Dolores Reyes-Duarte

Parameters affecting productivity in the lipase-catalysed synthesis of sucrose palmitate

The industrial application of lipases for the synthesis of sucrose esters is usually limited by its low productivity, as we need a medium where a polar reagent (the sugar) and a non-polar fatty acid donor are soluble and able to react in the presence of the biocatalyst. In this work, we have studied the problems encountered when trying to increase the volumetric productivity of sucrose esters. The synthesis of sucrose palmitate was performed in 2-methyl-2-butanol:dimethylsulfoxide mixtures by transesterification of different palmitic acid donors with sucrose, catalysed by the immobilized lipase from Candida antarctica B (Novozym 435). A protocol for substrate preparation different from that previously reported was found to improve the reaction rate. Several parameters, such as sucrose and acyl donor loadings, the percentage of DMSO in the mixture and the nature of acyl donor, were investigated. Under the best experimental conditions (15% DMSO, 0.1 mol l−1 sucrose, 0.3 mol l−1 vinyl palmitate), a maximum of 45 g l−1 sucrose palmitate was obtained in 120 h. Using methyl or ethyl palmitate, the highest productivity was 7.3 g l−1 in 120 h using 20% DMSO with 0.2 mol l−1 sucrose and 0.6 mol l−1 acyl donor. The formation of free fatty acid, and the effect of the percentage of DMSO on the monoester/diester selectivity were also studied. To our knowledge, this is the first report on enzymatic synthesis of sucrose esters of long fatty acids using alkyl esters as acyl donors.

Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters.

Herein, we applied a community genomic approach using a naphthalene‐enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β‐hydrolase family. The protein shares low‐to‐medium identity (≤ 57%) with known esterase/lipase‐like proteins. The enzyme is most active at 25–30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold‐adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β‐hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200–21 000 units g−1 protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio‐selectivity (1.0–55 000 units g−1 protein), including (±)‐menthyl‐acetate, (±)‐neomenthyl acetate, (±)‐pantolactone, (±)‐methyl‐mandelate, (±)‐methyl‐lactate and (±)‐glycidyl 4‐nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad‐spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo‐assembly (i.e. through ester bonds) of multi‐aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available.

Catalytic role of conserved HQGE motif in the CE6 carbohydrate esterase family

An acetylxylan esterase (R.44), belonging to the carbohydrate esterase family 6 (CE6), retrieved from bovine rumen metagenome was analyzed. Molecular modelling and site‐directed mutagenesis indicated that the enzyme possesses a catalytic triad formed by Ser14, His231 and Glu152. The catalytic Ser and His have been identified in highly conserved sequences GQSX and DXXH in the CE6 family, respectively, and the active‐site glutamate was part of a highly conserved sequence HQGE. This motif is situated near to the so‐called Block III in the CE6 family and its role in catalysis has not been identified so far.

Novel Hybrid Esterase-Haloacid Dehalogenase Enzyme

The existence of different catalytic mechanisms (or reaction types) in the same active site is an example of catalytic promiscuity. 2] The promiscuity can result from natural evolution of an enzyme, which enhances organism metabolic flexibility and environmental fitness, or from laboratory evolution and can be exploited in numerous synthetic applications. a/b-Hydrolasefold proteins belong to one of the largest protein superfamilies within the a/b class of folds and exhibit enormous sequence diversity, 4] fold plasticity, and activities. f] As they also exhibit high conservation of tertiary structures and catalytic triads, they have been suggested to have evolved from a common protein ancestor, from which divergent evolution led to the emergence of a large number of promiscuous enzymes. 6] Serine esterases and haloacid dehalogenases are a/b-type hydrolases that differ in their topological features, the nature/ position of the nucleophile, and the geometry of their catalytic scaffolds, while esterases (EC 3.1.1.1) preferentially hydrolyze water-soluble simple esters and contain a Ser/Asp(Glu)/His catalytic triad, haloacid dehalogenases (HAD; EC 3.8.1.2) catalyze the conversion of haloacid compounds into the corresponding alcohols and hydrogen halides by means of an Asp/Asp/His catalytic triad. Although, it has been suggested that dehalogenases are evolutionarily related to esterases, so far no protein, either naturally occurring or laboratory generated, has been reported to possess both activities. We describe here a multifunctional a/b-hydrolase-fold enzyme, designated REBr, mined from a metagenome library established from the DNA of a microbial community from seawater contaminated with crude oil. The protein showed a novel hydrolytic phenotype, namely the cleavage of both common p-nitrophenyl (pNP) and short aliphatic esters, and organic haloalkanoates. The existence of these two activities in a single protein is remarkable as they involve distinct catalytic mechanisms (for details see Figure S1 in the Supporting Information). The rEBr gene (933 bp), encodes a protein (310 AA, Mr = 33 852 Da) that exhibits high homology (up to 63 % identity, 75 % similarity) with a number of a/b-fold hydrolases (see Figure S2). Not only does this protein hydrolyze a series of commercially available common pNP and nonactivated short fatty acid esters as propyl propionate and ethyl butyrate, but also haloacids: [(kcat/Km)]ester/[(kcat/Km)]haloacid factor of ~4:1 for the best substrates, optimally at 40 8C and pH 8.0–8.5 (Figure S3; Tables 1 and S2). Weak though measurable activity with haloalkanes was detected and no epoxide tested was hydrolyzed. The enzyme cleaved a full set of halides at both terminal and subterminal positions, with catalytic efficiencies increasing in the order bromide (1-fold), fluoride (1.1-fold), chloride (12-fold),

