A New Lipase Isolated from Oleaginous Seeds from <Emphasis Type="Italic">Pachira aquatica</Emphasis> (Bombacaceae)

A new lipase from seeds of Pachira aquatica was purified to homogeneity by SDS-PAGE obtaining an enzyme with a molecular weight of approximately 55 kDa. The purified lipase exhibited maximum activity at 40 degrees C and pH 8.0, for an incubation time of 90 min. Concerning temperature stability, at the range from 4 to 50 degrees C, it retained approximately 47% of its original activity for 3 h. The enzyme activity increased in the presence of Ca(++) and Mg(++), but was inhibited by Hg(++), Mn(++), Zn(++), Al(+++) and various oxidizing and reducing agents. The lipase was highly stable in the presence of organic solvents, and its activity was stimulated by methanol. The values of K(m) and V(max) were 1.65 mM and 37.3 micromol mL(-1) min(-1), respectively, using p-nitrophenylacetate as substrate. The enzyme showed preference for esters of long-chain fatty acids, but demonstrated significant activity against a wide range of substrates.     

Transesterification of Waste Cooking Oil by an Organic Solvent-Tolerant Alkaline Lipase from Streptomyces sp. CS273

The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0?～?9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo₄, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis.     

Purification and Properties of a New Thermostable Cyclodextrin Glucanotransferase from Bacillus pseudalcaliphilus 8SB

A new cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was studied in respect to its γ-cyclizing activity. An efficient conversion of a raw corn starch into only two types of cyclodextrins (β- and γ-CD) was achieved by the purified enzyme. Crude enzyme obtained by ultrafiltration was purified up to fivefold by starch adsorption with a recovery of 62% activity. The enzyme was a monomer with a molecular mass 71 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The CGTase exhibited two pH optima, at pH 6.0 and 8.0, and was at most active at 60 °C and pH 8.0. The enzyme retained more than 80% of its initial activity in a wide pH range, from 5.0 to 11.0. The CGTase was strongly inhibited by 15 mM Cu(2+), Fe(2+), Ag(+), and Zn(2+), while some metal ions, such as Ca(2+), Na(+), K(+), and Mo(7+), exerted a stimulating effect in concentration of 5 mM. The important feature of the studied CGTase was its high thermal stability: the enzyme retained almost 100% of its initial activity after 2 h of heating at 40-60 °C; its half-life was 2 h at 70 °C in the presence of 5 mM Ca(2+). The achieved 50.7% conversion of raw corn starch into 81.6% β- and 18.4% γ-CDs after 24 h enzyme reaction at 60 °C and pH 8.0 makes B. pseudalcaliphilus 8SB CGTase industrially important enzyme for cyclodextrin production.     

Isolation, Characterization, and Catalytic Properties of a Novel Lipase Which Is Activated in Ionic Liquids and Organic Solvents

A novel extracellular lipase with organic solvent tolerance was isolated from a local Pseudomonas species. The lipase gene was cloned and expressed in Escherichia coli as a heterologous host and purified by affinity chromatography. The activity of purified lipase was investigated in the presence of imidazolium-based ionic liquids (ILs) such as EMIM[Cl], BMIM[Cl], and HMIM[Cl]. It has been found that the activity of treated lipase with ILs was higher than untreated control in the hydrolysis reaction. Also, the results indicated that the enzymatic activity strongly depends on IL concentration in reaction media. The best concentration of the IL was 30%, 45%, and 50% (v/v) for HMIM[Cl], BMIM[Cl], and EMIM[Cl], respectively. Additionally, the enzyme exhibited excellent stability in the presence of 25% of n-hexane, toluene, acetone, and t-butanol. The optimum values of pH and temperature were determined 10 and 55 °C, respectively. The K (m) and V (max) values were calculated 0.4 mM and 1.92 U/ml, respectively, using p-nitrophenyl palmitate as substrate. With respect to the biochemical properties of the newly isolated lipase such as high-level stability and noticeable activity in the presence of organic solvents and ionic liquids, the newly isolated lipase seems to be a good candidate for environmental and industrial processes carried out in non-aqueous media.     

