Cooperativity and substrate specificity of an alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1

The saccharifying alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1 hydrolyzes both α-(1,4)- and α-(1,6)-glycosidic linkages of different linear and branched polysaccharides. The following observations were made concerning the analysis of the coexpressed amylase and neopullulanase enzymes. Even though the enzymes were subjected to a rigorous purification protocol, the activities could not be separated, because both the enzymes were found to migrate in a single peak. By contrast, two independent bands of amylolytic activity at 70 kDa and pullulanolytic activity at 53 kDa were evident by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reducing and nonreducing PAGE, and zymographic analysis on different polysaccharides. Preferential chemical modification of the enzyme and concomitant high-performance thin-layer chromatographic analyses of the saccharides liberated showed that amylase is sensitive to 1-(dimethylamino-propyl)-3-ethyl carbodiimide-HCl and cleaved α-(1,4) linkages of starch, amylose, and amylopectin producing predominantly maltotriose. On the other hand, formalin-sensitive neopullulanase acts on both α-(1,4) and α-(1,6) linkages of pullulan and starch with maltotriose and panose as major products. It is understood that neopullulanase exhibits dual activity and acts in synergy with amylase toward the hydrolysis of α-(1,4) linkages, thereby increasing the overall reaction rate; however, such a synergism is not seen in zymograms, in which the enzymes are physically separated during electrophoresis. It is presumed that SDS-protein intercalation dissociated the enzyme complex, without altering the individual active site architecture, with only the synergism lost. The optimum temperature and pH of amylase and neopullulanase were 60°C and 8.0, respectively. The enzymes were found stable in high alkaline pH for 24 h. Therefore, the saccharifying alkaline amylase and neopullulanase of M. halobius OR-1 evolved from tapioca cultivar shows a highly active and unique enzyme complex with several valuable biochemical features.     

Lactobacillus plantarum amylase acting on crude starch granules

The microheterogeneous native amylolytic complex secreted by the isolate A6 of Lactobacillus plantarum revealed a selective enzyme specificity loss when submitted to a limited proteolysis under a suboptimum pH condition. A clear electrophoretic profile change toward just one shorter, more acidic, and equally active polypeptide fragment resulted from the pronase E pretreatment. Although the whole enzyme activity remained apparently unaffected for soluble starch, the native parallel activity on intact and nongelatinized starch granules either from cereals or tubers was dramatically reduced. This phenomenon was more clearly documented by scanning electron microscopy using the easiest accessible native substrate: wheat starch granules. The anion-exchange-purified native enzymes from L. plantarum displayed a different optimum pH curve when compared with the thermotolerant α-amylase from Bacillus licheniformis. The α-amylases from the lactic-acid-producing A6 isolate presented an electrophoretic profile easily distinguishable from those from B. liqueniformis and B. subtilis species.     

Ligand-exchange chromatography of amino acid enantiomers and its application to the biotransformation of DL-aspartic acid byPseudomonas dacunhae

Summary The separation of the D and L enantiomers of eighteen essential α amino acids has been investigated by ligand-exchange chromatography (LEC). The effect of column temperature on the retention times and resolution of individual amino acid enantiomers has been studied by varying the temperature from 25 to 50 °C for a mobile phase containing Cu²⁺ ions. By use of a temperature of 50 °C and Zn²⁺ in the mobile phase, eight of the eighteen amino acid enantiomers can be resolved sufficiently well for practical application. Only phenylalamine, tyrosine, and tryptophan can be separated by use of Ni²⁺ as complexation metal at 50 °C. LEC has been used to monitor the decarboxylation of racemic DL-aspartic acid byPseudomonas dacunhae. Analysis of DL amino acid enantiomers in different media was performed at column temperatures of 30 and 50°C by addition of 0.125 mM Cu²⁺ to the aqueous mobile phase. It was found that the analytical performance is most dependent on the identity of the metal used for complexation; the concentration of the metal was of secondary importance and the column temperature less important still.     

