Lipase from a Brazilian strain ofPenicillium citrinum

A lipases (glycerol ester hydrolases E. C. 3.1.1.3) from a brazilian strain ofPenicillium citrinum has been investigated. When the microorganism was cultured in the simple medium (1.0% olive oil and 0.5% yeast extract), using olive oil in as carbon source in the inocula, the enzyme extracted showed maximum activity (409 IU/mL). In addition, decrease of yeast extract concentration also reduces the lipase activity. Nevertheless, when yeast extract was replaced by ammonium sulfate, no activity was detected. Purification by precipitation with ammonium sulfate showed best activity in the 40–60% fraction. The optimum temperature for enzyme activity was found in the range of 34–37°C. However, after 30 min at 60°C, the enzyme was completely inactivated. The enzyme showed optimum at pH 8.0. The dried concentrated fraction (after dialysis and lyophilization) maintained its lipase activity at room temperature (28°C) for 8 mo. This result in lipase stability suggests an application of lipases fromP. citrinum in detergents and other products that require a high stability at room temperature.     

Comparison of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase Immobilization Yield of Entrapment,Adsorption, and Covalent Bond Techniques

The purpose of this study was to immobilize lipase from Yarrowia lipolytica using three methods including inclusion, adsorption, and covalent bond to study enzyme leaching, storage, and catalytic properties. Sodium alginate and chitosan were the polymers selected to immobilize lipase by inclusion. The beads of each polymer were dried by freeze drying and fluidization. The results show that chitosan was more adapted to the inclusion of lipase. Even though freeze dried, bead activity was low compared to that of fluidized beads. The freeze-drying process seems to produce suitable beads for storage at 4 and 20 degrees C. The immobilization by adsorption was carried out on both celite and silica gel. Maximum immobilization yield of 76% was obtained with celite followed by 43% in silica gel. The enzyme adsorbed on the two supports exhibited greater stability at a certain temperature (50 degrees C) and in no polar solvents (Isooctane, n-heptane, and n-hexane). In addition, the lipase immobilized by covalent bond retained residual activity equitable to 70%. It was demonstrated that the enzyme immobilized by covalent bond showed greater activity (80%) after 5 months of storage.     

Effect of Sol–Gel Encapsulation on Lipase Structure and Function: A Small Angle Neutron Scattering Study

The application of small angle neutron scattering (SANS) to the characterisation of sol–gel hosts containing biomolecules offers the opportunity to explore the relationship between gel structure and catalyst. A model system involving the immobilisation of Candida antarctica lipase B (CALB) was investigated.Gels were produced by fluoride-catalysed hydrolysis of fixed ratios of tetramethylorthosilicate (TMOS) and methyltrimethoxysilane (MTMS). Phase separation between the enzyme and the evolving sol–gel matrix was minimised by incorporating glycerol into the sol–gel precursor solution. The potential stabilising effect of the NaF catalyst upon the enzyme was also investigated. Scattering studies were conducted on both immobilised lipase, and lipase in free solution. Scattering studies on free enzyme provided evidence of multiple populations of enzyme aggregates and showed that choice of solvent affected the degree of aggregation. Both NaF and glycerol affected neutron scattering, indicating changes in lipase conformation. Increasing glycerol concentration increased the degree of aggregation and produced differences in solvent packing on the surface of protein molecules. Initial evidence from SANS data indicated that the presence of the enzyme during gel formation conferred structural changes on the gel matrix. Modelling the effect of sol–gel encapsulation on lipase requires comparison of data from free enzyme to the immobilised form. Removal of the enzyme from the sol–gel structure, post gelation, is necessary to better characterise the modified matrix. This methodological problem will be the subject of future investigations.     

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.     

A stable lipase fromCandida lipolytica

Although Upases have been intensively studied, some aspects of enzyme production like substrate uptake, catabolite repression, and enzyme stability under long storage periods are seldom discussed in the literature. This work deals with the production of lipase by a new selected strain ofCandida lipolytica. Concerning nutrition, it was observed that inorganic nitrogen sources were not as effective as peptone, and that oleic acid or triacylglycerides (TAG) were essential carbon sources. Repression by glucose and stimulation by oleic acid and long chain TAG (triolein and olive oil) were observed. Extracellular lipase activity was only observed at high levels at late stationary phase, whereas intracellular lipase levels were constant and almost undetectable during the cultivation period, suggesting that the produced enzyme was attached to the cell wall, mainly at the beginning of cultivation. The crude lipase produced by this yeast strain shows the following optima conditions: pH 8.0–10.0, temperature of 55°C. Moreover, this preparation maintains its full activity for at least 370 d at 5°C.     