Serum albumin targeted, pH-dependent magnetic resonance relaxation agents

The objective of this work was the synthesis of serum albumin targeted, Gd(III)-based magnetic resonance imaging (MRI) contrast agents exhibiting a strong pH-dependent relaxivity. Two new complexes (Gd-glu and Gd-bbu) were synthesized based on the DO3A macrocycle modified with three carboxyalkyl substituents?α to the three ring nitrogen atoms, and a biphenylsulfonamide arm. The sulfonamide nitrogen coordinates the Gd in a pH-dependent fashion, resulting in a decrease in the hydration state, q, as pH is increased and a resultant decrease in relaxivity (r(1)). In the absence of human serum albumin (HSA), r(1) increases from 2.0 to 6.0?mM(-1) s(-1) for Gd-glu and from 2.4 to 9.0?mM(-1) s(-1) for Gd-bbu from pH?5 to 8.5 at 37?°C, 0.47?T, respectively. These complexes (0.2?mM) are bound (>98.9?%) to HSA (0.69?mM) over the pH range 5-8.5. Binding to albumin increases the rotational correlation time and results in higher relaxivity. The r(1) increased 120?% (pH?5) and 550?% (pH?8.5) for Gd-glu and 42?% (pH?5) and 260?% (pH?8.5) for Gd-bbu. The increases in r(1) at pH?5 were unexpectedly low for a putative slow tumbling q=2 complex. The Gd-bbu system was investigated further. At pH?5, it binds in a stepwise fashion to HSA with dissociation constants K(d1)=0.65, K(d2)=18, K(d3)=1360?μM. The relaxivity at each binding site was constant. Luminescence lifetime titration experiments with the Eu(III) analogue revealed that the inner-sphere water ligands are displaced when the complex binds to HSA resulting in lower than expected r(1) at pH?5. Variable pH and temperature nuclear magnetic relaxation dispersion (NMRD) studies showed that the increased r(1) of the albumin-bound q=0 complexes is due to the presence of a nearby water molecule with a long residency time (1-2?ns). The distance between this water molecule and the Gd ion changes with pH resulting in albumin-bound pH-dependent relaxivity.     

Single-Step Purification and Characterization of Recombinant Aspartase of Aeromonas media NFB-5

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b(+) expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5?mM) induction for 5?h at 37?°C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni?CNTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH?8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35?°C. The enzyme showed apparent K _m and V _max values for L-aspartate as 2.01?mM and 114?U/mg, respectively. The enzyme was stable in pH range of 6.5?C9.5 and temperature up to 45?°C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51?kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.     

Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection

A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ～ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.     

Catalytic and Thermodynamic Properties of a Tannase Produced by Aspergillus niger GH1 Grown on Polyurethane Foam

Tannase is an inducible enzyme with important applications in the food and pharmaceutical industries. This enzyme was produced by the fungus Aspergillus niger GH1 under solid-state fermentation using polyurethane foam as solid support and tannic acid as sole carbon source and tannase inducer. Physicochemical properties of A. niger tannase were characterized, and the kinetic and thermodynamics parameters on methyl gallate hydrolysis were evaluated. The enzyme was stable in a pH range of 2-8 and a functional temperature range of 25-65 °C. The highest k(cat) value was 2,611.10 s(-1) at 65 °C. Tannase had more affinity for methyl gallate at 45 °C with a K(M) value of 1.82 mM and an efficiency of hydrolysis (k(cat)/K(M)) of 330.01 s(-1) mM(-1). The lowest E(a) value was found to be 21.38 kJ/mol at 4.4 mM of methyl gallate. The lowest free energy of Gibbs (ΔG) and enthalpy (ΔH) were found to be 64.86 and 18.56 kJ/mol, respectively. Entropy (ΔS) was -0.22 kJ/mol K. Results suggest that the A. niger GH1 tannase is an attractive enzyme for industrial applications due its catalytic and thermodynamical properties.     

