José M. Sánchez-Montero

Thermal stabilization of immobilized lipase B from Candida antarctica on different supports: Effect of water activity on enzymatic activity in organic media

Abstract Covalent immobilization of C. antarctica lipase B (CALB) on sepharose, alumina, and silica was undertaken. The thermal stability of these covalently immobilized catalysts were studied and compared to adsorbed derivatives from Novo Nordisk at 50°C under wet conditions. Native enzyme and Novozym 435 follow a deactivation model E → E 1 whereas covalently immobilized derivatives and SP435A follow the model E → E 1 → E 2 . This different behavior is related to the nature of the support and the immobilization methodology. Water absorption isotherms of dry solid biocatalysts in air or isooctane were used to predict the optimum preequilibrium a w value to obtain the highest rate in the esterification of ( r,s )-ibuprofen.

Novel microbial lipases: catalytic activity in reactions in organic media.

2,000 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Culture broths were initially tested with 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufinyl ester, a chromogenic substrate specific for lipases. Fourteen lipase-producing microorganisms were selected and their taxonomic identification was carried out. Hydrolysis of tributyrin or olive oil and the esterification of oleic acid with heptanol were selected to preliminary evaluate the catalytic activity of these lipases. All the selected lipases catalysed this esterification reaction with good yields. Resolution of (R,S)-2-(4-isobutylphenyl) propionic acid, (R,S)-1-phenylethanol, (R,S) 1-phenylethylamine and of (R) or (S) glycidol were performed to evaluate the stereoselectivity of these novel enzymes as biocatalysts in reactions in organic media. Lipases from the fungi Fusarium oxysporum and Ovadendron sulphureo-ochraceum gave the best yields and enantioselectivities in the resolution of racemic ibuprofen and 1-phenylethanol. Several lipases displayed a high stereoselectivity in the resolution of chiral amines by an alcoxycarbonylation reaction.

Multi-Target-Directed Ligands and other Therapeutic Strategies in the Search of a Real Solution for Alzheimer's Disease

The lack of an adequate therapy for Alzheimers Disease (AD) contributes greatly to the continuous growing amount of papers and reviews, reflecting the important efforts made by scientists in this field. It is well known that AD is the most common cause of dementia, and up-to-date there is no prevention therapy and no cure for the disease, which contrasts with the enormous efforts put on the task. On the other hand many aspects of AD are currently debated or even unknown. This review offers a view of the current state of knowledge about AD which includes more relevant findings and processes that take part in the disease; it also shows more relevant past, present and future research on therapeutic drugs taking into account the new paradigm “Multi-Target-Directed Ligands” (MTDLs). In our opinion, this paradigm will lead from now on the research toward the discovery of better therapeutic solutions, not only in the case of AD but also in other complex diseases. This review highlights the strategies followed by now, and focuses other emerging targets that should be taken into account for the future development of new MTDLs. Thus, the path followed in this review goes from the pathology and the processes involved in AD to the strategies to consider in on-going and future researches.

Effect of Walnut-Enriched Restructured Meat in the Antioxidant Status of Overweight/Obese Senior Subjects with at Least One Extra CHD-Risk Factor

Background: A number of recent studies indicate that antioxidants reduce the oxidative stress associated with the development of coronary heart diseases (CHD). Objective: (i) To investigate whether the erythrocyte catalase (CAT), superoxide dismutase (SOD), total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSG), and lipid peroxidation (LPO), and serum uric acid and paraoxonase-1 (PON1) are modified at increased CHD-risk individuals consuming walnut-enriched meat (WM), (ii) to evaluate whether these changes were influenced by basal serum cholesterol, body mass index or smoking habit. Design: The study was a non blinded, cross-over, placebo-controlled trial in which 22 volunteers (60% overweight and 40% obese) with increased CHD-risk were randomly assigned to receive WM or control meat (CM) during two different periods of 5 weeks. Results: A significant interaction time*treatment (p < 0.05) was observed in all enzymes and substrates tested except HDL-C, uric acid and LPO. The treatment significantly increased CAT activity, total glutathione and GSSG (p < 0.05). Significant gender*time*treatment interaction (p = 0.043) for total glutathione was found increasing at the end of the WM period in male but not changing in female. Total glutathione and GSH/GSSG ratio (p < 0.05) were lower in smokers. Hypercholesterolemics presented higher uric acid (p < 0.05) but no enzyme activities or substrate concentrations were different from those of normocholesterolemics. Conclusions: The WM tested appears to be a functional food as it improved the antioxidant status of increased CHD-risk volunteers. Despite its high energy content, it also appears adequate for overweight and obese people because did not exert negative effect upon body weight.

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.

