PURIFICATION AND CHARACTERIZATION OF AN ALKALINE CELLULASE PRODUCED BY Bacillus subtilis (AS3)

An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6–10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.     

A Thermostable Xylanase from a Thermophilic Acidophilic Bacillus sp.

Bacillus sp. 11-IS, a strain of thermophilic acidophilic bacteria, produced an extracellular xylanase during growth on xylan. The enzyme purified from the culture supernatant solution was homogeneous on disc-gel electrophoresis. The molecular weight was calculated to be 56,000 by SDS-gel electrophoresis. The enzyme had a pH optimum for activity at 4.0, and its stability range was pH 2.0 ~ 6.0. The temperature optimum was 80°C (10-min assay); however, the enzyme retained full activity after incubation at 70°C for 15 min. The enzyme acted on carboxymethyl cellulose (CMC) and cellulose, as well as on xylan. The Michaelis constants for larchwood xylan and CMC were calculated to be 1.68 mg xylose eq/ml and 0.465 mg glucose eq/ml, respectively. The predominant hydrolysis products from larchwood xylan were xylobiose, xylotriose, and xylose; the release of arabinose from rice-straw arabinoxylan was not detected. CMC was cleaved to cellobiose and larger oligosaccharides. Thus, the enzyme is considered to be an endoenzyme which degrades the β-1,4-glycosyl linkages in xylan and cellulose.     

Biochemical Studies on Cycasin

Purification of β-glucosidase from the seeds of Japanese cycad, and properties of the purified preparation are described. The enzyme activity was determined by colorimetry using ONPG as substrate. Crude preparation was obtained easily by adsorption on fibrous CMC pulp. It was further purified by chromatography on CMC powder, and a preparation which showed an activity of 135-folds of the original extract was obtained. Influences of pH, temperature, and substrate concentration upon the enzyme activity were examined. Michaelis constant of the enzyme for ONPG was 3.3×10^–3M.     

CMCase production by Spicellum roseum in liquid and solid culture

Summary CMCase was produced by 7 strains of Spicellum roseum in both liquid and wheat bran solid substrate cultures. No growth occurred above 35°C. Maximum enzyme production occurred at 30°C, whereas best enzyme activity occurred at pH 5.0 and 50°C. In liquid cultures of S. roseum, NRRL strains 13103, 13104, and 13106 produced activities of ca. 1.1, 1.5, and 1.5 mg glucose per hr/ml culture supernate at 1 week and 2.9, 1.5, and 2.1, respectively at 3 weeks compared to Trichoderma reesei NRRL 11236 (MCG77), which produced activities of 2.8 and 1.3 at 1 and 3 weeks.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

A constitutive endoglucanase (CMCase) from Bacillus licheniformis-1

Summary Bacillus licheniformis-1 elaborated a constitutive extracellular CMCase. The temperature and pH optima of the enzyme were 55°C and 6.1 respectively. Its production was enhanced in the presence of easily metabolisable sugars and was lower when cellulosic sources were used.Abbreviations used CMC Carboxy Methyl Cellulose. - CP-60 Cellulose Powder 60 - CP-100 Cellulose Powder 100 - CP-X Cellulose Powder X     

Purification and characterization of an endoxylanase from the culture broth of <Emphasis Type="Italic">Bacillus cereus</Emphasis> BSA1

An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE cellulose chromatography and followed by gel filtration through Sephadex-G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55°C and at pH 7.0 and remained reasonably stable in a wide range of pH (5.0–8.0) and temperature (40–65°C). The K _m and V _max values were found to be 8.2 mg/ml and 181.8 μmol/(min mg), respectively. The enzyme had no apparent requirement of cofactors, and its activity was strongly inhibited by Cu²⁺, Hg²⁺. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhibited xylanase activity. Since the enzyme was active over wide range of pH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.     

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material

Bacillus subtilis TD6 was isolated from Takifugu rubripes, also known as puffer fish. Cellulase from this strain was partially purified by ammonium sulphate precipitation up to 80% saturation, entrapped in calcium alginate beads, and finally characterized using CMC as the substrate. For optimization, various parameters were observed, including pH maximum, temperature maximum, sodium alginate, and calcium chloride concentration. pH maximum of the enzyme showed no changes before and after immobilization and remained stable at 6.0. The temperature maximum showed a slight increase to 60 °C. Two percent sodium alginate and a 0.15 M calcium chloride solution were the optimum conditions for acquisition of enzyme with greater stability. K (m) and V (max) values for the immobilized enzyme were slightly increased, compared with those of free enzyme, 2.9 mg/ml and 32.1 μmol/min/mL, respectively. As the purpose of immobilization, reusability and storage stability of the enzyme were also observed. Immobilized enzyme retained its activity for a longer period of time and can be reused up to four times. The storage stability of entrapped cellulase at 4 °C was found to be up to 12 days, while at 30 °C, the enzyme lost its activity within 3 days.     

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn²⁺, Hg²⁺, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K _m and V _max of the enzyme for CMC were 3.4 mg ml^–1 and 44.7 mol min^–1 (mg protein)^–1, respectively.     

