Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): a metagenomic approach.

The esterases and lipases from the α/ß hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46'N, 2°59'W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated.

Application of commercial and non-commercial immobilized lipases for biocatalytic production of ethyl lactate in organic solvents.

This study explores the potential for enhancing the production of ethyl lactate (EL), a green solvent, through enzymatic esterification. Different solvents were compared as organic media for conversion of lactate and ethanol into EL, catalyzed by Novozym 435. Chloroform and hexane were the most effective in low acid concentrations (0.01-0.1M) exhibiting maximum EL yields of 88% and 75% respectively. The yield of EL improved as the solvent's LogP increased up to a value of 2. Non-commercial immobilized biocatalysts consisting heterologous Rhizopous oryzae (rROL) and Candida rugosa (CRL) lipases immobilized on hydrophobic supports were compared to commercial biocatalysts clarifying that Novozym 435 and Lipozyme RM IM could be efficiently applied. Operational stability tests were conducted using Novozym 435, which retained higher activity in chloroform as compared to hexane. Although non-commercial biocatalysts were not competitive in esterification, they exhibited significant activity towards hydrolysis constituting a valuable alternative to higher-cost options.

Exploring substrate specificities of a recombinant Rhizopus oryzae lipase in biodiesel synthesis.

The alcoholysis of triolein was used to explore the specific features of a recombinant Rhizopus oryzae lipase (rROL) for biodiesel synthesis. For this purpose, different acylglycerols were compared as substrates in lipase-catalysed transesterification. rROL was shown to exhibit a higher specificity towards 1-monoolein than triolein compared to other R. oryzae lipases, being more than 4-fold more specific; in contrast, rROL did not accept 2-monoolein as substrate, concluding that it is highly 1,3-positional specific. Comparing ethanol and methanol as acyl-acceptors, it was observed that the latter caused more lipase inactivation. Regarding alcohols, it was also demonstrated that acyl migration occurred in moderate alcohol concentrations.

Utilization of discard bovine bone as a support for immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris.

In this study the possibility of using discard bovine bone as support for immobilization of Rhizopus oryzae lipase expressed in Pichia pastoris was analyzed. Discard bovine bone were milled and then subjected to a chemical treatment with acetone in order to remove lipids and blood traces. Two types of supports were evaluated: bovine bone and calcined bovine bone for 2 h at 600°C. Supports were characterized by: ICP, SEM, XRD, FTIR, XPS, and N2 adsorption isotherms. Calcined bovine bone presented appropriate characteristics for the lipase immobilization due to the removal of collagen: high porosity, large surface area and suitable porous structure. Biocatalysts were prepared with different initial enzyme load. For the equilibrium adsorption studies, the Langmuir isotherm was used to fit the data results. The immobilization occurs in monolayer to a value of 35 UA mg-1 . The activities of biocatalysts were tested in transesterification reaction of olive oil. For the enzyme load used in the test, a final yield percentage of 49.6 was achieved after six methanol additions and 180 min of reaction, similar values were obtained using Relizyme as support. Therefore, the bovine bone discard is an economical and appropriate choice for use support immobilization of enzymes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1246-1253, 2016.

Enzymatic biodiesel synthesis from yeast oil using immobilized recombinant Rhizopus oryzae lipase.

The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.