Hydrolysis of used frying palm olein and sunflower oil catalyzed by porcine pancreatic lipase

The enzymatic hydrolysis of frying used vegetable oils with different degrees of alteration were measured using porcine pancreatic lipase (acylglycerol acylhydrolase EC 3.1.1.3). Successive frying of potatoes significantly increased the level of total polar lipid content in the palm olein from 9.3±0.1 mg/100 mg oil to 26.4±0.3 mg/100 mg oil after 90 fryings, and from 4.0±0.1 mg/100 mg oil to 27.7±0.3 mg/100 mg oil in sunflower oil after 60 fryings. Triacylglycerol polymers, triacylglycerol dimers, and oxidized triacylglycerols also increased 37-, 7.9-, and 7.5-times in palm olein, respectively, and 56-, 22-, and 4.7-times in sunflower oil, respectively. However, diacylglycerols and free fatty acid levels related to hydrolytic alteration did not increase with the number of fryings in both oils. The substrate concentration in the reactor was determined by calculating the molecular weight of each oil showing a different degree of alteration. We compared the methodology used by us and that used by other authors. The results show that the methods are reproducible and that the values obtained are in concordance with theoretical values. The kinetic parameters apparent Michaelis-Menten constant (KMapp) and apparent maximum velocity of hydrolysis (Vmaxapp) were different in unused palm olein (5.1±0.7 and 166±7.6, respectively) than in sunflower oil (2.2±0.3 and 62±2.2, respectively). However, changes inKMapp andVmaxapp were not related to the degree of alteration of the oils.

Water adsorption isotherm as a tool to predict the preequilibrium water amount in preparative esterification

SummaryThe cross point of the water adsorption isotherms of the lyophilised enzyme in air and of the lyophilised enzyme in the organic solvent gives a criterium to predict the equilibrium aw value to obtain the maximum yield in an esterification reaction in preparative conditions. The esterification of several (R,S) 2-arylpropionic acids is tested.

Contribution to the study of the alteration of lipase activity ofCandida rugosa by ions and buffers

A semipurifiedC. rugosa lipase (LS) has been prepared from commercial lipase (LC) using an economical procedure. The presence of sugars and glycopeptides has been detected in LS and LC. Pure lipase only has covalently bonded sugars. The hydrolysis of olive oil catalyzed by LS and commercial lipase (LC) is sensitive to the presence of cations Na(I), Mg(II), Ca(II), and Ba(II) and to the nature of buffer. Highest enzyme activity is obtained with 0.1M Tris/HCl buffers and the combination of NaCl 0.11M and CaCl2 0.11M. Fluorescence spetroscopy analysis of LC, LS, and both pure isoenzymes lipases A and B, was used to analyze the interaction of the lipase with these effecttors. Inorganic cations Na or Ca do not interact with pure enzyme LA but do interact with LC and LS and do so slightly with LB. The organic cations (morfolinium ortris) interact with pure lipases. We postulate that the increase in the lipase activity produced by Na(I) or Ca(II) is related with interfacial phenomena, but the increase might be more specific in the hydrolysis of olive oil in the presence of Tris-HCl or morfoline-HCl buffer, owing to enzyme-buffer interaction.

Effect of seaweed and cholesterol-enriched diets on postprandial lipoproteinaemia in rats.

High postprandial lipaemia increases cardiovascular risk. Algae consumption may affect postprandial lipoproteinaemia. The effects of dietary alga and cholesterol supplementation on postprandial lipaemia and lipoproteinaemia and arylesterase (AE) activity in growing male Wistar rats were tested in the present study. Six groups of ten rats were fed a casein-based diet for 3 weeks. Three of the diets contained 2.4 % cholesterol-raising agent (Chol), while the other three did not (NChol). Seven percentage of the control diets (NChol-C and Chol-C) consisted of a cellulose-wheat starch mix (35:65), while the Nori alga diets (NChol-N and Chol-N) and Konbu diets (NChol-K and Chol-K) contained 7 % of each respective freeze-dried alga. Postprandial plasma was obtained after a 3 h diet withdrawal. Supplementary cholesterol and alga type significantly affected (at least P < 0.05) the cholesterol, TAG, phospholipid and protein contents of the various lipoprotein fractions. AE enzyme activity increased (P < 0.05) in NChol rats given Nori and Konbu diets. NChol-K, but not NChol-N, rats displayed higher (P < 0.05) plasma cholesterol, TAG and phospholipid levels than NChol-C animals. NChol-K rats presented higher TAG, phospholipid, protein and lipoprotein mass values than their NChol-C counterparts. Inclusion of algae in Chol diets decreased (P < 0.001) the postprandial hypertriacylglycerolaemia. The Chol-N diet affected most lipoprotein fraction contents. Chol-N rats had lower postprandial cholesterolaemia and a better lipoprotein profile (fewer LDL and a tendency toward more HDL and fewer cholesterol-enriched VLDL) than Chol-K rats, suggesting that Nori is the alga of choice in dietary treatment of hypercholesterolaemia.

Heptyl oleate synthesis as useful tool to discriminate between lipases, proteases and other hydrolases in crude preparations

Heptyl oleate synthesis is a good test to compare crude lipases. Being a reaction selective for lipases, esterases and proteases are not active in this esterification. The heptyl oleate synthesis has been carried out with crude lipases and other hydrolases (such as proteases and esterases) from different suppliers and origins, in order to explore the methodology scope. While the initial rate of esterification seems to be mainly dependent on the concentration of lipases in the sample tested, the final synthetic yield depends on the total amount of water in the reactor. The standard protocol to evaluate crude lipases is described.