J.V. Sinisterra

Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases

A total of 969 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Ten lipase-producing microorganisms were selected and their taxonomic identification was carried out. From these strains Achremonium murorum, Monascus mucoroides, Arthroderma ciferri, Fusarium poae, Ovadendron sulphureo-ochraceum and Rhodotorula araucariae are described as lipase-producers for the first time. Hydrolysis activity of the crude lipases against both tributyrin and olive oil was measured. Heptyl oleate synthesis was carried out to test the activity of the selected lipases as biocatalysts in organic medium. All the selected lipases were tested as biocatalysts in several organic reactions using unnatural substrates. Lipases from the fungi Fusarium. oxysporum and O. sulphureo-ochraceum gave the best yields and enantioselectivities in the esterification of carboxylic acids. F. oxysporum and Penicillium chrysogenum lipases were the most active ones for the acylation of alcohols without steric hindrance. A. murorum lipase is very useful for the esterification of menthol. F. oxysporum and Fusarium. solani lipases were very stereoselective in the synthesis of carbamates.

Immobilization of lipase from Candida cylindracea on inorganic supports

Abstract Lipase from Candida cylindracea has been covalently immobilized on trichlorotriazine activated supports (alumina, silica and two types of controlled pore glass). The optimum conditions of the activation process have been determined (pretreatment, solvent, gram of activating agent/gram of support ratio and reaction time). The influence of the enzyme concentration and of the temperature on the immobilization process has been evaluated. The immobilized derivatives on silica and alumina exhibited greater residual activity and were more resistant to inactivation by temperature (50°C) than their immobilized counterpart on controlled pore glasses. The derivatives obtained on alumina and silica have been used in the hydrolysis of ( R,S ) ethyl 2-phenylpropionate only yielding the S (+) acid. The influence of Na I and Ca II on the lipase activity is discussed. The immobilized derivative on silica, stored at 50°C, was 37 times more stable than the native enzyme and displayed 80% residual activity after 336 h of operating time

Modification of purified lipases from Candida rugosa with polyethylene glycol: a systematic study

Abstract Semipurified lipase and pure isoenzymes [lipase A (CRLA) and lipase B (CRLB)] of Candida rugosa were chemically modified using pNPCF-PEG. The modified enzymes can be stored at 4°C for 6 months without losing activity. The chemically modified lipases were more stable than the native enzymes and were stored at 50°C in isooctane. The chemically modified enzymes were used in i) hydrolysis of triolein; ii) esterification of oleic acid; and iii) enantioselective esterification of ( r,s ) ibuprofen. Lipase activity was less than esterase activity as a result of the chemical modification of the lipase. The influence of purification and chemical modification degrees in the i) storage stability; ii) catalytic activity; iii) stability with respect to isooctane; and iv) stereoselectivity is discussed. We modulated the hydrophobicity of the biocatalyst by changing the modification degree of the lipase. This effect allowed us to select the optimum biocatalyst to achieve the maximum yield for esterification in different organic solvents. Only the purification of C. rugosa lipase increased the activity and enantioselectivity. Purification and chemical modification did not change the enantiopreference of the lipase.

Treatment of Candida rugosa lipase with short-chain polar organic solvents enhances its hydrolytic and synthetic activities

Following a simple and quick treatment based on dissolving the crude lipase from Candida rugosa in different percentages (v/v) of several polar organic solvents (methanol, ethanol, 1 and 2-propanol, 1 and 2-butanol and acetone) followed by dialysis, different preparations with enhanced activities were obtained. The opening of the lid covering the active site is proposed as the reason for explaining the activity enhancement, both in aqueous and anhydrous organic media.

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Abstract Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts (t1/2≈ 5h) with respect to the native pure lipases (t1/2≈ 0.28h). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO 2 (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO 2 (hydrophobic support) using the hydrolysis of the esters of ( R ) or ( S ) 2-chloropropanoic and of ( R , S ) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.

