Aline Machado de Castro

Production and Use of Lipases in Bioenergy: A Review from the Feedstocks to Biodiesel Production

Lipases represent one of the most reported groups of enzymes for the production of biofuels. They are used for the processing of glycerides and fatty acids for biodiesel (fatty acid alkyl esters) production. This paper presents the main topics of the enzyme-based production of biodiesel, from the feedstocks to the production of enzymes and their application in esterification and transesterification reactions. Growing technologies, such as the use of whole cells as catalysts, are addressed, and as concluding remarks, the advantages, concerns, and future prospects of enzymatic biodiesel are presented.

PRODUÇÃO, PROPRIEDADES E APLICAÇÃO DE CELULASES NA HIDRÓLISE DE RESÍDUOS AGROINDUSTRIAIS

PRODUCTION, PROPERTIES AND APPLICATION OF CELLULASES IN THE HYDROLYSIS OF AGROINDUSTRIAL RESIDUES. Cellulases have been intensively studied in the past few years, due to the interests in biofuels production from lignocellulosic materials, since they permit maintaining mild conditions during the conversion process. These enzymes can be produced by a broad variety of naturally occurring microorganisms, such as from genera Aspergillus, Trichoderma, Penicillium and Humicola. Targeting the increasing of expression levels, molecular biology tools have been used for heterologous genes insertion in host cells, e. g., Pichia pastoris and Escherichia coli. Enzymes from fungal cellulolytic complex usually act best at pH between 4 and 5 under temperatures from 40 to 60 °C and can be used for either sequential (SHF) or simultaneous (SSF) hydrolysis together alcoholic fermentation. In this review, the main raw materials for production of cellulases are identified, as well as the state of art of enzymes’ properties, production and main applications.

Economic Analysis of the Production of Amylases and Other Hydrolases by Aspergillus awamori in Solid-State Fermentation of Babassu Cake

Amylases are one of the most important industrial enzymes produced worldwide, with their major application being in ethanol manufacturing. This work investigated the production of amylases by solid-state fermentation of babassu cake, using the filamentous fungus Aspergillus awamori IOC-3914. Lab-scale experiments were carried out to generate input data for simulations of an industrial plant for amylase production. Additionally to the target enzymes, other hydrolases (cellulases, xylanases, and proteases) were also produced, enriching the final product. The most suitable fermentation time was 144 hours, when exoamylase and endoamylase activities of 40.5 and 42.7 U g−1 were achieved, respectively. A first evaluation showed a large impact of the inoculum propagation medium on production costs. Therefore, five propagation media were compared, and PDA medium presented the best cost-benefit ratio. The credits obtained from sales of fermented cake as a coproduct enabled a significant decrease in the production cost of the enzyme product, down to 10.40 USD kg−1.

Enzyme Surface Glycosylation in the Solid Phase: Improved Activity and Selectivity of Candida Antarctica Lipase B

Tailor‐made oligosaccharides and polymers were investigated for a specific surface glycosylation of Candida antarctica lipase (fraction B) (CAL‐B) already immobilized on octyl‐Sepharose by interfacial activation. The chemical modification was performed in the N‐terminal amino acid enzyme residue by using low oxidized aldehyde–dextran polymers through a reductive amination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. Circular dichroism experiments revealed interesting conformational changes in secondary and tertiary structures of the protein after modification. The formed immobilized glycosylated lipase biocatalysts were more stable, active, and selective toward different substrates than unmodified CAL‐B. These immobilized conjugates were compared with a genetically glycosylated version of CAL‐B expressed in Pichia pastoris immobilized in the same way. Enzyme thermostability was improved after chemical modification with Dextran‐1500 and also by the genetic glycosylation, retaining 90–96 % activity after 24 h at 55 °C. The catalytic activity of CAL‐B was improved by the incorporation of dextran polymers (Mw=1500 or 6000) more than twofold in the hydrolysis of p‐nitrophenylbutyrate and more than threefold in the hydrolysis of methyl mandelate at pH 7. However, the activity of the genetically glycosylated CAL‐B was threefold lower in the hydrolysis of both substrates. The enantioselectivity of CAL‐B increased for all formed bioconjugates, with the Dextran‐1500–CAL‐B conjugate being the most selective in the hydrolysis of racemic methyl mandelate (up to 88.1 % ee at pH 5). This glycosylated CAL‐B also demonstrated the highest synthetic activity in the transesterification of methyl butyrate with glycerol, with 80 % yield of monoglyceryl ester at 100 % conversion compared to 57 % yield obtained with unmodified CAL‐B or other polymer–lipase conjugates.