Partial purification and characterization of two proteases fromAgave fourcroydes

Large amounts of wastes are produced by the henequen industry from which it may be possible to obtain products of commercial importance. Our laboratory has detected proteolytic activity inAgave fourcroydes (henequen) juice. The proteolytic activity is a result of two proteases of 14,500 and 12,000 daltons, respectively. Both enzymes are very stable at -20°C., when freeze-dried and in the presence of EDTA and cysteine. They differ in pH optimum and thermal stability.     

Partial Deuterium Labeling of Dimethacrylate Monomers

Summary. Methacrylic acid-d₅ was prepared in a yield of 30% with 98.6% deuterium incorporation using a two step synthesis. A solution of acetone-d₆ and KCN in D₂O was treated with glacial acetic acid to give the cyanohydrin of acetone-d₆. The latter compound was then dehydrated in anhydrous sulfuric acid at 120°C and subsequently hydrolysed in water at 90°C to form methacrylic acid-d₅. Hydrolysis of commercial nonaethyleneglycol dimethacrylate gave a mixture of ethylene glycols. These glycols were combined with methacrylic acid-d₅ in the presence of p-TsOH in benzene to form nonaethyleneglycol dimethacrylate-d₁₀ with ∼21% deuterium incorporation. Deuterated bisGMA was also prepared from methacrylic acid-d₅ and diglycidyl ether of bisphenol-A. Present address: Boron Molecular Pty Ltd, PO Box 756, Noble Park, VIC 3174, Australia     

Partial purification and characterization of protease enzyme from Bacillus subtilis megatherium

Bacteria of genus Bacillus are active producers of extracellular proteases, and characteristics of enzyme production by Bacillus species have been well studied. The aim of this experimental study is isolation and partial purification of protease enzyme from the Bacillus subtilis megatherium bacteria species. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species on suitable media. The partial purification was reali-zed by applying successively ammonium sulfate precipitation, dialysis, DEAE-cellulose ion exchange chromatography to the supernatant. In this study, the effect of substrate concentration, reaction time, the effect of inhibitor and activator on the optimum pH, optimum temperature, pH stability, and temperature stability was determined. Molecular weight of the obtained enzyme was investigated by SDS-PAGE. In this study, the specific activity of the supernatant, which was partially purified from Bacillus subtilis megatherium bacteria, was 10.4 U/mg, specific activity of supernatant was 13.5 U/mg after 80% ammonium sulfate fractionation. The final enzyme preparation was 1.1-fold purer than the crude homogenate. Molecular weight of the protease was determined, and it was found that the weight of enzyme was 45 kDa by using SDS-PAGE.     

Purification and characterization of a microbial dehydrogenase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M _r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD⁺ less effectively than NADP⁺. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD⁺ or NADP⁺. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl₂. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD⁺ and NADP⁺ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD⁺ or NADP⁺. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.     

Effect of C∶N molar ratio on monomer composition of polyhdroxyalk anoates produced by Pseudomonas mendocina 0806 and Pseudomonas pseudoalkaligenus YS1

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by bacteria. In this study, the effect of C∶N molar ratio on the monomer composition of PHAs was investigated, including medium chain length PHA produced by Pseudomonas mendocina 0806 and PHA blends consisting of monomers of 3-hydroxybutyrate and medium chain length hydroxyalkan⇘te produced by Pseudomonas pseudoalkaligenus YS1. It was observed that there were some fixed ranges of C∶N molar ratio that affect the monomer composition of PHA independently of the substrate. For strain 0806, the ranges were C∶N <20, 20<C∶N<200, and C∶N>200. The monomer composition was constant among these ranges when using glucose and octanoate as the sole substrate. For strain YS1, the ranges were C∶N<20, 20<C∶N<45, and C∶N>45. These results are useful for controlling monomer composition in PHA production.     

20-Hydroxyecdysone from Stemmacantha uniflora Subsp. satzyperovii

The dynamics of 20-hydroxyecdysone content in vegetative and generative organs of Stemmacantha uniflora subsp. satzyperovii (Soskov) Dittrich, which is distributed over Primorskii Krai, were investigated. A high content of 20-hydroxyecdysone during the plant vegetative period is characteristic of growing organs. The amount is maximal for young leaves during development of racemes (7.8 g/mg) and for ripe achenes (11.15 g/mg).     

