Production and characterization of polygalacturonase fromGeotrichum candidum

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The K_m and V_max values for polygalacturonic acid were 4.2 mg/ml and 0.19mm galacturonic acid/min, respectively. The enzyme was not substrate inhibited.     

Thermostable enzymes for industrial applications

Summary The variety of thermostable (TS) enzymes has been steadily increasing for use in industrial applications, mainly as replacements for thermolabile (TL) enzymes. For example, TS amylases fromBacillus licheniformis andBacillus stearothermophilus have replaced TL amylases fromBacillus subtilis. TS enzymes also have advantages in new areas such as cyclodextrin production. The TS cyclodextrin glycosyl transferase (CGTase) fromThermoanaerobacter sp. (95°C optimum) gives a higher productivity than the CGTase fromBacillus macerans (55°C optimum). In the area of enzymatic bleach boosting of wood pulps, a TS xylanase (Myceliophera thermophila) would be advantageous over a TL xylanase (Trichoderma reesei), due to the high temperature of the incoming pulp. Not all TS enzymes are from thermophiles; the mesophileCandida antarctica produces a TS lipase which has a temperature optimum of 90°C when immobilized. The characterization of these enzymes will be described along with comparisons to some newly described TS enzymes.     

Extracellular mannanases and galactanases from selected fungi

Summary Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of -mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.     

Parameters influencing asymmetric synthesis of (R)-mandelonitrile by a novel (R)-hydroxynitrile lyase from Eriobotrya japonica

(R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a hydroxynitrile lyase from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. The effects of pH, temperature, organic solvent, substrate concentration and enzyme concentration on the initial activity and enantioselectivity of the enzyme were studied. Both pH and temperature had a large effect on the initial velocity and enantiomeric excess (e.e.) of the product, (R)-mandelonitrile. High enantiomeric purity of the product was observed at low pH and temperature because the non-enzymatic reaction producing racemates of mandelonitrile was almost suppressed. The optimum pH and temperature to obtain high e.e. were pH 4.0 and 10 °C, respectively. Surprisingly, the organic solvents had a significant influence on the initial velocity of the reaction but less influence on the enantiomeric purity of product. The EjHNL was very stable in ethyl acetate, diethyl ether, methyl-t-butyl ether, diisopropyl ether, dibutyl ether and hexane for 12 h. The best solvent for the highest initial velocity and e.e. was diethyl ether with an optimum aqueous phase content of 50% (v/v). The initial reaction rate increase as the aqueous phase content rose, but when the content was more than 50%, a reduction of e.e. was observed. Increasing the concentration of the substrates accelerated the initial velocity, but caused a slight decrease in the e.e. of the product. Under the optimized conditions, the conversion and e.e. of (R)-mandelonitrile for 3 h were 40 and 99%, respectively. The aqueous phase containing the enzyme also showed considerably efficient reusability for 4 batch reactions.     

Origin of intestinal disaccharidases of Ascaris suum

Disaccharidases from the gut of Ascaris suum were investigated to determine whether they were synthesized by the worm or whether they were host enzymes adsorbed to the worms' intestinal cells. Alpha-d-glucoside glucohydrolase (maltase) (EC 3.2.1.20), Beta-d-fructofuranoside fructohydrolase (invertase) (EC 3.2.1.26) and 1-glucohydrolase (trehalase) (EC 3.2.1.28) from Ascaris were studied in both a membrane (brush border)-bound and solubilized form with regard to temperature stability and pH optima. Data collected were compared to similar data on hog intestinal enzymes. Worm maltase and trehalase were relatively heat labile, whereas the hog enzymes were more stable to heat inactivation. Worm invertase was heat stable in comparison to the hog enzyme. The pH optima for Ascaris maltase and invertase were different from those of hog disaccharidases, whereas the pH optimum for trehalase from both parasite and host were similar. Tissue homogenates of second-stage larvae contained measurable maltase, but not sucrase, or trehalase activity. Results suggested that Ascaris intestinal disaccharidases represent three distinct enzymes of parasite rather than host origin.     

Catalysis by enzymes entrapped into hydrated surfactant aggregates having lamellar or cylindrical (hexagonal) or ball-shaped (cubic) structure in organic solvents

Instead of aqueous solutions, universally recognized in enzymology, ternary systems of the water/organic solvent/surfactant type are suggested as liquid-crystalline media for enzymatic reactions. Two systems, water/octane/Aerosol OT and water/cyclohexane/Brij 96, have been used to solubilize acid and alkaline phosphatases and peroxidase. The enzymes under study do function in liquid-crystalline mesophases having lamellar, cylindrical (reversed hexagonal) and ball-shaped (reversed cubic) packing of the surfactant molecules. A significant result is that the phase transition from one liquid-crystalline structure to another entails, as a rule, a reversible change in the catalytic activity of the solubilized enzyme.     