Expression,Purification and Characterization of Human α‐l‐Fucosidase

α‐l ‐Fucosidases (EC 3.2.1.51) are exo‐glycosidases. On the basis of the multi‐alignment of amino acid sequence, α‐l ‐fucosidases were classified into two families of glycoside hydrolases, GH‐29 and GH‐95. They are responsible for the removal of l ‐fucosyl residues from the non‐reducing end of glycoconjugates. Deficiency of α‐l ‐fucosidase results in Fucosidosis due to the accumulation of fucose‐containing glycolipids, glycoproteins and oligosaccharides in various tissues. Recent studies discovered that the fucosylation levels are increased on the membrane surfaces of many carcinomas, indicating the biological function of α‐l ‐fucosidases may relate to this abnormal cell physiology. Although the gene of human α‐l ‐fucosidase (h‐fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a soluble and active from. We report herein that, with carefully control on the growing condition, an active human α‐l ‐fucosidases (h‐Fuc) was successfully expressed in Escherichia coli for the first time. After a series steps of ion‐exchange and gel‐filtration chromatographic purification, the recombinant h‐Fuc with 95% homogeneity was obtained. The molecular weight of the enzyme was analyzed by SDS‐PAGE (～50 kDa) and confirmed by ESI mass (50895 Da). The recombinant h‐Fuc was stable up to 55 °C with incubation at pH 6.8 for 2 h; the optimum temperature for h‐Fuc is approximately 55 °C. The enzyme was stable at pH 2.5–7.0 for 2 h; the enzyme activity decreased greatly for pH greater than 8.0 or less than 2.0. The K_m and k_cat values of the recombinant h‐Fuc (at pH 6.8) were determined to be 0.28 mM and 17.1 s^?1, respectively. The study of pH‐dependent activity showed that the recombinant enzyme exhibited optimum activity at two regions near at pH 4.5 and pH 6.5. These features of the recombinant h‐Fuc are comparable to the native enzyme purified directly from human liver. Studies on the transfucosylation and common intermediate of the enzymatic reaction by NMR support that h‐Fuc functions as a retaining enzyme catalyzing the hydrolysis of substrate via a two‐step, double displacement mechanism.     

Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08

The gene encoding a glycoside hydrolase family 43 beta-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-beta-D: -xylopyranose (4NPX) and p-nitrophenyl-alpha-L: -arabinofuranose (4NPA), and it was found that the ratio k (cat)/K (m) 4NPA/k (cat)/K (m) 4NPX was approximately 7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k (cat)/K (m) muA/k (cat)/K (m) muX was approximately 5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K (i) of 6.8 +/- 0.62 mM and xylose K (i) of 76 +/- 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 degrees C, with a t (1/2) of 35 min at 57.5 degrees C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.     

Kinetic Stability Modelling of Keratinolytic Protease P45: Influence of Temperature and Metal Ions

The activity and kinetic stability of a keratinolytic subtilisin-like protease from Bacillus sp. P45 was investigated in 100 mM Tris-HCl buffer (pH 8.0; control) and in buffer with addition of Ca(2+) or Mg(2+) (1-10 mM), at different temperatures. Addition of 3 mM Ca(2+) or 4 mM Mg(2+) resulted in a 26% increment on enzyme activity towards azocasein when compared to the control (100%; without added Ca(2+) or Mg(2+)) at 55 °C. Optimal temperature for activity in the control (55 °C) was similar with Mg(2+); however, temperature optimum was increased to 60 °C with 3 mM Ca(2+), displaying an enhancement of 42% in comparison to the control at 55 °C. Stability of protease P45 in control buffer and with Mg(2+) addition was assayed at 40-50 °C, and at 55-62 °C with Ca(2+) addition. Data were fitted to six kinetic inactivation models, and a first-order equation was accepted as the best model to describe the inactivation of protease P45 with and without metal ions. The kinetic and thermodynamic parameters obtained showed the crucial role of calcium ions for enzyme stability. As biocatalyst stability is fundamental for commercial/industrial purposes, the stabilising effect of calcium could be exploited aiming the application of protease P45 in protein hydrolysis.     

亲水梳状环氧聚合物载体柔性固定化脂肪酶

以氯乙酰化聚苯乙烯微球载体为大分子引发剂,以甲基丙烯酸缩水甘油酯和亲水性丙烯酰胺为共聚接枝单体,以氯化亚铜及2,2'-联吡啶为催化体系,采用原子转移自由基聚合法接枝合成了具有柔性链的亲水梳状环氧聚合物载体PS-acyl-P(AM-co-GMA),并将其用于耐有机溶剂YCJ01脂肪酶的共价柔性固定化.结果表明,固定化酶催...     

Effect of Aeration and Agitation Regimes on Lipase Production by Newly Isolated Rhodotorula mucilaginosa–MTCC 8737 in Stirred Tank Reactor Using Molasses as Sole Production Medium

