a thermostable glucoamylase from Talaromyces emersonii was cloned and, subsequently, heterologously expressed in Aspergillus niger. This glucoamylase gene encodes a 618 amino acid long protein with a calculated molecular weight of 62,827Da. T. emersonii glucoamylase fall into glucoside hydrolase family 15, showing approximately 60% sequence similarity to glucoamylase from A. niger. The expressed enzyme shows high specific activity towards maltose, isomaltose, and maltoheptaose, having 3-6-fold elevated k(cat) compared to A. niger glucoamylase. T. emersonii glucoamylase showed significantly improved thermostability with a half life of 48h at 65 degrees C in 30% (w/v) glucose, compared to 10h for glucoamylase from A. niger. The ability of the glucoamylase to hydrolyse amylopectin at 65 degrees C is improved compared to A. niger glucoamylase, giving a significant higher final glucose yield at elevated temperatures. The increased thermal stability is thus reflected in the industrial performance, allowing T. emersonii glucoamylase to operate at a temperature higher than the A. niger enzyme.     

Chitinolytic activity of filamentous fungi

The chitinolytic activity of nine species of filamentous fungi, classified with seven genera (specifically, Aspergillus, Penicillium, Trichoderma, Paecilomyces, Sporotrichum, Beaueria, and Mucor), was studied. When cultured in liquid medium containing 1% crystalline chitin, all fungi produced extracellular chitosans with activity varying from 0.2 U/mg protein (Sporotrichum olivaceum, Mucor sp., etc.) to 4.0-4.2 U/mg protein (Trichoderma lignorum, Aspergillus niger).     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by β-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.     

Purification and characterization of an organic solvent-tolerant alkaline cellulase from a halophilic isolate of Thalassobacillus

An extracellular cellulase from Thalassobacillus sp. LY18 was purified 4.5-fold with a recovery of 21 % and a specific activity of 52.4 U mg(-1) protein. Its molecular mass was 61 kDa estimated by SDS-PAGE. It was an endoglucanase for soluble cellulose with optimal activity was at 60 °C and pH 8 with 10 % (w/v) NaCl. It was stable from 30 to 80 °C and from pH 7 to 11 with NaCl from 5 to 17.5 % (w/v). EDTA inhibited activity indicating it was a metalloenzyme. Inhibition by diethyl pyrocarbonate and β-mercaptoethanol suggested that histidine residues and disulfide bonds may play important roles in its catalytic function. The cellulase was highly active in non-ionic surfactants and was stable in water-insoluble organic solvents with log P (ow) ≥ 2.13.     

Energy-driven uptake of humic acids by Aspergillus niger.

The uptake of humic acids by mycelia of Aspergillus niger was demonstrated to be energy-dependent with a sensitivity to sodium azide and to 2,4-dinitrophenol. Greater uptake of humic acids by submerged mycelium occurred at pH 3.0 and at 32 degrees C. The rate of uptake was influenced by the concentration of humic acids with an apparent Km of 0.2 grams/ml and with a Vmax of 0.13 mg humic acids per gram mycelial dry weight.10 min-1. In the absence of added energy source, Vmax of 0.05 mg humic acids per gram mycelial dry wt.10 min-1 was obtained; however, the affinity for humic acids by this uptake system was the same as for the energy-driven process. Apparent binding of humic acids to cell structures was indicated because only 41.8% of the humic acids taken up by the energy-dependent system could be recovered.