Mixed reverse micelles facilitated downstream processing of lipase involving water‐oil‐water liquid emulsion membrane

Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water‐oil‐water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0‐fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1–17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream processing of enzymes/proteins. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1084–1092, 2014     

Characterization of the amylolytic system ofCandida strains

Some physical and chemical properties ofα-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) produced in semisolid fermentation byCandida fennica FTPT-1829,C. famata FTPT-1539 andC. fennica FTPT-8903 were determined. The optimum temperature values were 42, 44, 48 °C and 60, 50, 50 °C forα-amylase and glucoamylase excreted byC. fennica 8903,C. fennica 1829 andC. famata 1539, respectively. The optimum pH values for all strains were 5.0 and 6.0 forα-amylase and glucoamylase, respectively. The degradation of pullulan by all the yeast species indicates debranching activity. This research was carried out with financial support fromPrPq/UFMG. The second author received grants fromRHAE/CNPq.     

Comparative studies on the in vitro properties of phytases from various microbial origins

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.     

Selective precipitation and recovery of xylanase using surfactant and organic solvent

The selective precipitation of xylanase from an aqueous phase containing mixtures of xylanase and cellulase was studied using an ionic surfactant as precipitating ligand and a polar organic solvent as recovery solvent. Of four ionic surfactants tested, sodium di-(2-ethylhexyl) sulfosuccinate (AOT) showed a complete removal of xylanase at pH 4.5. The recovery of xylanase from the xylanase-AOT complex was a strong function of the type and the volume of the polar solvent and of the concentration of sodium acetate buffer in the final aqueous solution used to solubilize the recovered xylanase. With ethanol as a recovery solvent, a recovery of xylanase activity of 78 +/- 10% was obtained. The cellulase activity in the recovered xylanase was below the detection limit. The results demonstrate that an ionic surfactant can recover enzymes from aqueous solutions without loss in their activity.     

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log P_octanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between ? 1.1 and 8.8 were examined. Organic solvents with log P_octanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL^?1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.     

PARTIAL CHARACTERIZATION OF SOLUBLE ACETYLCHOLINESTERASE ISOENZYMES OF THE RAT BRAIN

(1) The 105,000 g supernatant fluid obtained from rat brain was separated by agar-gel electrophoresis. (2) Three isoenzymes, capable of hydrolysing acetylthiocholine, one of them also hydrolysing butyrylthiocholine, were detected. (3) The pH optima and K_m for hydrolysis of acetyl- and butyrylthiocholine by the supernatant fluid were determined. (4) After extraction of acetylcholinesterase isoenzymes from the gel, individual isoenzymes were characterized by pH optima and K_m values. (5) Two of the enzymes were characterized as acetylcholinesterase (EC 3.1.1.7) and one as butyrylcholinesterase (EC 3.1.1.8).     

<Emphasis Type="Italic">Candida rugosa</Emphasis> lipase-catalyzed polyurethane degradation in aqueous medium

Candida rugosa lipase (EC 3.1.1.3) was used to degrade commercially-available solid poly(ester)urethane (Impranil) in an aqueous medium under different temperature, pH, enzyme and substrate concentrations. A mathematical model was developed and applied to represent the degradation kinetics of the solid polyurethane. Reaction optima were found to be pH 7 and 35°C. Diethylene glycol, a degradation byproduct, generation rate was measured to be 0.12 mg/l min and the activation energy was calculated as 9.121 kcal/gmol K. This information will be useful in developing bioreactors for practical applications to manage polyurethane wastes using lipase.     

Semicontinuous cellulase production in an aqueous two-phase system with Trichoderma reesei rutgers C30

Cellulases [see 1,4(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma reesei, Rutgers C30, can be semicontinuously produced in an aqueous two-phase system composed of dextran and poly(ethylene glycol) using Solka Floc BW 200 as substrate. When substrate was intermittently added along with fresh top phase, which replaced the withdrawn top phase containing the produced enzymes, a yield of 1740 U endo-β-d-glucanase/g cellulose and 59.3 FPU/g cellulose was extracted with the top phase. Without fresh substrate added, a yield of 3920 U endo-β-d-glucanase/g cellulose and 127.7 FPU/g cellulose was extracted after five runs.     