Effect of temperature, moisture, and carbon supplementation on lipase production by solid-state fermentation of soy cake by Penicillium simplicissimum

The production of lipases by Penicillium simplicissimum using solid-state fermentation and soy cake as substrate was investigated. The effects of temperature, cake moisture, and carbon supplementation on lipase production were studied using a two-level experimental plan. Moisture, pH, and lipase activity were followed during fermentation. Statistical analysis of the results was performed to evaluate the effect of the studied variables on the maximum lipase activity. Incubation temperature was the variable that most affected enzyme activity, showing a negative effect. Moisture and carbon supplementation presented a positive effect on activity. It was possible to obtain lipase activity as high as 21 U/g of dry cake in the studied range of process variables.     

New methodology for tosylation of hydroxylic supports as exemplified by the immobilization of micrococcal endonuclease on agarose

Improvement have been made in a simplified procedure we previously reported (J.Mol.Catal. (1986),38,227 for the activation of tosyl chloride of supports possessing primary hydroxyl groups. The method is simple, can be completed in less than 90 min, yields a broad range of activation degrees, and, since it involves no toxic reagents, may be used for preparing immobilized enzymes to be utilized in food manufacturing and processing. The immobilization ofStophylococcal Nuclease has been carried out by this method. The insolubilized derivatives are more active than the native enzyme in the hydrolysis of DNA. The thermal stability of nuclease derivatives is greater than that of the native enzyme. These derivatives remain active at 50°C, and the native enzyme, 39°C. The insolubilized nuclease is more stable against organic solvents such as, dimethylsulfoxide (DMSO) or tetrahydroduran (THF) than the native enzyme.     

Immobilization of Candida antarctica Lipase B by Adsorption to Green Coconut Fiber

An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t _1/2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction.     

Wood cellulignin as an alternative matrix for enzyme immobilization

The objective of this work was to select an efficient methodology for preparing active samples of Candida rugosa lipase immobilized in wood cellulignin, to be applied in hydrolysis and ester reactions. For this purpose, lipase was immobilized in the matrix by physical adsorption (pure cellulignin) and covalent binding (activated cellulignin with glutaraldeyde or carbonyldiimidazole [CDI]) in the presence or absence of polyethylene glycol (PEG) (Molecular mass of 1500 Daltons) as stabilizing agent. The activating agent and the presence of PEG-1500 in the immobilization procedure showed a strong influence on enzyme retention in the support. The values for enzyme retention ranged from 20 to 68%, and the highest yield was obtained when the enzyme was immobilized in cellulignin activated with CDI in the presence of PEG-1500. This immobilized derivative presented high hydrolytic (193.27 μM/[mg·min]) and synthetic (522.92 μM/[g·min]) activities when compared with those obtained by other techniques. The superiority of this immobilized system was confirmed by additional analyses, such as infrared spectroscopy and elemental analysis, which demonstrated an appropriate enzyme fixation and the highest level of protein incorporation in the support. Further information on the immobilized derivative was obtained by assessing the recycle potential in both aqueous and nonaqueous media.     

Preparation and characterization of pancreatic lipase immobilized in Eudragit-matrix

Pancreatic lipase (EC 3.1.1.3) was immobilized by entrapping in a commercial preparation of acrylic/methacrylic acid ester-based copolymer (Eudragit E 30 D). The activity of the immobilized lipase beads with a diameter of 1.5-2.0 mm was found to be lower than that of the free lipase. The optimum pH was shifted to the alkaline region and the thermal stability increased, whereas the optimum temperature level remained unchanged. The most important reason for the decreased activity was diffusion limitations. The diffusion of the substrate and products became more pronounced, and lipolytic activity increased upon addition of n-hexane into the reaction medium. The storage and operational stabilities of the immobilized lipase were investigated, and both characteristics were found to be increased when compared to the free enzyme. Furthermore, mechanical or magnetic stirring during the operation were found to have no influence on the carrier-matrix as determined by nephelometric measurements.     