CelF of Orpinomyces PC-2 has an intron and encodes a cellulase (CelF) containing a carbohydrate-binding module

A cDNA, designated celF, encoding a cellulase (CelF) was isolated from the anaerobic fungus Orpinomyces PC-2. The open reading frame contains regions coding for a signal peptide, a carbohydrate-binding module (CBM), a linker, and a catalytic domain. The catalytic domain was homologous to those of CelA and CelC of the same fungus and to that of the Neocallimastix patriciarum CELA, but CelF lacks a docking domain, characteristic for enzymes of cellulosomes. It was also homologous to the cellobiohydrolase IIs and endoglucanases of aerobic organisms. The gene has a 111-bp intron, located within the CBM-coding region. Some biochemical properties of the purified recombinant enzyme are described. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Purification and characterization of two low molecular weight endoglucanases produced by Penicillium occitanis mutant pol 6

Two endoglucanases (EGs), EG A and EG B, were purified to homogeneity from Penicillium occitanis mutant Pol 6 culture medium. The molecular weights of EG A and EG B were 31,000 and 28,000 kDa, respectively. The pI was about 3 for EG A and 7.5 for EG B. Optimal activity was obtained at pH 3.5 for both endoglucanases. Optimal temperature for enzyme activity was 60 degrees C for EG A and 50 degrees C for EG B. EG A was thermostable at 60 degrees C and remained active after 1 h at 70 degrees C. EGs hydrolyzed carboxymethylcellulose, phosphoric acid swollen cellulose, and beta-glucan efficiently, whereas microcrystalline cellulose (Avicel) and laminarin were poorly hydrolyzed. Only EG B showed xylanase activity. Furthermore, these EGs were insensitive to the action of glucose and cellobiose but were inhibited by the divalent cations Hg2+, Co2+, and Mn2+.     

Partial Molar Volumes of Tetraalkylammonium Ions in N, N-Dimethylformamide

The densities of tetraalkylammonium bromide, R₄NBr (R = Et, Pr, Bu, Hex, Hep, Oct), solutions in dimethylformamide have been measured for the composition range (0.05–0.4) mol-kg⁻¹ at 25 ^∘C. Apparent molar V_φ and limiting partial molar volumes ⫫₂^o of the electrolytes have been evaluated. Using the extrapolation values, the limiting partial molar volumes of the tetraalkymammonium ions (⫫_i^o) have been calculated. Analysis of different contributions to the ionic ⫫_i^o indicated partial penetration of solvent molecules into the van der Waal’s volume of tetraalkylammonium (TAA) ions.     

Lignin biotransformations by an aromatic aldehyde oxidase produced byStreptomyces viridosporus T7A

Streptomyces viridosporus produces an intracellular aromatic aldehyde oxidase that oxidizes aromatic and α, β-unsaturated aromatic aldehydes to their corresponding acids. It also produces extracellular oxidase as shown by zones of clearing when grown on agar containing insoluble dehydrodivanillin (DHDV). This extracellular form may be responsible for oxidizing aldehyde groups in lignin. The extracellular oxidase was expressed maximally after 3 d growth in medium containing only yeast extract. However, higher levels were produced when lignocellulose was in the medium. The enzyme was partially purified and its molecular weight was approximated to be about 80,000 daltons. Mutant cultures that had lost the ability to produce zones of clearing on DHDV-containing agar solubilized smaller quantities of lignin as compared to the wild type, except for one strain. A partially purified oxidase preparation was shown to oxidize a natural lignocellulose substrate.     

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.     

Evidence for a novel mechanism of microbial cellulose degradation

There are two well studied mechanisms that are used by cellulolytic microorganisms to degrade the cellulose present in plant cell walls and a third less well studied oxidative mechanism used by brown rot fungi. The well studied mechanisms use cellulases to hydrolyze the β-1,4 linkages present in cellulose, however the way in which cellulases are presented to the environment are quite different for each mechanism. Most aerobic microorganisms secrete a set of cellulases outside the cell (free cellulase mechanism) while most anaerobic microorganisms produce large multi enzyme complexes on their outer surface (cellulosomal mechanism). Their genomic sequences suggest that the aerobic bacterium, Cytophaga hutchinsonii and the anaerobic bacterium, Fibrobacter succinogenes, do not use either of these mechanisms for degrading cellulose, as these organisms only code for normal endocellulases not for processive cellulases like exocellulases and processive endocellulases which are used in both of the well studied mechanisms.     

