J.M. Sánchez-Montero

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.

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.

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.

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.

Modification of hydrophilicity/hydrophobicity of the microenvironment of lipase of Candida rugosa by dextrans

Replacing the lactose used in the commercial preparation of lipase from Candida rugosa by dextrans with different molecular weight, several preparations with enhanced activities in esterification of (R,S)-ibuprofen in organic medium were obtained. The presence of carbohydrates modifies the microenvironment of the enzyme and maintains the hydration of the biocatalyst. We can modulate the hydrophilic/hydrophobic balance on the surface of the biocatalyst creating non covalent enzyme-dextran complexes.

Regioselective resolution of 1,n-diols catalysed by lipases: a rational explanation of the enzymayic selectivity

The regioselective acylation of different phenylalkanediols catalysed by porcine pancreatic lipase (PPL) was used for modelling the enzymatic substrate recognition. Thus, different racemic or prochiral (1,n)-diols, with n ranging from 2 to 6, were resolved via transesterification with vinyl acetate, and the results obtained (yield, reaction rate, enantioselectivity) were explained according to the microcrystalline enzyme structure. A logical model for explaining the enzyme regio-and stereoselectivity is proposed, based on literature data reporting similar recognition patterns for some other lipases; our model is built on three residues of the active site (Ser153, Phe216 and His264), which turned out to be crucial for the substrate binding and transformation. Furthermore, some other tentative models proposed for PPL recognition are explained with our criterion.

Improved stability of the lipase from Candida rugosa in different purification degrees by chemical modification

Semipurified lipase of Candida rugosa and pure isoforms (lipase A and lipase B) have been chemically modified using two methodologies based on polyethyleneglycol (PEG). The activation of PEG with p-NO2-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG in the enzymatic activity of the lipase. The chemical modification increases the stability of pure lipases in isooctane at 50 °C.

Simple determination of the water content in lyophilized isoenzymes of Candida rugosa lipase

Thermogravimetric and differential thermal analysis have shown that lyophilized Candida rugosa lipase A has more water bonded to it than isoenzyme lipase B: respectively 522 and 220 molecules of water per molecule of lipase. This may explain the different thermal deactivation behaviours of these enzymes in isooctane at high temperature.

Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.