Production of biodiesel by immobilized Candida sp. lipase at high water content

A new process for enzymatic synthesis of biodiesel at high water content (10–20%) with 96% conversion by lipase from Candida sp. 99–125 was studied. The lipase, a no-position-specific lipase, was immobilized by a cheap cotton membrane and the membrane-immobilized lipase could be used at least six times with high conversion. The immobilized lipase could be used for different oil conversion and preferred unsaturated fatty acids such as oleic acid to staturated fatty acids such as palmitic acid. The changes in concentration of fatty acids, diglycerides, and methyl esters in the reaction were studied and a mechanism of synthesis of biodiesel was suggested: the triglycerides are first enzymatically hydrolyzed into fatty acids, and then these fatty acids are further converted into methyl esters.     

Multicomponent thermosensitive systems for biocatalysts

Composite matrices based on macroporous silica modified by N-vinylcaprolactam copolymers with diallyldimethylammonium chloride and with 2-hydroxyethyl methacrylate were obtained. Lipase from Pseudomonas fluorescens was immobilized on the obtained materials. The temperature dependence of the hydrolytic activity of the immobilized lipase preparations in the triacetin hydrolysis was investigated. The hydrolytic activity of lipase immobilized on the matrix modified by the N-vinylcaprolactam copolymer with 2-hydroxyethyl methacrylate can be regulated by varying the temperature of the reaction medium. The temperature dependence of the hydrolytic activity of the immobilized enzyme has a maximum at 40 °C, the activity of the immobilized lipase being ∼3.5 times higher compared to that at 20 °C. After immobilization on these composite materials, lipase retained the activity in the acetylation of 1-(RS)-phenylethanol with vinyl acetate in Bu^tOMe.__________Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 443–448, February, 2005.     

Production of lipase by a newly isolated <Emphasis Type="Italic">Bacillus coagulans</Emphasis> under solid-state fermentation using melon wastes

Summary An extracellular lipase was produced by Bacillus coagulans by solid-state fermentation. Solid waste from melon was used as the basic nutrient source and was supplemented with olive oil. The highest lipase production (78,069 U/g) was achieved after 24h of cultivation with 1% olive oil enrichment. Enzyme had an optimal activity at 37°C and pH 7.0, and sodium dodecyl sulfate increased lipase activity. NH ₄NO₃ increased enzyme production, whereas organic nitrogen had no effect. The effect of the type of carbon sources on lipolytic enzyme production was also studied. The best results were obtained with starch and maltose (148,932 and 141,629 U/g, respectively), whereas a rather low enzyme activity was found in cultures grown on glucose and galactose (approx 118,769 and 123,622 U/g, respectively). Enzyme was inhibited with Mn⁺² and Ni⁺² by 68 and 74%, respectively. By contrast, Ca⁺² enhanced enzyme production by 5%.     

界面活化的溶胶凝胶包埋Candida rugosa脂肪酶催化合成维生素E琥珀酸酯

采用界面活化的溶胶凝胶包埋Candida rugosa脂肪酶(CRL)催化合成了维生素E琥珀酸酯.考察了影响溶胶凝胶包埋固定化CRL的因素,获得的最佳固定化条件为:丙基三甲氧基硅烷/正硅酸四乙酯摩尔比为1/1,水与硅烷前体摩尔比为15,酶的添加量为0.5mg/ml,PEG400的添加量为12μl/ml溶胶. 溶胶凝胶包埋的CRL在50℃,18h后其活性仍然保持了70.58%,是游离酶的2.6倍,且稳定性得到了明显的改善.基于CRL的界面特性,采用五种表面活性剂对其进行界面活化.结果表明,采用橄榄油活化的溶胶凝胶包埋的CRL合成维生素E琥珀酸酯的酯化活力最高,相比原酶和未界面活化的溶胶凝胶包埋酶分别提高了6.7和1.43倍.     

