Purification and Characterization of a Lipase from Candida curvata Lodder and Kreger — van Rij CBS 570

The purification of the lipase from Candida curvata CBS 570 was achieved steps. Its optimum pH is 6 and its optimum temperature range is Iff C to 60°C. This enzyme is thermoresistant and only loses 20% of its activity when heated at 50°C during 30 minutes. Its activation energy is 144 kcal/mole and its inactivation energy 22 kcal/mole. Its molecular weight was determined to be 195000. EDTA, p-chloromercuri benzoate, N-ethyl- and iodoacetamide have no influence on the activity of this enzyme, whereas Cu⁺⁺ and Zn⁺⁺ show strong inhibitory effects. The lipase activity is induced by the presence of triglycerides and inhibited by the presence of glucose. This enzyme strongly attacks triolein and trilinolein molecules, however it only hydrolyzes a little tristearin and trilinolenin.     

Partial purification and conversion of the particulate-bound diglyceride kinase ofEscherichia coli to a water soluble,detergent free state

Alkali (pH 11.5) treatment of a washed, ammonium sulfate-precipitated residue derived from a triton-X-100 extract of a 30,000 Xg particulate diglyceride kinase preparation ofE. coli converts much of the enzyme to a 100,000 Xg soluble, detergent free state. This procedure improves the enzyme as an analytical tool for the structural analysis of triglycerides, and may be applicable for the solubilization of the many other glyceride- and phosphoglyceride-forming enzymes which have all resisted further purification because of insoluble, particulate properties.     

The preparation of an immobilised glucose isomerase II. Immobilisation and properties of the immobilised enzyme

Glucose isomerase ex Lactobacillus brevis was successfully immobilised on microcrystalline cellulose, using the transition metal-link method. Immobilisation could be performed over a pH range of 5 to 9, and usually resulted in an apparent specific activity increase. The immobilised glucose isomerase generally displayed properties similar to those of the soluble enzyme, with the exception of the following differences:

• (i) a pH optimum at pH = 6, an acid shift of 0.5 units on immobilisation;
• (ii) an optimum reaction temperature at 50 °C, lower than that for the soluble enzyme;
• (iii) on incubation at 4 °C, a retention of 53% of the initial specific activity, when stored in 0.02 M, pH = 7, Tris buffer, after 8 weeks, compared with an apparent activation of the soluble enzyme after 10 and 19 weeks' storage.

Storage properties of the immobilised enzyme at 4 °C in Tris were apparently improved by the presence of Mn⁺⁺ and Co⁺⁺, although associated with some protein release. Storage at 4 °C in water alone, as opposed to Tris, resulted in a more rapid activity loss.     

Activation and solubilization by triton X-100 of membrane-bound phospholipase D of rat brain

In the present study, we examined the ability of detergents to stimulate and solubilize phospholipase D (PLD) of a particulate fraction of rat brain. PLD activity was assayed by measuring the [³H]choline produced from the exogenous substrate dipalmitoyl phosphatidyl[³H]choline (dipalmitoyl [³H]PC). In the absence of detergents, PLD activity was not detectable. Of the detergents examined, Triton X-100 was found to markedly enhance PLD activity, whereas other detergents including sodium doexycholate, sodium cholate, CHAPS and Lubrol-PX caused only a small, if any increase in activity. Enhancement by Triton X-100 was maximal at 0.1–0.2% (w/v) and decreased at higher concentrations. The optimal pH was 7.1–7.3. Both Ca²⁺ and Mg²⁺ inhibited enzyme activity stimulated by Triton X-100 in a concentration-dependent manner. Triton X-100 effectively solubilized PLD from the particulate fraction of rat brain; more than 70% of the activity of the particulate fraction was extracted by 0.5–1.0% (w/v) Triton X-100. Furthermore, when the PLD activities in brains of three different species (rat, rabbit and bovine) were measured under optimal conditions, the activities were found to differ greatly. PLD activity was highest in rabbit brain, followed by rat and bovine brains; the activity in bovine brain was extremely low compared to the activities in rat and rabbit brains. We conclude that Triton X-100 is potentially useful for the purification of PLD and that rabbit and rat brains are the preferred sources.     