Stabilization and functional properties of Escherichia coli penicillin G acylase by covalent conjugation of anionic polysaccharide carboxymethylcellulose

The stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (G _i values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower K _m and higher k _cat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme.     

Preparation of Triglyceride Hydroperoxides

Corticium rolfsii AHU 9627, which we isolated from a tomato stem, is one of the most promising producers of a raw starch saccharifying enzyme. The effects of the cultural conditions and medium components on the enzyme production were investigated. The enzyme production was improved by increasing both the concentrations of carbon sources and organic nutrients in the medium. Under the optimum cultural conditions, the enzyme activity of the culture supernatant against raw starch reached a maximum after 8-days incubation at 27°C and the activity reached 80 units per ml (when determined at 40°C and pH 4.0). The optimal pH and temperature for the enzyme reaction were 4.0 and 65°C, respectively. The saccharifying reaction was scarcely inhibited even with a high substrate concentration, and raw starch was rapidly hydrolyzed into glucose.     

Hydrolysis of inulin from Jerusalem artichoke by inulinase immobilized on aminoethylcellulose

Purified inulinase (inulase, 2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) of Kluyveromyces fragilis has been immobilized on 2-aminoethyl-cellulose by treatment with 2% glutaraldehyde in 0.05 m phosphate buffer, pH 7.0, for 2 h at room temperature. The immobilized enzyme preparation had 39.3 units inulinase activity per gram dried matrix, with 53.4% recovery yield of activity, and showed good operational stability in the presence of substrate, inulin or the tuber extract of Jerusalem artichoke. Optimum pH and temperature were 5.5 and 45°C, respectively. In a batch reactor, the conversion was 90% ($\text{d}\text{-fructose/}\text{d}\text{-glucose}\text{= 76/24}$) and 34 mg d-fructose per ml was produced from the artichoke tuber extract by the immobilized inulinase in 20 h. In column reactor packed with 28 ml immobilized enzyme, the following conditions were found to be optimal: height/diameter ratio of column, 10.3; space time, 3.8 h; temperature, 40°C. Operation under these conditions gave 90% conversion of a 7% inulin solution and the productivity was 102 mmol l^?1 h^?1.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Cherry fruit development: The use of a microassay for studying indoleacetic acid oxidase

An improved procedure is described for extraction and assay of indoleacetic acid oxidase from seeds of sour cherry (Prunus cerasus L.). The extraction procedure was optimized for pH, buffer, polyvinylpolypyrrolidone (PVP) and tissue: buffer ratio. Greatest extraction efficiency was obtained at pH 4.0, 0.2 M acetate buffer, tissue: PVP ratio of 1:2.5 and tissue: buffer ratio of 50 ml per g of seed. The enzyme was assayed at 30°C using indoleacetic acid-1-¹⁴C as substrate and radioassaying the ¹⁴CO₂ evolved. Mn²⁺ and 2,4-dichlorophenol enhanced enzyme activity but were not obligatory. A minimum substrate concentration of 60 M was needed for quantitative evaluation. This assay was sensitive and reproducible, enzyme activity being demonstrated in as little as 0.8 mg of seed tissue with a coefficient of variation of 1 to 9%.     

Augmented cellulase production by Bacillus subtilis strain MU S1 using different statistical experimental designs

The production of cellulase by Bacillus subtilis MU S1, a strain isolated from Eravikulam National Park, was optimized using one-factor-at-a-time (OFAT) and statistical methods. Physical parameters like incubation temperature and agitation speed were optimized using OFAT and found to be 40?°C and 150?rpm, respectively, whereas, medium was optimized by statistical tools. Plackett-Burman design (PBD) was employed to screen the significant variables that highly influence cellulase production. The design showed carboxymethyl cellulose (CMC), yeast extract, NaCl, pH, MgSO₄ and NaNO₃ as the most significant components that affect cellulase production. Among these CMC, yeast extract, NaCl and pH showed positive effect whereas MgSO₄ and NaNO₃ were found to be significant at their lower levels. The optimum levels of the components that positively affect enzyme production were determined using response surface methodology (RSM) based on central composite design (CCD). Three factors namely CMC, yeast extract and NaCl were studied at five levels whilst pH of the medium was kept constant at 7. The optimal levels of the components were CMC (13.46?g/l), yeast extract (8.38?g/l) and NaCl (6.31?g/l) at pH 7. The maximum cellulase activity in optimized medium was 566.66?U/ml which was close to the predicted activity of 541.05?U/ml. Optimization of physical parameters and medium components showed an overall 3.2-fold increase in activity compared to unoptimized condition (179.06?U/ml).     

