María L. Rúa

Purification, Immobilization, and Stabilization of a Lipase from Bacillus thermocatenulatus by Interfacial Adsorption on Hydrophobic Supports

A lipase from Bacillus thermocatenulatus (BTL2) cloned in E. coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton. Only one band was detected by SDS‐PAGE. The pure enzyme was immobilized using different methodologies. BTL2 adsorbed on a hydrophobic support (octadecyl‐Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity. The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl‐Sepabeads‐BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 °C or 30% of dioxane and 45 °C after several days of incubation. The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively. The adsorption of this thermophilic lipase on octadecyl‐Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 °C.

Reactivity of Pure Candida rugosa Lipase Isoenzymes (Lip1, Lip2, and Lip3) in Aqueous and Organic Media. Influence of the Isoenzymatic Profile on the Lipase Performance in Organic Media

Three pure isoenzymes from Candida rugosa lipase (CRL: Lip1, Lip2, and Lip3) were compared in terms of their stability and reactivity in both aqueous and organic media. The combined effect of temperature and pH on their stability was studied applying a factorial design. The analysis of the response surfaces indicated that Lip1 and Lip3 have a similar stability, lower than that of Lip2. In aqueous media, Lip3 was the most active enzyme on the hydrolysis of p‐nitrophenyl esters, whereas Lip1 showed the highest activity on the hydrolysis of most assayed triacylglycerides. The highest differences among isoenzymes were found in the hydrolysis of triacylglycerides. Thus, a short, medium, and long acyl chain triacylglyceride was the preferred substrate for Lip3, Lip1, and Lip2, respectively. In organic medium, Lip3 and Lip1 provided excellent results in terms of enantioselectivity in the resolution of ibuprofen (EF value over 0.90) and conversion, whereas initial esterification rate was higher for Lip3. However, the use of Lip2 resulted in lower values of conversion, enantiomeric excess, and enantioselectivity. In the case of trans‐2‐phenyl‐1‐cyclohexanol (TPCH) resolution, initial esterification rates were high except for Lip3, which also produced poor results in conversion and enantiomeric excess. The performance of the pure isoenzymes in the enantioselectivity esterification of these substrates was compared with different CRL crude preparations with known isoenzymatic content and the different results could not be explained by their isoenzymatic profile. Therefore, it can be concluded that other factors can also affect the catalysis of CRL and only the reproducibility between powders can ensure the reproducibility in synthesis reactions.

Strategies for improving extracellular lipolytic enzyme production by Thermus thermophilus HB27

In Thermus thermophilus HB27 cultures the localisation of lipolytic activity is extracellular, intracellular and membrane bound, with low percentage for the former. Therefore, the extracellular secretion must be increased in order to simplify the downstream process and to reduce the economic cost. This study focuses on the design of an innovative operational strategy to increase extracellular lipolytic enzyme production by T. thermophilus HB27 at bioreactor scale. In order to favour its secretion, the effect of several operational variables was evaluated. Among them, the presence of oils in the culture medium leads to improvements in growth and lipolytic enzyme activity. Sunflower oil is the most efficient inducer showing better results when added after 10h of growth. On the other hand, although surfactants lead to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. Thus, by addition of surfactants at the stationary phase, a release of intracellular and membrane enzyme which increases the extracellular enzyme proportion is detected. Based on these results, strategies with successive addition of oil and surfactant in several culture phases in shake flask are developed and verified in a laboratory scale stirred tank bioreactor.

Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pI, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.

A controlled fed-batch cultivation for the production of new crude lipases from Candida rugosa with improved properties in fine chemistry

A controlled constant feeding rate fed-batch strategy using oleic acid as inducer produced a crude lipase preparation from Candida rugosa (CRL-UAB) with higher protein content, carbohydrate content and lipase activity than commercial Sigma type VII CRL. CRL-UAB was partially characterised and tested in selective biotransformations of chiral compounds in aqueous (2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE)) and organic media (2-phenyl propionic acid and ketoprofen). CRL-UAB showed higher substrate specificity and enantioselectivity in aqueous media compared to Sigma CRL. Also, higher specific initial rates with 2-phenyl propionic acid and ketoprofen were observed in organic media. The influence of water on the esterification of ketoprofen was not relevant with CRL-UAB under the conditions tested, whereas a dramatic influence was observed in Sigma CRL. Different CRL-UAB batches obtained under the same cultivation controlled conditions were identical from the point of view of chromatographic behaviour, immobilisation rates and catalytic properties, indicating that a reproducible C. rugosa lipase extract had been obtained.

Lipases and Esterases from Extremophiles: Overview and Case Example of the Production and Purification of an Esterase from Thermus thermophilus HB27

Extremophiles are organisms that have evolved to exist in a variety of extreme environments. They fall into a number of different classes that include thermophiles, halophiles, acidophiles, alkalophiles, psychrophiles, and barophiles (piezophiles). Extremophiles have the potential to produce uniquely valuable biocatalysts that function under conditions in which usually the enzymes of their nonextremophilic counterparts could not. Among novel enzymes isolated from extremophilic microorganisms, hydrolases, and particularly lipases and esterases are experiencing a growing demand. Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) catalyze the cleavage of ester bounds in aqueous media and the reverse reaction in organic solvents. Both lipolytic enzymes have relevant applications in food, dairy, detergent, biofuel, and pharmaceutical industries. Here, we summarize the properties of lipases and esterases from the main extremophile groups: thermophiles and hyperthermophiles, psychrophiles, halophiles, alkalophiles/acidophiles, and solvent-resistant microorganisms.We report the biomass and lipolytic activity production by Thermus thermophilus HB27 in 5-L stirred-tank bioreactor at 70°C. Suitability of thermal spring water for culture media formulation is shown. In addition, a protocol to isolate and purify a cell-bound esterase from this microorganism is described.