The influence of media and process parameters (aeration and agitation) on fermentation broth rheology and biomass formation has been studied in 1.5-l stirred tank reactor for lipase production using Rhodotorula mucilaginosa MTCC 8737. Molasses, as sole production medium, is used for lipase production by varying aeration (1, 2, and 3 vvm) and agitation speeds (100, 200, and 300 rpm). Maximum lipase activity of 72 U/ml was obtained during 96 h of fermentation at 2 vvm, 200 rpm, pH 7, and 25 +/- 2 degrees C temperature. Lipase production kinetics with respect to dry cell weight of biomass showed Y (P/S) of 25.71 U/mg, specific product formation of 10.9 U/mg DC, and Y (X/S) 2.35 mg/mg. Maximum lipase activity (MC 2) of 56 U/ml was observed at 1% molasses, and a further increase in the molasses concentration of (%) 1.5 and 2 inhibited the product formation of lipase with 15 and 8.5 U/ml, respectively. The production kinetics of molasses media showed Y (P/X) was 14 U/mg DC, Y (P/S) 16 U/mg, and Y (X/S) 1.14 mg/mg during 96 h of bioreactor operation. The k(L)a values for all batches (MC 1-MC 4) at 96 h of fermentation were 32, 28, 21, and 19/h, and the |oxygen transfer rate were 54.4, 56, 35.7, and 17.29 mg/l h, respectively. Increase in molasses concentration resulted in decreased lipase activity by increase in viscosity of the fermentation broth.     

Development and characterization of an immobilized enzyme reactor based on glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been extensively studied as a target for new drugs to be used in the treatment of various parasitic diseases. The standard approach to the determination of GAPDH activity utilizes solubilized free enzyme and is limited by the enzyme's low stability. In the current study the stability of GAPDH was significantly increased through the covalent immobilization of the enzyme on a wide-pore silica support containing glutaraldehyde (Glut-P). The optimal conditions for the immobilization were: 100 mg Glut-P stationary phase, approximately 150 microg of enzyme dissolved in pyrophosphate buffer (15 mM, pH 8.5). The mixture was gently agitated for 6 h at 4 degrees C. Under these conditions 91.3% of protein was immobilized on 100 mg of Glut-P support with retention of 2.97% of the initial enzymatic activity. The activity of the immobilized GAPDH was stable for over 30 days. The GAPDH-Glut-P stationary phase was packed into a glass column to produce a GAPDH immobilized enzyme reactor (GAPDH-IMER). The activity and kinetic parameters of the GAPDH-IMER were investigated and the results demonstrated that the enzyme retained its activity and sensitivity to the competitive inhibitor agaric acid.     

Highly relaxing gadolinium based MRI contrast agents responsive to Mg2+ sensing

A Gd complex based on a polyphosphonated pyridyl ligand shows a very high stability in aqueous solution (log?K(EuL) = 25.7), a high relaxivity (8.5 mM(-1) s(-1) at 25 °C and 20 MHz) and a marked and selective relaxivity enhancement (37%) in the presence of Mg(2+), opening interesting perspectives for the design of cation responsive contrast agents.     

A beta-glucosidase from Sclerotinia sclerotiorum: biochemical characterization and use in oligosaccharide synthesis

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one beta-glucosidase (beta-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that beta-glu x may be a homodimer. For p-nitrophenyl beta-d-glucopyranoside hydrolysis, apparent Km and Vmax values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55-60 degrees C and pH 5.0, respectively. beta-Glu x was strongly inhibited by Fe2+ and activated about 35% by Ca2+. beta-Glu x possesses strong transglucosylation activity in comparison with commercially available beta-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50 degrees C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 3C nuclear magnetic resonance spectroscopy.     

Lipase from a Brazilian StrainPenicillium citrinum Cultured in a Simple and Inexpensive Medium st]Heat-Denaturation, Kinetics, and pH Stability

This work is a study of lipase production by a Brazilian strain ofPenicillium citrinum using an inexpensive and simple medium without organic nitrogen sources and of some important industrial properties, including thermostability in relation to ionic strength. The maximal lipase activity (1585 U/L) was obtained whenPenicillium citrinum was cultured on 0.75% ammonium sulfate complemented with minerals salts instead of yeast extract. Although this activity was about 55% lower than that produced in medium with yeast extract (2850 U/L), the specific activity (7.8 U/mg proteins) was higher than that obtained with the yeast extract (4.9 U/mg proteins). The morphology of fungus changed totally, with yeast extract there are smooth, solid, and spherical pellets whereas on ammonium sulfate there are small “hairy” pellets uniformly suspended in the medium. The effect of ferrous (Fe⁺⁺) ions was carried out using medium MA with and without Fe⁺⁺ ions. Lipase production byPenicillium citrinum in medium MA requires Fe⁺⁺ ions, the absence of which caused a decreased of about 50% in the specific activity (3.5 U/mg proteins). The utilization of commercial, locally available oils as carbon sources, such as soybean oil (236 U/L) and corn oil (74 U/L) resulted in lower activity compared to olive oil, showing that lipase production byPenicillium citrinum is specifically induced by olive oil. Potassium concentration in the medium can effects the production of lipase (1 mM (1585 U/L), 10 mM (1290 U/L), and 30 mM (1238 U/L), 50 mM (195 U/L), and 100 mM (2 U/L). The crude culture filtered was susceptable to thermal deactivation. It was stable at pH 6.0, but was not stable at the optimum pH (8.0-8.5) at 50 mM. At the low ionic concentration (1-25 mM) this lipase was stable at low pH (3.5-4.0). The activation energy was 22.4 ±2.2 Kcal. mol 1.     