Purification of L-asparaginase from a bacteria Erwinia carotovora and effect of a dihydropyrimidine derivative on some of its kinetic parameters

L-Asparaginase shows antileukemic activity and is generally administered in the body in combination with other anticancer drugs like pyrimidine derivatives. In the present study, L-asparaginase was purified from a bacteria Erwinia carotovora and the effect of a dihydropyrimidine derivative (1-amino-6-methyl-4-phenyl-2-thioxo, 1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester) was studied on the kinetic parameters Km and Vmax of the enzyme using L-asparagine as substrate. The enzyme had optimum activity at pH 8.6 and temperature 35 degrees C, both in the absence and presence of pyrimidine derivative and substrate saturation concentration at 6 mg/ml. For the enzymatic reaction in the absence and presence (1 to 3 mg/ml) of dihydropyrimidine derivative, Km values were 7.14, 5.26, 4.0, and 5.22 M, and Vmax values were 0.05, 0.035, 0.027 and 0.021 mg/ml/min, respectively. The kinetic values suggested that activity of enzyme was enhanced in the presence of dihydropyrimidine derivative.     

The properties of immobilized whole cell ofHumicola spp. with rifamycin oxidase activity

Summary The whole cell ofHumicola spp. ATCC 20620 with rifamycin oxidase activity was immobilized by copolymerization with acrylamide. The whole cell was defatted by treatment with acetone to reduce the diffusional resistance through the cell membrane. The recovery of enzyme activity after the immobilization step was about 50%. The acetone-defatted cell showed the maximum activity at pH 7.5 for both free and the immobilized forms. No appreciable activity loss could be detected when stored at 4 °C and pH 7.8 for one month, while the half life at 40 °C and pH 8 was decreased to about 8 days. The apparent Km values of rifamycin oxidase for the free and immobilized acetonedefatted cells were 0.3mM and 0.6mM, respectively. The enzyme demonstrated substrate inhibition, but the degree of substrate inhibition was different between two forms of the enzyme preparation. A complete substrate inhibition was observed for the immobilized cell, whereas the enzyme activity was partially inhibited at high substrate concentration in the acetone-defatted cells.     

Cloning, expression, purification, and properties of an endoglucanase gene (glycosyl hydrolase family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris

A gene coding for an endoglucanase (EglA), of the glycosyl hydrolase family 12 and derived from Aspergillus niger VTCC-F021, was cloned and sequenced. The cDNA sequence, 717 bp, and its putative endoglucanase, a 238 aa protein with a predicted molecular mass of 26 kDa and a pI of 4.35, exhibited 98.3-98.7% and 98.3-98.6% identities, respectively, with cDNA sequences and their corresponding endoglucanases from Aspergillus niger strains from the GenBank. The cDNA was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 1.59 U/ml culture supernatant, after 72 h of growth in a YP medium induced with 1% (v/v) of methanol. The molecular mass of the purified EglA, determined by SDS-PAGE, was 33 kDa, with a specific activity of 100.16 and 19.91 U/mg toward 1% (w/v) of beta-glucan and CMC, respectively. Optimal enzymatic activity was noted at a temperature of 55°C and a pH of 5. The recombinant EglA (rEglA) was stable over a temperature range of 30- 37°C and at pH range of 3.5-4.5. Metal ions, detergents, and solvents tested indicated a slightly inhibitory effect on rEglA activity. Kinetic constants (K(m), V(max), k(cat), and k(cat)/ K(m)) determined for rEglA with beta-glucan as a substrate were 4.04 mg/ml, 102.04 U/mg, 2,040.82 min-1, and 505.05, whereas they were 10.17 mg/ml, 28.99 U/mg, 571.71 min-1, and 57.01 with CMC as a substrate, respectively. The results thus indicate that the rEglA obtained in this study is highly specific toward beta-glucan. The biochemical properties of rEglA make it highly valuable for downstream biotechnological applications, including potential use as a feed enzyme.     

Production and partial purification of invertase using <Emphasis Type="Italic">Cympopogan caecius</Emphasis> leaf powder as substrate

The present study investigates the efficiency of Aspergillus niger to produce invertase, an industrially important enzyme by using powdered stem of Cympopogan caecius (Lemon grass) as sole substrate and sole carbon source for the microorganism. The molecular weight of invertase was estimated to be 66–70 kDa by sodium do decyl sulphate poly acrylamide gel electrophoresis (SDS PAGE). The production of the enzyme was studied at different pH scales ranging from pH 4.0 to 7.0 at a constant temperature of 30°C and 2% substrate concentration. The maximum production of invertase (specific activity −0.0516 μk/mg protein) was obtained at pH 5.5 at 30°C temperature, and incubation for 48 h. The activity was found to be stable at pH 5.5 for 30 min. The enzyme was found to be stable in the temperature range of 20–55°C. The effect of divalent metal ions Cu²⁺, Fe²⁺, Co²⁺ on the activity of the enzyme invertase showed that these ions affected the activity by a certain factor. The study can be further industrially exploited in a country-like India where lemon grass is found in plenty and can be used as substrate for enzyme production. Moreover, the preparation of the substrate is also a simple process.     

Production of glucose isomerase by different strains ofStreptomyces

Summary The glucose isomerase activity ofStreptomyces haeochromogenes strains 1 and 2 varies considerably with the assay conditions (pH, glucose concentration,etc.). Nine other species of streptomyces were tested under conditions optimal forS.phaeochromogenes 2. The highest enzyme activity was found inS.nigrificans 3014.