Microbial synthesis and characterization of physiochemical properties of polyhydroxyalkanoates (PHAs) produced by bacteria isolated from activated sludge obtained from the municipal wastewater works in Hong Kong

The first objective of this study was the measurement of physical properties of P(3HB-co-3HV) copolymers with different (hydroxybutyrate) HB to (hydroxyvalerate) HV ratios produced by Bacillus cereus (TRY2) isolated from activated sludge. The 3HV PHBV copolymers were 0.05, 22.6, 39.2, 54.1, and 69.1 mol%, respectively. The second objective was to study possible wastewater treatment and production of PHAs at the same time by B. cereus (TRY2) and Pseudomonas spp. (TOB17) (both were isolated from activated sludge), recombinant Bacillus DH5α, and a combination of the above three bacteria. The results were satisfactory; the maximum COD and TOC of the sewage sludge reduced were 53.5% and 67.5%, respectively.     

A group theoretical study of f n systems in an environment of approximately icosahedral symmetry

Group theoretical methods are developed to determine in a generalised format for an f ⁿ ion in a crystal field environment of icosahedral symmetry, the effect on the energy level scheme when a crystal field distortion is considered parallel to any direction. As an illustration, the effect on the g-tensor components are examined as a function of the magnitude and the direction of the crystal field distortion. All appropriate reduced matrix elements in group theoretical terminology are evaluated for the f ⁿ -ion ground states. Specific results are given for the f ³-ion case and compared with electron paramagnetic resonance, optical, and magnetic susceptibility data.     

Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.     

Performance of an internal-loop airlift bioreactor for treatment of hexane-contaminated air

Hexane is a toxic volatile organic compound that is quite abundant in gas emissions from chemical industries and printing press and painting centers, and it is necessary to treat these airstreams before they discharge into the atmosphere. This article presents a treatment for hexane-contaminated air in steady-state conditions using an internal-loop airlift bioreactor inoculated with a Pseudomonas aeruginosa strain. Bioprocesses were conducted at 20-mL/min, a load of 1.26 g/m³ of C₆H₁₄, and a temperature of 28°C. The results of hexane removal efficiencies were presented as a function of the inoculum size (approx 0.07 and 0.2 g/L) and cell reuse. Bioprocess monitoring comprises quantification of the biomass, the surface tension of the medium, and the hexane concentration in the fermentation medium as well as in the inlet and outlet airstreams. The steady-state results suggest that the variation in inoculum size from 0.07 to 0.2 g/L promotes hexane abatement from the influent from 65 to 85%, respectively. Total hydrocarbon removal from the waste gas was achieved during experiments conducted using reused cells at an initial microbial concentration of 0.2 g/L.     

Contribution to the analytical characterization of coatings produced by thermal spraying

This paper reports on the use of Auger electron spectroscopy (AES)/ depth profile analysis for the investigation of plasma-sprayed coatings. Prior to spraying the St 37 substrates are heated to 300 °C or 500 °C for ceramic or metallic layers, respectively. Studies of the starting materials and of the interfaces are important if the adhesion mechanism is to be understood. Therefore the initial components—the unheated and heated substrates and the powder particles NiCrAl, Al₂O₃ and ZrO₂-7.25Y₂O₃—are analyzed. Depth profiles obtained from two coatings St 37/NiCrAl and St 37/Al₂O₃ show the influence of plasmaspraying on substrate surfaces and sprayed particles. Plasma-spraying mainly causes a decrease of superficial carbon contamination for both coating layers. In the case of St 37/NiCrAl incorporation of carbon in the sprayed layer is observed. The whole layer is almost completely oxidized except for some areas where substrate and particle material are present. It is assumed that these areas are identical with so-called adherence zones.Dedicated to Professor Günther Tölg on the occasion of his 60th birthday