Functional Stabilization of Cellulase from Aspergillus niger by Conjugation with Dextran-aldehyde

The covalent conjugates of cellulase from Aspergillus niger were prepared with various molar ratios by using dextran. The conjugate (n_E/n_D: 1/5) showed higher activity than purified enzyme at all temperatures after 1 h of incubation and its activity could also be measured at higher temperature. Also, this conjugate lost only 60% activity in 2 h at 70°C in comparison to the purified enzyme, which lost all its activity. In addition, conjugation protected cellulase against denaturation in the presence of sodium dodecylsulfate (residual activity of about 80%) and inactivation by air bubbles (residual activity of about 50% for 4 h).     

Separation of enzymes by sequential macroaffinity ligand-facilitated three-phase partitioning

Pectinase and cellulase were separated from a commercial enzyme preparation called Pectinex Ultra SP-L. This was carried out using a process called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this method, a water-soluble polymer is floated as an interfacial precipitate by adding ammonium sulfate and tert.-butanol. The polymer (appropriately chosen) in the presence of an enzyme for which it shows affinity, selectively binds to the enzyme and floats as a polymer-enzyme complex. In the first step, pectinase was purified (with alginate as the polymer) 13-fold with 96% activity recovery. In the second MLFTPP step, using chitosan, cellulase was purified 16-fold with 92% activity recovery. Both preparations showed a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis. This illustrative example shows that the strategy of sequential MLFTPP can be used to separate important biological activities from a crude broth.     

Production,purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60 degrees C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0, retaining >80% of its original activity within this range. Half-lives of 150 min at 50 degrees C and 6.5 min at 60 degrees C were found. K(m) and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birchwood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-D-glucuronoxylan with a K(m) of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

纤维素酶的二步分离纯化新工艺

 以普通定性滤纸为底物 ,经碱处理后 ,研究其对纤维素酶的亲和吸附作用。结果表明 ,普通定性滤纸对纤维素酶具有比较强的特异性吸附作用 ,能够从粗酶液中分离出纤维素酶 ,再经POROS 2 0HQ阴离子交换柱纯化后即可得到电泳纯的纤维素酶。该法大大简化了传统的纤维素酶纯化工艺 ,所得的纤维素酶活力极高 ,比活达 35 0U/mg以上 ,滤纸一步吸附后纤维素酶的纯化倍数为 9 5 5 ,活性回收率在 10 %左右。纯化后的纤维素酶为内切 β 葡聚糖酶 ,相对分子质量为 6 0 0 0 0 ,最佳 pH为 4 0 ,最佳温度为 70℃。     

Interaction between indigo and adsorbed protein as a major factor causing backstaining during cellulase treatment of cotton fabrics

A model microassay system was developed to measure indigo backstaining on cotton fabrics in the presence of enzymes on a small laboratory scale. Backstaining indexes for 11 cellulase samples were measured, and the enzymes were ranked from lower to higher backstaining. Two multienzyme cellulase preparations were separated into fractions using chromatofocusing on a Mono P column. Adsorption ability and backstaining properties of purified enzyme fractions were studied. Evidence was obtained that protein adsorption on cotton fabrics is a crucial parameter causing backstaining (both for crude cellulase samples and purified enzyme components).     

Production and Optimization of Physicochemical Parameters of Cellulase Using Untreated Orange Waste by Newly Isolated <Emphasis Type="Italic">Emericella variecolor</Emphasis> NS3

Cellulase enzymes have versatile industrial applications. This study was directed towards the isolation, production, and characterization of cellulase enzyme system. Among the five isolated fungal cultures, Emericella variecolor NS3 showed maximum cellulase production using untreated orange peel waste as substrate using solid-state fermentation (SSF). Maximum enzyme production of 31 IU/gds (per gram of dry substrate) was noticed at 6.0 g concentration of orange peel. Further, 50 °C was recorded as the optimum temperature for cellulase activity and the thermal stability for 240 min was observed at this temperature. In addition, the crude enzyme was stable at pH 5.0 and held its complete relative activity in presence of Mn²⁺ and Fe³⁺. This study explored the production of crude enzyme system using biological waste with future potential for research and industrial applications.     