Lipase-catalyzed production of optically active acids via asymmetric hydrolysis of esters

It has been found that lipase fromCandida cylindracea hydrolyzes octyl R(+)- but not S(-)-2-chloropropionate. At the same time, the enzyme exhibits no appreciable stereoselectivity in the hydrolysis of the methyl ester of the same acid. Solubility determination experiments showed that at the concentrations used, methyl 2-chloropropionate was completely dissolved in water, whereas the octyl ester existed as an emulsion in water. It is therefore speculated that in order to express its stereoselectivity the lipase needs to adsorb on the substrate—water interface. R,S-2-chloropropionic acid was preparatively resolved via yeast lipase-catalyzed asymmetric hydrolysis of its octyl ester. Gram quantities of R(+)-chloropropionic acid and octyl S(-)-2-chloropropionate of high optical purity were readily prepared.     

Purification and partial characterization ofRhizomucor miehei lipase for ester synthesis

A commercialRhizomucor miehei lipase was purified by ammonium sulfate precipitation. Phenyl Sepharose 6 Fast Row hydrophobic interaction chromatography, and DEAE Sepharose Fast Flow anion-exchange chromatography. The recovery of lipase activity was 32% with a 42-fold purification. The molecular size of the purified enzyme was 31,600 Dalton and the pI 3.8. The enzyme was stable for at least 24 h within a pH range of 7.0-10.0, and 96.8% of the enzyme activity remained when kept at 30‡C for 24 h. Further, about 10–30% of the lipase activity was inhibited by K⁺, Li⁺, Ni⁺, Co²⁺, Zn²⁺, Mg²⁺, Sn²⁺, Cu²⁺, Ba²⁺, Ca²⁺, and Fe²⁺ ions and by SDS, but EDTA had no effect. Under the experimental conditions, the optimum temperature for the hydrolysis of olive oil was 50‡C (pH 8.0), and for the synthesis of 1-butyl oleate, 37‡C. It was concluded that hydrolytic activity of lipase alone is not a sufficient criterion for its synthetic potential. The optimal molar ratio of oleic acid and 1-butanol was 2:1 for 1-butyl oleate synthesis. The 1-butyl oleate yield was unaffected by purification of the enzyme after 12 h.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

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.     

Reductive alkylation of lipase

Candida rugosa lipase was modified via reductive alkylation to increase its hydrophobicity to work better in organic solvents. The free amino group of lysines was alkylated using propionaldehyde with different degrees of modification obtained (49 and 86%). Far-ultraviolet circular dichroism (CD) spectroscopy of the lipase in aqueous solvent showed that such chemical modifications at the enzyme surface caused a loss in secondary and tertiary structure that is attributed to the enzyme unfolding. Using molecular modeling, we propose that in an aqueous environment the loss in protein structure of the modified lipase is owing to disruption of stabilizing salt bridges, particularly of surface lysines. Indeed, molecular modeling and simulation of a salt bridge formed by Lys-75 to Asp-79, in a nonpolar environment, suggests the adoption of a more flexible alkylated lysine that may explain higher lipase activity in organic solvents on alkylation.     

Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E

The bifunctional activities of α-amylase and pullulanase are found in the cloned recombinant amylopullulanase. It was encoded in a 2.9-kb DNA fragment that was amplified using polymerase chain reaction from the chromosomal DNA of Thermoanaerobacter ethanolicus 39E. An estimated 109-kDa recombinant protein was obtained from the cloned gene under the prokaryotic expression system. The optimum pH of the recombinant amylopullulanase was 6.0. The most stable pH for the α-amylase and pullulanase activity was 5.5 and 5.0, respectively. The optimum temperature for the α-amylase activity was 90°C, while its most stable temperature was 80°C. Regarding pullulanase activity, the optimum temperature and its most stable temperature were found to be 80 and 75°C, respectively. Pullulan was found to be the best substrate for the enzyme. The enzyme was activated and stabilized by the presence of Ca²⁺, whereas EDTA, N-bromosuccinimide, and α-cyclodextrin inhibited its bifunctional activities. A malto-2–4-oligosac-charide was the major product obtained from the enzymatic reaction on soluble starch, amylose, amylopectin, and glycogen. A single maltotriose product was found in the pullulan hydrolysis reaction using this recombinant amylopullulanase. Kinetic analysis of the enzyme indicated that the K _m values of α-amylase and pullulanase were 1.38 and 3.79 mg/mL, respectively, while the V _max values were 39 and 98 μmol/(min · mg of protein), respectively.