Poly(hydroxyethyl methacrylate‐co‐glycidyl methacrylate) reactive membrane utilised for cholesterol oxidase immobilisation

Poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) p(HEMA–GMA) membrane was prepared by UV‐initiated photopolymerisation of 2‐hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) in the presence of an initiator, azobisisobutyronitrile (AIBN). Cholesterol oxidase was immobilised directly on the membrane by forming covalent bonds between its amino groups and the epoxide groups of the membrane. An average of 53 µg of enzyme was immobilised per cm² of membrane, and the bound enzyme retained about 67% of its initial activity. Immobilisation improved the pH stability of the enzyme as well as its temperature stability. The optimum temperature was 5 °C higher than that of the free enzyme and was significantly broader. The thermal inactivation rate constants for free and immobilised preparations at 70 °C were calculated as k_{i (free)} 1.06 × 10^?1 min^?1 and k_{i (imm)} 2.68 × 10^?2 min^?1, respectively. The immobilised enzyme activity was found to be quite stable in the repeated experiments. © 2002 Society of Chemical Industry     

Purification and properties of an extracellular lipase fromPythium ultimum

An extracellular triacylglycerol lipase (EC 3.1.1.3) fromPythium ultimum strain No. 144 was purified by ammonium sulfate precipitation, and by diethylaminoethyl Sepharose CL-6B and Sephacryl S−200 chromatography. The purified enzyme preparation showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the protein was estimated at 270 kD using gel filtration on Sephacryl S−200, and 68 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicating that the enzyme may be a tetramer. The optimum pH and temperature for activity of the enzyme were 8.0 and 30°C, respectively. Activity was reduced by Co²⁺, Fe²⁺, Sn²⁺ and Mn²⁺ and stimulated by Ca²⁺, Mg²⁺, Na⁺, K⁺ and surfactants such as taurocholic acid, Triton X−100,n-octyl glucoside,n-dodecyl-β-D-maltoside, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate(CHAPS), and 3-[-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate. The apparent maximum specific activity was 42 μmole/min/mg in the absence of CHAPS and 77 μmole/min/mg in its presence. The reaction rate was progressively higher with increasing number of double bonds in the substrate, and the enzyme showed a preference for triacylglycerols containing fatty acids having thecis double bond configuration. AAES publication No. 6-933419.     

Purification and enzymatic properties of the extracellular and constitutive chitosanase produced by Bacillus subtilis RKY3

BACKGROUND: Purification and enzymatic properties of a chitosanase from Bacillus subtilis RKY3 have been investigated to produce a chitooligosaccharide. The enzyme reported was extracellular and constitutive, which was purified by two sequential steps including ammonium sulfate precipitation and ion exchange chromatography. RESULTS: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified chitosanase revealed one single band corresponding to a molecular weight of around 24 kDa. The highest chitosanase activity was found to be at pH 6.0 and at 60 °C. Although the mercaptide forming agents such as Hg²⁺ (10 mmol L^?1) and p‐hydroxymercuribenzoic acid (1 mmol L^?1, 10 mmol L^?1) significantly or totally inhibited the enzyme activity, its activity was enhanced by the presence of 10 mmol L^?1 Mn²⁺. The enzyme showed activity for hydrolysis of soluble chitosan and glycol chitosan, but colloidal chitin, carboxymethyl cellulose, crystalline cellulose, and soluble starch were not hydrolyzed. The analysis of chitosan hydrolysis by thin‐layer chromatography and viscosity variation revealed that the purified enzyme should be endosplitting‐type chitosanase. CONCLUSION: The chitosanase produced by Bacillus subtilis RKY3 was a novel chitosanlytic enzyme with relatively low molecular weight, which is a versatile enzyme for chitosan hydrolysis because it could hydrolyze soluble chitosan into a biofunctional oligosaccharide at a high level. Copyright © 2011 Society of Chemical Industry     