Optimization,Purification, and Characterization of Extracellular Mesophilic Alkaline Cellulase from Sponge-Associated <Emphasis Type="Italic">Marinobacter</Emphasis> sp. MSI032

Marinobacter sp. (MSI032) isolated from the marine sponge Dendrilla nigra was optimized for the production of extracellular cellulolytic enzyme (CMCase) by submerged fermentation. Initial experiments showed that the culture medium containing 1% maltose as carbon source and 1% peptone and casein as nitrogen source supported maximal enzyme production at 27 °C and at a pH of 9.0. Further optimization carried out showed the maximal enzyme production was supported by the presence of 2% NaCl and 10 mM Zn²⁺ ions in the production media. The production of enzyme cellulase occurred at 48 h of incubation which proved the importance of this strain for cellulase production in large scale. Further, the enzyme was purified to 12.5-fold with a 37% yield and a specific activity of 2,548.75 U/mg. The purified enzyme displayed maximum activity at mesophilic temperature (27–35 °C) and at a broad pH range with optimal activity at pH 9.0. The purified enzyme was stable even at a higher alkaline pH of 12.0 which is greater than the pH stability that has not been reported in any of the cellulolytic isolates studied so far. Thus, from the present study, it is crucial that, instead of exploring the thermophilic resource that is limited in natural environments, the mesophilic bacteria that occurs commonly in nature can be added up to the database of cellulolytic bacteria. Thus, it is possible that a wide diversity of mesophilic bacteria associated with marine sponges opens up a new doorstep for the degradation of cellulosic waste material for the production of liquid fuels. This is the first report elucidating the prospects of sponge-associated marine bacterium for the production of extracellular alkaline cellulase.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

Pretreatment of sugar cane bagasse hemicellulose hydrolyzate for ethanol production by yeast

The cellulase system ofBacillus circulans F-2 effectively hydrolyzed carboxymethyl cellulose (CMC), xylan, avicel, cellobiose, filter paper, cotton, andp-nitrophenyl-Β-D-cellobioside, and the crude enzyme produced mainly glucose from digestion of avicel. Two major and one minor peaks of enzyme activities were eluted on DEAE ion-exchange chromatography, and designated cellulase complex I(C-I) and complex II(C-II) for the two major peaks, and cellulase-III for a minor peak. C-I and C-II were further purified on gel filtration column of a TSK-Gel SW G3000 ×L. The molecular masses of C-I and C-II were estimated to be about 669 and 443 kDa, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the C-I and C-II complexes showed that the C-I complex was present as a multiple protein complex, consisting of at least five CMCases and two xylanases, and that the C-II complex was consisted of at least three CMCase and four xylan ases. C-I showed high activities of cellohydrolase, CMCase, xylanase, and Β-glucosidase, whereas C-II showed high activities of CMCase, xylanase, avicelase, and Β-glucosidase. The outstanding property of the C-II was its high hydrolytic activity toward filter paper, a highly resistant substrate against enzymatic degradation. However, cellulaseIII showed only strong avicelase activity. These results indicated that the cellulase system of the strain exists as multiple complex forms.     

Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60°C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0 retaining >80% of its original activity within this range. Half-lives of 150 min at 50°C and 6.5 min at 60°C were found. K _m and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birch wood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-d-glucuronoxylan with a K _m of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

表面活性剂对脂肪酶活性和选择性的影响

考察了几种表面活性剂对lipaseOF粗酶和纯化酶催化拆分酮基布洛芬的影响。除吐温-80,吐温-60和壬基酚聚氧乙烯醚外,大部分表面活性剂对酶活性有抑制作用,其中只有吐温-80能显著提高酶的立体选择性。酶的活性和选择性与表面活性剂浓度有关。在表面活性剂浓度为最佳(20mg/mL吐温-80或30mg/mL壬基酚聚氧乙烯醚)时lipaseOF粗酶的活性可分别提高13和15倍。加入80mg/mL吐温-80,粗酶和纯化酶的对映体选择率(E值)分别由1.1和8.0增至6.7和>100。     

Water Hyacinth as Carbon Source for the Production of Cellulase by <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Water hyacinth (Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation, and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity of 0.22 ± 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend.     

Solid-phase synthesis of a farnesylated CaaX peptide library: inhibitors of the Ras CaaX endoprotease

A solid-phase method, based on Kaiser's p-benzophenone oxime resin, was developed for the synthesis of a series of N-acetyl-S-(E, E-farnesylated) Ca(1)a(2)X tetrapeptides as potential inhibitors of recombinant Ras and a-factor converting enzyme (RCE). N-Acetyl-S-(E, E-farnesyl)-L-cysteine was coupled to resin-bound a(1)a(2) dipeptide using HOBt/DCC activation in conjunction with N-BOC chemistry. The protected farnesylated tripeptide was cleaved from the resin with simultaneous addition of the X residue by treating the resin-bound farnesylated Ca(1)a(2) tripeptide with L-amino acid benzyl ester tosylates under mildly acidic conditions. The benzyl ester was saponified, and the resulting carboxylate precipitated by ether to afford a library of tetrapeptides as a mixture of diastereomers at the cysteine center. The peptides were evaluated as inhibitors of recombinant yeast RCE endoprotease (yRCE) to obtain information about the affinity of the enzyme for the a(1)a(2)X portion of the Ca(1)a(2)X moiety.     

Efficient Production of γ-GABA Using Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ～47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg^?1. The purified cellulase retained its activity over a wide range of temperature (50–70 °C) and pH (3–7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.     