Lipase-Catalyzed Modification of Phenolic Antioxidants

The chemical acylation of natural antioxidants may improve their oxidative and thermal stability, as well as modify their hydrophile-lipophile balance (HLB). These processes are generally carried out under harsh conditions using strongly corrosive acids. In contrast, lipase-catalyzed acylation is characterized by mild reaction conditions, low energy requirements, and a minimization of side reactions. We report the one-step enzymatic acylation of a phenolic antioxidant (α-tocopherol) and a polyphenol (resveratrol) by lipase-catalyzed transesterification. In particular, the regioselectivity of resveratrol acylation can be controlled by an adequate selection of the biocatalyst.

High Throughput Screening of Esterases, Lipases and Phospholipases in Mutant and Metagenomic Libraries: A Review

Nowadays, enzymes can be efficiently identified and screened from metagenomic resources or mutant libraries. A set of a few hundred new enzymes can be found using a simple substrate within few months. Hence, the establishment of collections of enzymes is no longer a big hurdle. However, a key problem is the relatively low rate of positive hits and that a timeline of several years from the identification of a gene to the development of a process is the reality rather than the exception. Major problems are related to the time-consuming and cost-intensive screening process that only very few enzymes finally pass. Accessing to the highest possible enzyme and mutant diversity by different, but complementary approaches is increasingly important. The aim of this review is to deliver state-of-art status of traditional and novel screening protocols for targeting lipases, esterases and phospholipases of industrial relevance, and that can be applied at high throughput scale (HTS) for at least 200 distinct substrates, at a speed of more than 105 - 108 clones/day. We also review fine-tuning sequence analysis pipelines and in silico tools, which can further improve enzyme selection by an unprecedent speed (up to 1030 enzymes). If the hit rate in an enzyme collection could be increased by HTS approaches, it can be expected that also the very further expensive and time-consuming enzyme optimization phase could be significantly shortened, as the processes of enzyme-candidate selection by such methods can be adapted to conditions most likely similar to the ones needed at industrial scale.

Catalytic profiles of lipolytic biocatalysts produced by filamentous fungi

Abstract The aim of this work is to propose and evaluate a catalytic profile of biocatalysts with lipase activity based on four characteristic reactions of lipases. For that, the catalytic profiles of three lipolytic biocatalysts from strains of the genus Rhizopus produced by solid-state fermentation (SSF) were determined based on acyl-, regio-, enantio- and chemoselectivity. Four commercial lipases were also evaluated according to their catalytic selectivity. p-nitrophenol esters from C-4 to C-16 were used to evaluate the acyl selectivities of the biocatalysts. The three biocatalysts produced by SSF presented the highest hydrolytic activity on triglycerides of medium chain length (C-8). The use of high performance thin layer chromatography (HPTLC) allowed us to demonstrate the biocatalysts produced by SSF presented sn-1,3-regioselectivity. Based on the Quick E method, the biocatalysts preferentially acted on the S-enantioisomer of glycidyl butyrate esters. Although their E values were insufficiently high (< 5), they demonstrated 1,3-sn selectivity hydrolytic activity, and the ability to synthesise ester and amide bonds (benzyl oleate and N-benzyloleamide, respectively), validating them as promising biocatalysts for these types of applications.