Multiple forms of the enzyme glycerophosphodiesterase are present in human brain

Brain levels of glycerophosphodiesters, including glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), are altered in many human central nervous system disorders. Although much information is available on the enzymes responsible for the formation of these phospholipid metabolites, little information is known regarding their catabolism, by glycerophosphodiesterases, in human brain. In both autopsied and biopsied temporal cortex, a phosphocholine-producing lycerophosphodiesterase activity was observed. In the presence of 1 mM EDTA, the enzyme possessed a pH optimum of 9.0, while the addition of 5 mM zinc acetate shifted the pH optimum to 10.5. When assayed at pH 9.0 in the absence of zinc acetate, the K_m and V_max were 104±2 μM and 77±18 nmol/h/mg protein, respectively, while assaying at pH 10.5 in the presence of 5 mM zinc acetate yielded a K_m of 964±56 μM, and a V_max of 534±114 nmol/h/mg protein. Furthermore, whereas submillimolar concentrations of zinc acetate stimulated the activity of the enzyme in a dose-dependent manner when assayed at pH 10.5 (EC₅₀=20.3±3.0 μM), this did not result in a reciprocal inhibition of glycerophosphocholine phosphodiesterase (GPC PD) activity when assayed at a more acidic pH. This may suggest that human brain contains two phosphocholine-producing GPC PD activities, differentiable by their sensitivity to zinc ions. An activity capable of hydrolyzing GPE to form phosphoethanolamine could not be detected in either biopsied or autopsied brain. However, a choline/ethanolamine-producing glycerophosphodiesterase activity could be readily detected in biopsied, but not autopsied brain. This novel enzyme possessed a neutral pH optimum and was dependent upon divalent cations for activity. In conclusion, human brain contains at least two different glycerophosphodiesterases, a phosphocholine, and a choline/ethanolamine-producing activity, only one of which can be detected in autopsied tissue. The results of previous studies measuring brain glycerophosphodiesterase activity in degenerative brain conditions may need to be reevaluated in the light of these observations.     

活性物质降解烟叶中淀粉的工艺研究

对固定化酶乳状液添加剂与水溶液酶添加剂降低低次烟叶中淀粉的质量分数进行了研究,考察了两种方法降解烟叶淀粉的工艺条件,并通过实验得出了各自的最佳工艺条件,固定化方法为:α-淀粉酶16 u/g(烟叶),糖化酶80 u/g(烟叶),降解温度50~55℃,作用时间15 h,w(H2O)=20%~25%;水溶液酶为:α-淀粉酶16 u/g,糖化酶80 u/g,温度40~45℃,时间9 h,w(H2O)=20%~25%。结果表明,固定化酶处理烟叶,淀粉降解率为30.25%,好于水溶液酶的25.25%,特别在抽吸品质方面,前者处理明显好于后者处理。     

Effect of clay/water ratio during bentonite clay organophilization on the characteristics of the organobentonites and its polypropylene nanocomposites

A sodium bentonite (montmorillonite‐based layered silicate clay) was organically modified with cetyl trimethyl ammonium bromide (cetrimide), using different clay/water ratios—but the same clay/cetrimide ratio—to suspend the bentonite clay and perform its organophilization. Infrared spectroscopy and thermogravimetric analysis indicated the incorporation of organic modifier into the bentonite. Wide‐angle X‐ray scattering showed that the incorporation of surfactant significantly increased the interlayer spacing in the bentonite for all concentrations studied. It was found that clay/water ratio employed during the modification affects neither the amount of organic salt incorporated nor the interlayer spacing in the organobentonites, but influences its degree of structural disorder. Lower clay/water ratios resulted in a more disorderly structure, as established by the decrease of the areas under the X‐ray peaks as the clay/water ratio diminishes. The modified bentonites were melt compounded with maleic anhydride‐grafted polypropylene. X‐ray diffraction patterns of the hybrids revealed that the more disorderly organobentonites were better dispersed in the polymer matrix, indicating that, in the present system, the structure of polymer nanocomposites obtained were affected by the clay/water ratio used in organobentonite preparation. POLYM. ENG. SCI., 2009. © 2009 Society of Plastics Engineers     

Pigment removal from canola oil using chlorophyllase

Frost-damaged or prematurely harvested canola seed (rapeseed) may yield oil with a high chlorophyll content (50–60 μg/ml). Enzymatic hydrolysis of chlorophyll, added to buffer/surfactant, buffer/acetone or buffer/acetone/canola oil, to produce water-soluble chlorophyllide (green pigment) was studied using a crude chlorophyllase preparation (acetone-dried chloroplasts) from 15 to 20-day-old sugar beet seedlings. In buffer/surfactant, the optimum pH for enzyme activity was temperature dependent. At 30 C and 0.24% Triton X-100 (or 30% acetone), chlorophyllase showed maximum activity toward a crude chlorophyll preparation over the range of pH 8–10. At 60 C, the activity was more than twofold higher, with a sharp maximum at ∼pH 8. Mg²⁺ enhanced the activity with an optimal concentration of 50 mM. At pH 7.5, 50 C and in the presence of only 6% acetone, the enzyme showed high affinity for chlorophyll (Km=15μM or 13.5 μg/ml), suggesting that the natural chlorophyll concentrations found in green canola oils might facilitate high enzymatic efficiencies. The crude enzyme was stable in buffer/acetone at pH 7.5 and 50 C for at least two hr. With acetone concentrations as low as 6%, maximum enzyme activities in buffer and buffer/canola oil required intensive mixing (homogenization) of the various substrate, enzyme and liquid phases. In general, the rate and extent of chlorophyll hydrolysis were greater in buffer than in buffer/oil. In both reaction systems, chlorophyll hydrolysis slowed down with time due to accumulation of phytol, which proved to be a competitive inhibitor (K_i=11 μM or 3.3 μg/ml). The other hydrolysis product, chlorophyllide, did not affect enzymatic activity. Crude canola oil used in the reconstitution of green oil did not support enzymatic chlorophyll hydrolysis without prior degumming and desoaping. The optimum buffer/oil ratio of the reaction mixtures was above 2/1 (v/v).     