Structural basis for the kinetics of Candida rugosa Lip1 and Lip3 isoenzymes

Abstract The fungus Candida rugosa produces several lipase isoenzymes and the 3D structure of two were solved (Lip1 and Lip3). We have isolated homodimers of Lip3 isoenzyme, maintained by hydrophobic interactions, which were stable in aqueous solutions. Under kinetic conditions, the dimers were still functional and showed an increased capability to hydrolysed soluble triacylglycerides compared to the monomeric counterpart. The kinetic properties of Lip1 were more similar to those of monomeric Lip3 although they were activated by the substrate interface in a different degree. The structural basis to interpretate the kinetic differences between these lipases is proposed to be related to the conformational flexibility of their flaps that regulate the entrance of the substrate to the active site.

Crystallization and preliminary X‐ray diffraction studies of two different crystal forms of the lipase 2 isoform from the yeast Candida rugosa

The yeast Candida rugosa produces several closely related lipases which show a high degree of sequence identity (between 77 and 88% for pairs of proteins). Despite this high sequence identity, they exhibit markedly different substrate specificities, indicating that subtle structural differences may produce significant functional changes. Isoform 2 (lip2) has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality crystals have been obtained from two different experimental conditions (designated A and B, respectively). Type A crystals belong to space group P1 and have unit-cell parameters a = 62.15, b = 91.14, c = 108.46 A, alpha = 90.78, beta = 106.31, gamma = 86.91 degrees; type B crystals are monoclinic with a nearly hexagonal topology, with unit-cell parameters a = 116.11, b = 225.55, c = 116.06 A, beta = 119.89 degrees, and belong to space group P2(1). Diffraction data were collected to a resolution of 1.97 A at a synchrotron facility from type A crystals and to 2.65 A on an in-house rotating-anode generator from type B crystals. Whereas the triclinic crystal reveals monomeric lip2, the monoclinic crystal contains dimeric lip2.

Modelling the enzymatic activity of two lipases isoenzymes commonly used in the food industry Modelado de la actividad enzimática de dos isoenzimas lipasas comúnmente utilizadas en la industria alimentaria

An in-depth analysis of the kinetics of two lipases isoenzymes (Lip1 and Lip2) in triacetin hydrolysis in absence and in presence of hexane was carried out. The addition of hexane led to an increase in enzymatic activities of both enzymes for all triacetin concentrations, and the kinetic data described a hyperbola which was consistent with the classical Michaelis–Menten model. Without hexane, the time-course of the triacetin hydrolysis by Lip1 and Lip2 did not follow a Michaelian behaviour. In this case, a first phase of low enzymatic activity (at triacetin concentrations from 0 to 250 mM) was followed by a rapid increase in velocity at triacetin concentrations ≥250 mM. The Michaelis–Menten model was unable to describe the first phase due to the linear (nonhyperbolic) relationship between the velocity and the triacetin concentration, meanwhile the logistic model provided a satisfactory description of the experimental data corresponding to the second phase of activity. En este trabajo se llevó a cabo un profundo análisis de la cinética de dos isoenzimas lipasas (Lip1 y Lip2) en la hidrólisis de triacetina, en ausencia y en presencia de hexano. La adición de hexano a la mezcla de reacción incrementó las actividades enzimáticas de ambas enzimas para todas las concentraciones de triacetina, obteniéndose una relación hiperbólica compatible con el modelo clásico de Michaelis-Menten. En ausencia de hexano, la actividad de Lip1 y Lip2 no mostró un comportamiento Michaeliano, observándose una fase inicial de baja velocidad a concentraciones de triacetina entre 0–250 mM, seguida de un rápido incremento en la actividad enzimática ([triacetina] ≥ 250 mM). El modelo de Michaelis-Menten no pudo ser utilizado para describir la primera fase debido al incremento lineal (no hiperbólico) de la velocidad con la concentración de triacetina, mientras el modelo logístico describió adecuadamente la cinética de hidrólisis en la segunda fase.

Contribution of the Oligomeric State to the Thermostability of Isoenzyme 3 from Candida rugosa

Thermophilic proteins have evolved different strategies to maintain structure and function at high temperatures; they have large, hydrophobic cores, and feature increased electrostatic interactions, with disulfide bonds, salt-bridging, and surface charges. Oligomerization is also recognized as a mechanism for protein stabilization to confer a thermophilic adaptation. Mesophilic proteins are less thermostable than their thermophilic homologs, but oligomerization plays an important role in biological processes on a wide variety of mesophilic enzymes, including thermostabilization. The mesophilic yeast Candida rugosa contains a complex family of highly related lipase isoenzymes. Lip3 has been purified and characterized in two oligomeric states, monomer (mLip3) and dimer (dLip3), and crystallized in a dimeric conformation, providing a perfect model for studying the effects of homodimerization on mesophilic enzymes. We studied kinetics and stability at different pHs and temperatures, using the response surface methodology to compare both forms. At the kinetic level, homodimerization expanded Lip3 specificity (serving as a better catalyst on soluble substrates). Indeed, dimerization increased its thermostability by more than 15 °C (maximum temperature for dLip3 was out of the experimental range; >50 °C), and increased the pH stability by nearly one pH unit, demonstrating that oligomerization is a viable strategy for the stabilization of mesophilic enzymes.