A Novel Stepwise Recovery Strategy of Cellulase Adsorbed to the Residual Substrate after Hydrolysis of Steam Exploded Wheat Straw

Cellulase distribution between residual substrate and supernatant in the process of enzymatic hydrolysis of steam-exploded wheat straw was investigated. Subsequently, a novel stepwise recovery strategy with three successive steps was adopted to recover cellulase adsorbed to the residual substrate. The results showed that cellulase protein in the supernatant increased as the hydrolysis time increased. When hydrolysis ended, the cellulase remaining on the residual substrate accounted for 33–42% of the original added cellulase according to the different cellulase loading. To obtain the maximum cellulase recovery rate, the residual substrate was dealt with in three successive steps: washed with sodium acetate buffer (step 1), shaken with sodium acetate buffer (step 2), and then treated with 0.0015 mol/L, pH 10 Ca(OH)₂ (step 3). The total cellulase protein recovered by the three steps reached 96.70–98.14%. The enzyme activity of cellulase recovered by the first two steps was kept well. The ratios of the specific activity between the recovered cellulase and the original were 89–96%, which was by far higher than that using step 3 (the value was 48% ∼ 56%).     

Enzymatic kinetic of cellulose hydrolysis

The ethanol effect on the Trichoderma reesei cellulases was studied to quantify and clarify this inhibition type. To determine inhibition parameters of crude cellulase and purified exoglucanase Cel7A, integrated Michaelis-Menten equations were used assuming the presence of two inhibitors: cellobiose as the reaction product and ethanol as a possible bioproduct of cellulose fermentation. It was found that hydrolysis of cellulose by crude enzyme follows a model that considers noncompetitive inhibition by ethanol, whereas Cel7A is very slightly competitively inhibited. Crude cellulase is much more inhibited (K _iul=K _icl=151.9 mM) than exoglucanase Cel7A (K _icl=1.6 × 10¹⁵ mM). Also, calculated inhibition constants showed that cellobiose inhibition is more potent than ethanol inhibition both for the crude enzyme as well as exoglucanase Cel7A.     

Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization

A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase at 100°C was 60min. The enzyme was free from cellulase activity. K _m and V _max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min⁻¹ mg⁻¹, respectively. The enzyme was activated by Ag⁺, Co²⁺, and Cu²⁺; on the other hand, Hg²⁺, Ba²⁺, and Mn²⁺ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Purification and Biochemical Characterization of a Novel Thermo-stable Carboxymethyl Cellulase from Azorean Isolate Bacillus mycoides S122C

Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76?%, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62?kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by ??-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50?°C and pH?7.0, respectively. The purified CMcellulase was stable with about 60?% activity in broad pH ranges from 5 to 10 and temperature of 40 to 60?°C. However, purified CMcellulase was stable at about 70?% at 70?°C and also stable overall at 78?% for surfactants. CMcellulase activity was inhibited by ions such as HgCl₂, followed by CuSo₄, FeCl₂, and MnCl₂, while CoCl₂ activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.     

Purification and partial characterization of CMP-Neu5Ac synthetase from rat brain

N-Acetyl-neuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase), which catalyzes the formation of cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac) from cytidine-5′-triphosphate (CTP) and N-acetyl-neuraminic acid (Neu5Ac), was purified from rat brains aged 8-9 days, which presented the highest specific activity, and partially characterized. Partial protein fractionation in the crude extract was achieved by using 40-60% ammonium sulphate. Subsequently, CMP-Neu5Ac synthetase was purified by column chromatography on Sephacryl S-200 (gel filtration), Yellow-86-Agarose (affinity) and Phenyl-Sepharose (hydrophobic affinity). The pure enzyme had a specific activity of 3.6555 U/mg of protein and was purified 1662-fold, with an 18% yield. The purified CMP-Neu5Ac synthetase had a molecular weight of about 46 ± 1 kDa. Its purity was confirmed by sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). The active enzyme chromatographed on a gel filtration column at 190 kDa, suggesting it exists in its native form as a tetramer. The greatest activity of enzyme was observed a temperature of 40 °C for a period of 45 min of incubation, revealing a certain thermal stability. The enzyme was found to remain stable in the pH range 8.5-9.5 at 40 °C, specifically at pH 9.0 for a 45 min incubation period. The enzyme was blocked by thiol-modifying reagents and such heavy metal cations as Mn²⁺, Cu²⁺, Sn²⁺, Co²⁺, Zn²⁺ and Hg²⁺, but was not inhibited by thiol-containing reagents like reduced glutathione (GSH), mercaptoethanol and cysteine. Finally, in the presence of 0.01 M of dithiothreitol (DTT) or 0.06 M of NaF, the enzyme showed activity losses of approximately 20 and 17%, respectively.