Removal of Cadmium from Dilute Solutions by Hydroxyapatite. III. Flocculation Studies

Abstract

Synthetic hydroxyapatite in a fine particulate dispersion has been used previously for the removal of Cd²⁺ cations from dilute aqueous solutions. In the present work the flocculation of these hydroxyapatite particulates was examined with and without the presence of Cd²⁺ ions by applying the conventional inorganic flocculents ferric chloride and aluminum sulfate. An optical technique was applied in order to monitor the flocculation dynamics of the dispersed particles, complemented by electrophoretic and turbidity measurements. The relative size of aggregates formed during flocculation, expressed as a “flocculation index,” could be continuously measured by this technique. Results indicated that the flocculation index provided important information about flocculation mechanisms. The main parameters examined included flocculent dose, solution pH, and mixing intensity. The optimum conditions for efficient flocculation were determined.     

Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilization

Penicillin V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy‐activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55 °C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10‐fold increase in its half‐life at 40 °C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation. © 2001 Society of Chemical Industry     

Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-l-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba²⁺. This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.     

The effect of a perfluorochemical emulsion on enzyme reaction stability

A reaction system consisting of (oxygen-consuming) tyrosinase immersed within a perfluorochemical emulsion was investigated by comparing the activity and stability of the enzyme within the emulsion to that within pure buffer. The perfluorochemical emulsion contained 30 v/v% perfluorooctane, 70 v/v% aqueous buffer and pluronic-F68 as a surfactant. The catecholase activity of soluble mushroom tyrosinase was chosen for study and catechol was used as a substrate. A turnover parameter of 0.50 absorbance units and an inactivation rate constant of 0.012 s^?1 were found in the presence of a perfluorochemical emulsion and a turnover parameter of 0.53 absorbance units and an inactivation rate constant of 0.012 s^?1 were found in the absence of perfluorochemicals (pure buffer). The enzyme demonstrated no appreciable loss in performance within the perfluorochemical emulsion relative to its behaviour within aqueous buffer. This implies that the local concentrations of oxygen are the same under both systems and that under conditions when oxygen is limited in an aqueous system, tyrosinase performance would be improved within the perfluorochemical medium.     

Enhancement by ganglioside GT1b of annexin I phosphorylation in bovine mammary gland in the presence of phosphatidylserine and Ca2+

Ganglioside GT1b and, to a lesser extent, GD3, enhanced phosphorylation of a 36 kDa protein (the substrate of protein kinase C) in the particulate fraction from bovine mammary gland. Sialic acids, asialogangliosides, and GM3 were without effect, and GD1a conversely inhibited phosphorylation of the 36 kDa protein. The enhanced phosphorylation by GT1b required the simultaneous presence of phosphatidylserine (PS) and Ca²⁺. The 36 kDa protein reacted with anti-annexin I in Western blot analysis. Addition of purified annexin I to the reaction mixture containing the particulate fraction increased the extent of phosphorylated 36 kDa protein, and the phosphorylation was further enhanced by GT1b. The enhanced phosphorylation of annexin I by GT1b was also dependent on PS and Ca²⁺. When annexin I was phosphorylated by purified protein kinase C, GT1b inhibited the annexin I phosphorylation. Addition of epidermal growth factor or insulin to the particulate fraction had little effect on the enhancement. These results suggest that an enzyme or enzymes other than protein kinase C, epidermal growth factor receptor kinase, or insulin receptor kinase is responsible for the GT1b- and GD3-enhanced phosphorylation of annexin I in the presence of PS and Ca²⁺.     