Two Step Purification of <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. Lipase and Its Evaluation as a Detergent Additive at Low Temperatures

Acinetobacter sp. lipase was purified to homogeneity by a two-step process. The crude enzyme (along with biomass) was subjected to partial purification by aqueous two phase system (ATPS), avoiding centrifugation and filtration steps. Conditions for lipase partitioning by ATPS were optimized by response surface methodology (RSM) and a combination of 29.45% polyethylene glycol 8000, 15.5% phosphate, and a pH of 7.0 resulted in an optimal partition coefficient. Partially pure lipase was further purified by a modified batch process using Octyl Sepharose CL-4B in a vacuum filtration apparatus. This two-step process resulted in a purified lipase with a yield of 74.6% having a specific activity of 88.8 U/mg of protein and a purification fold of 14.92. The homogeneity of the lipase preparation obtained by the purification process was confirmed by reversed phase high performance liquid chromatography profile. The molecular weight of the purified lipase was found to be around 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified lipase exhibited pH and temperature optima of 8.5 and 37 degrees C, respectively. The lipase was active at low temperatures and it retained 86.8% activity at 10 degrees C. It also displayed other features such as stability over a broad range of pH (3.0-9.0) as well as stability in the presence of hydrogen peroxide and commercial detergents. Based on these characteristics, the potential of this lipase as an additive in laundry detergent formulation was evaluated under low temperature wash conditions. The results indicated that Acinetobacter sp. lipase increased the washing efficiency of the detergent Nirma by 21-24% at 15 degrees C-20 degrees C, respectively.     

Effective immobilisation of lipase to enhance esterification potential and reusability

A commercial lipase, “Lipolase T100”, was immobilised onto silica by means of physical adsorption. The silica-bound lipase was subsequently exposed to 1 vol. % glutaraldehyde (pentane-1,5-dial). The silica was loaded repeatedly with the Lipolase T100 in 0.05 M Tris buffer (pH 8.5) until saturation was achieved. During the 1st, 2nd, 3rd, 4th, and 5th cycles of loading of silica with the enzyme, the protein-binding on the silica achieved 51.73 %, 48.27 %, 26.92 %, 10.73 %, and 4.29 %, respectively. The synthesis of methyl salicylate (methyl 2-hydroxybenzoate) and linalyl ferulate (3,7-dimethylocta-1,6-dien-3-yl 4-hydroxy-3-methoxycinnamate) carried out at 45°C under shaking with mole ratios of 200 mM of acid and 500 mM alcohol in DMSO using 15 mg mL^?1 of hyper-activated biocatalyst resulted in yield(s) of 77.2 % of methyl salicylate and 65.3 % of linalyl ferulate in the presence of molecular sieves. The hyper-activated biocatalyst was more efficient than the previously reported silica-bound lipase with minimum leaching of the enzyme from the reaction mixture. The K _m and V _max of the free (0.142 mM and 38.31 μmol min^?1 mL^?1, respectively) and silica-bound lipase (0.043 mM and 26.32 μmol min^?1 mg^?1, respectively) were determined for the hydrolysis of p-NPP. During repeated esterification studies using silica-bound lipase, yields of 50.1 % of methyl salicylate after the 5th cycle, and 53.9 % of linalyl ferulate after the 7th cycle of esterification were recorded. In the presence of molecular sieves (30 mg mL^?1) in the reaction mixture, the maximum syntheses of methyl salicylate (77.2 %) and linalyl ferulate (65.3 %) were also observed. In a volumetric batch scale-up, when the reaction volume was increased to 50 mL, 44.9 % and 31.4 % yields of methyl salicylate and linalyl ferulate, respectively, were achieved.     

Purification and partial characterization of a lipase from Bacillus coagulans ZJU318

An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with 18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme activity was strongly inhibited by Ag⁺ and Cu²⁺, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly inhibit lipase activity.