Modification of the activities of two different lipases from Candida rugosa with dextrans

Abstract Semipurified lipase from C. rugosa Type VII and another semipurified lipase obtained by fed-batch controlled fermenter conditions of C. rugosa ATCC 14380 are modified using dextrans with different molecular weight (6,000; 10,000; 12,000; 25,000; 50,000 Da) Both lipases have different isoenzymes composition and sugar percentages. The lipase/dextran complexes are obtained by non-covalent or by covalent bonding of the sugar to the NH 2 groups of the protein. The modifications with dextrans increase the thermal stability of the biocatalyst compared to unmodified commercial lipase but not in the case of lipase obtained from C. rugosa ATCC 14380. This finding is related to the amount of sugar covalently bonded to the parent enzyme. The catalysts are used in the enantioselective esterification of ( R,S )-ibuprofen. The CRLS/dextran complexes are more active than the semipurified or commercial lipases from C. rugosa and they require a small amount of water to be active. The amount of water that must be added to obtain the maximum activity is different for each biocatalyst. The modification with dextrans is very interesting for semipurified lipase from C. rugosa but it is not useful for semipurified C. rugosa ATCC 14380 probably because this crude enzyme has high sugar content than commercial C. rugosa lipase.

Influence of the nature of modifier in the enzymatic activity of chemical modified semipurified lipase from Candida rugosa

Semipurified lipase of Candida rugosa (CRSL) was subjected to chemical modification, and the activities of the modified lipase, in hydrolysis and esterification reactions, were examined. The esterification reactions were carried out in the absence and presence of isooctane. When the enzyme was modified with polyethylene glycol (PEG), two methodologies were studied. The activation of PEG with p-NO(2)-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG. The chemical modification with PEG increases the stability of pure lipases in isooctane at 50 degrees C (extreme conditions). The chemically modified enzymes are useful for biotransformations in organic solvents. In addition the nitration of tyrosines with tetranitromethane was also studied. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 252-260, 1997.

Preparation of halohydrin β-blocker precursors using yeast-catalysed reduction

Abstract The preparation of halohydrin β-blocker precursors using yeast-catalysed reduction of α-haloketones was performed. The influence in the yield and e.e. of several process variables was analysed. The (S)-enantioselectivity observed with Saccharomyces cerevisiae can be changed to (R)-enantioselectivity using methyl vinyl ketone as selective inhibitor (25 mM). Using resting fresh cells better yields and e.e.s are observed than using growing cells. Yarrowia lipolytica 1240 resting cells gave 87% yield of (S)-1-chloro-3(1-naphthyloxy)propan-2-ol (99% e.e.). Pichia mexicana 11105 resting cells gave 85% yield of (R)-1-chloro-3(1-naphthyloxy)propan-2-ol (precursor of propranolol) (95% e.e). The reduction process is applied to other α-haloketones, a lower e.e. being obtained the closer the size of the ketone substituents.

Purine nucleoside synthesis from uridine using immobilised Enterobacter gergoviae CECT 875 whole cells

Abstract Biocatalysed purine nucleoside synthesis was carried out using immobilised Enterobacter gergoviae CECT 875. Similar yields (80–95%) in adenosine were obtained with both free and immobilised cells though in the last case a long reaction time was necessary. The immobilised cells can be reused at least for more than 30 times without significant loss of enzymatic activity. The immobilised biocatalyst in agarose is active in the synthesis of unnatural nucleosides.

Hydrolysis of (R,S)2-aryl propionic esters by pure lipase B from Candida cylindracea

Abstract Purified lipase B from Candida cylindracea (LB) has been obtained in large amounts. LB exhibits greater esterase and lipase activities than commercial lipase. The presence of divalent and/or monovalent cations increases the lipase activity with respect to the absence of external cations, using olive oil as substrate. LB is more active than commercial and semipurified lipases in the hydrolysis of ( R,S )2-arylpropionic ethyl esters. The presence of Na(I) or Ca(II) diminishes the enzymatic activity in the hydrolysis of these esters compared to that obtained in the absence of the external ions. LB is stereospecific in the hydrolysis of S (+)2-arylpropionate.