Chemical modification and immobilization of papain

Papain, an endolytic cysteine protease (EC: 3.4.22.2), from Carica papaya latex has been chemically modified using succinic anhydride. This reagent reacts with the amino group of the lysine residues in the enzyme, thereby changing its net charge from positive to negative. The resultant enzyme had its optimum pH shifted from pH 6 to 8 and there was no change in the temperature optima of 70 °C. The modified papain had a specific activity of about 62.8 IU mg^?1 of protein at pH 8.0 at 30 °C, whereas for the native enzyme it was 46.57 IU mg^?1 under same conditions. Stability of the modified papain was further increased by entrapping in alginate/starch beads. The immobilized papain retained its activity even after six cycles of hydrolysis. The wet beads, when dried at 50 ± 2 °C, increased the storage stability of the immobilized enzyme. The succinylated papain is active in various organic solvents and hence can be successfully used in biotransformations as well as being used as a proteolytic component in detergents. Copyright © 2004 Society of Chemical Industry     

Purification and Biochemical Characterization of Lipase from Tunisian Euphorbia peplus Latex

Lipases from vegetable sources have been the focus of intense and growing research. The use of enzymes from plants has the advantage of employing industrial waste products. The lipase activity of Euphorbia peplus L. (Euphorbiaceae) was investigated for the first time. The Euphorbia peplus latex lipase (EpLL) was purified after ammonium sulfate fractionation and anion exchange chromatography on a DEAE-Cellulose column leading to 12.57-fold purification. The EpLL displayed a probable molecular weight of about 40 kDa. The lipase activity was optimum at a temperature of 40 °C and pH 8, the specific activities of EpLL were found to be 249 ± 12.45 and 161.4 ± 8.07 U/mg when tributyrin (TC₄) and olive oil were used as substrate respectively. The enzyme retained 80 % of its activity when incubated for 1 h at 50 °C. The EpLL was strongly destabilised by divalent metal ions (Fe²⁺, Mg²⁺, Zn²⁺ and Cu²⁺). Lipase was slightly stimulated by Triton X-100 and Tween-80, while strongly inhibited by sodium dodecyl sulfate. A good stability of the enzyme in the presence of organic solvents was reveled suggesting its industrial utility.     

The biosynthesis of cholesterol and other sterols by brain tissue: I. Subcellular biosynthesis in vitro

The biosynthesis of cholesterol by subcellular particles from rat brain was studied with several labeled cholesterol precursors as substrates. Rats from two age groups were used for preparation of the subcellular fractions: 15-day-old and adult. Microsomes and a soluble fraction were required for maximum biosynthesis of¹⁴C-nonsaponifiable material. The latter was synthesized in good yield by subcellular fractions from both age groups, but 90% or more was present as¹⁴C-squalene, when either U-¹⁴C-glucose, 2-¹⁴C-sodium acetate or 2-¹⁴C-mevalonic acid was the radioactive substrate. Neither³H-squalene oxide nor¹⁴C-lanosterol was converted to sterol when incubated with microsomal+soluble preparations, but some 4% of¹⁴C-desmosterol was converted to cholesterol by adult preparations. Thus a metabolic block, largely between squalene to desmosterol, exists in isolated microsomal+soluble preparations from both 15-day-old and adult rat brain. One of 12 papers to be published from the Sterol Symposium presented at the AOCS Meeting, New Orleans, April 1970.     

Preparation of microporous activated carbon from Aegle marmelos fruit shell by KOH activation

Activated carbon (AC) is well‐known for its unique properties; hence, the search for new precursors and the investigation of new methods for the preparation of AC is still drawing attention of many researchers. In the present work, microporous AC was prepared from Aegle marmelos fruit shell (AMFS) by potassium hydroxide (KOH) activation. The effect of process parameters such as impregnation ratio, carbonisation temperature and holding time on porous characteristics was investigated. The porous characteristics of prepared AC samples were analysed by N₂ adsorption–desorption isotherms, and it was found that the isotherms obtained resemble typical microporous solids (Type‐I). The Langmuir surface area and total pore volume of the sample prepared at optimum conditions were found to be 937 m²/g and 0.33 cm³/g, respectively. The contribution of micropores to the porous characteristics of the prepared AC is very much appreciable, and about 97% of the total surface area and pore volume is attained by micropores. Pore size distribution (PSD) by Dubinin–Astakhov (DA) and micro‐pore (MP) methods confirmed the presence of micropores to a great extent with insignificant mesoporosity. © 2013 Canadian Society for Chemical Engineering