Thermodynamic theory explains the temperature optima of soil microbial processes and high Q10 values at low temperatures

Our current understanding of the temperature response of biological processes in soil is based on the Arrhenius equation. This predicts an exponential increase in rate as temperature rises, whereas in the laboratory and in the field, there is always a clearly identifiable temperature optimum for all microbial processes. In the laboratory, this has been explained by denaturation of enzymes at higher temperatures, and in the field, the availability of substrates and water is often cited as critical factors. Recently, we have shown that temperature optima for enzymes and microbial growth occur in the absence of denaturation and that this is a consequence of the unusual heat capacity changes associated with enzymes. We have called this macromolecular rate theory – MMRT (Hobbs et al., 2013 , ACS Chem. Biol. 8:2388). Here, we apply MMRT to a wide range of literature data on the response of soil microbial processes to temperature with a focus on respiration but also including different soil enzyme activities, nitrogen and methane cycling. Our theory agrees closely with a wide range of experimental data and predicts temperature optima for these microbial processes. MMRT also predicted high relative temperature sensitivity (as assessed by Q₁₀ calculations) at low temperatures and that Q₁₀ declined as temperature increases in agreement with data synthesis from the literature. Declining Q₁₀ and temperature optima in soils are coherently explained by MMRT which is based on thermodynamics and heat capacity changes for enzyme‐catalysed rates. MMRT also provides a new perspective, and makes new predictions, regarding the absolute temperature sensitivity of ecosystems – a fundamental component of models for climate change.     

Properties of apyrase and inorganic pyrophosphatase inStreptomyces aureofaciens

Apyrase (ATP-diphosphohydrolase, EC 3.6.1.5) and inorganic pyrophosphatase (EC 3.6.1.1) were partially purified fromS. aureofaciens RIA 57 and characterized. Apyrase degrades, in addition to ATP, other nucleoside triphosphates and nucleoside diphosphates, diphosphate, thiamine diphosphate, phosphoenolpyruvate and oligophosphates of chain lengthn ≦ 90. The apyrase activity was detected in the membrane and supernatant fractions. Its properties (substrate specificity, effect of inhibitors, pH optimum and effect of Mg²⁺ ions) were similar in both fractions except for the effect of oligomycin that inhibited only the membrane fraction. Pyrophosphatase exhibited a strict substrate specificity, substrates other than diphosphate being degraded relatively slowly. Of other enzymes exhibiting the phosphatase activity acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1), trimetaphosphatase (EC 3.6.1.2) and exopolyphosphatase (EC 3.6.1.11) degrading oligophosphates of chain lengthn = 15, 40 and 60, were detected.     

Enantioselective synthesis of (S)‐naproxen using immobilized lipase on chitosan beads

S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane and Tween‐80 were 3:7 and 0.1% (v /v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

Comparative studies on glucoamylases from three fungal sources

Five commercial preparations of glucoamylases (three fromAspergillus niger, one each fromAspergillus foetidus andAspergillus candidus) were purified by ultrafiltration, Sepharose-gel filtration and DEAE-sephadex chromatography. Two forms of the enzyme, namely glucoamylase I and glucoamylase II were obtained from the fungi except from one strain ofA. Niger. All the enzymes appeared homogeneous by electrophoresis and ultracentrifugation. The specific activities varied between 85 and 142 units. The pH and temperature optima were between 4 and 5, and 60‡C respectively. The molecular weight as determined by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis ranged from 75,000 to 79,000 for glucoamylase I and 60,000 to 72,000 for glucoamylase II. OnlyA. niger glucoamylases contained phenylalanine at the N-terminal end. The amino acid composition of the enzymes was generally similar. However,A. niger andA. foetidus glucoamylases, in contrast toA. candidus enzymes, contained greater percentage of acidic than of basic amino acids. The enzymes contained 15 to 30% carbohydrate and 49 to 57 residues of monosaccharides per mol.A. niger enzymes contained mannose, glucose, galactose, xylose and glucosamine but theA. candidus enzyme lacked xylose and glucose and only xylose was absent inA, foetidus enzymes. Majority of the carbohydrate moieties were O-glycosidically linked through mannose to the hydroxyl groups of seline and threonine of the polypeptide chain.     

Physical properties of pectic polysaccharidases ofPseudomonas solanacearum from Nigeria

Pseudomonas solanacearum (obtained from Nigeria) produced certain pectic polysaccharidases when grown in aerobic batch cultures containing pectic substances. The pH optima of the enzymes were different. The optimum for polygalacturonase EC 3.2.2.15 was 5.5, and for pectate lyase EC 4.2.99.3 it was 8.5. The -1,4-glycosidic bonds between galacturonide units were cleaved at random, indicating the endo character of the enzymes. The pectic polysaccharidases were purified by (NH₄)₂SO₄ fractionation and by electrofocusing. Highest polygalacturonase activity and pectate lyase activity were obtained in the fractions at 41%–60% and 61%–80% (NH₄)₂SO₄ saturation, respectively. Polygalacturonase was resolved into two components with isoelectric points of 5.0 and 7.5; the isoelectric point of pectate lyase was 8.1.