Techno-economic evaluation of a complete bioprocess for 2,3-butanediol production from renewable resources

This study presents the techno-economic evaluation of 2,3-butanediol (BDO) production via fermentation using glycerol, sucrose and sugarcane molasses as carbon sources. Literature-cited experimental data were used to design the fermentation stage, whereas downstream separation of BDO was based on reactive extraction of BDO employing an aldehyde to convert BDO into an acetal that is immiscible with water. The selected downstream process can be used in all fermentations employed. Sensitivity analysis was carried out targeting the estimation of the minimum selling price (MSP) of BDO at different plant capacities and raw material purchase costs. In all cases, the MSP of BDO is higher than 1

Oriented irreversible immobilization of a glycosylated Candida antarctica B lipase on heterofunctional organoborane-aldehyde support

/kg that is considered as the target in order to characterize a fermentation product as platform chemical. The complex nutrient supplements, the raw material market price and the fermentation efficiency were identified as the major reasons for the relatively high MSP observed.

Consecutive lipase immobilization and glycerol carbonate production under continuous-flow conditions

The immobilization of Candida antarctica (fraction B) lipase expressed in Pichia pastoris, a selective glycosylated protein at Asn 74, on a new heterofunctional support consisted of phenylboronic acid and aldehyde groups (Borald) has been performed. This method occurs via a two step mechanism: first orientation by organoborane interaction at neutral pH and a consecutive multi-point covalent attachment by aldehyde reaction at alkaline pH. The enzyme was specifically immobilized on this support in 70% yield at pH 7, oriented by the reaction of the hydroxyl groups on the sugar moiety with boronic acid on the support, whereas commercial CAL-B from Novozymes (non-glycosylated) was hardly immobilized at this pH ( 99% final immobilization yield. The Borald-CAL-B preparation was a very stable biocatalyst in the presence of high amount of solvent or high temperature (e.g. more than 10 fold in the presence of 60% (v/v) acetonitrile). An improvement of the specific activity up to 5 fold for example in the hydrolysis of methyl phenyl acetate was obtained compared with a one-point covalent preparation. An ee of 89% towards R isomer was achieved with this new immobilized biocatalyst in the enantioselective hydrolysis of methyl mandelate.

Optimisation of Cellulase Production by Penicillium funiculosum in a Stirred Tank Bioreactor Using Multivariate Response Surface Analysis.

Several value-added products can be produced from glycerol and among these products glycerol carbonate (GC) has received much attention in recent years because of its physical properties and wide application in the chemical, pharmaceutical and food industries. As a continuation of our efforts to produce GC under environmentally acceptable conditions, we proposed a consecutive approach where lipase B from Candida antarctica was immobilized on Accurel MP1000 (CalBAcc) under continuous-flow conditions followed by glycerol carbonate production by a cascade process of triacylglycerol hydrolysis, biodiesel synthesis and esterification of the remaining glycerol with dimethyl carbonate towards GC production. Results of the immobilization of lipase B from Candida antarctica on a continuous-flow protocol demonstrated that a short residence time of 25 minutes could lead to 83% of protein loading. Screening of glycerol carbonate production in a packed bed reactor revealed that CalBAcc and N435 showed the best results of conversion and selectivity, with more selectivity by CalBAcc, that could lead to quantitative yields and excellent selectivities of the desired glycerol carbonate at residence times of 176 minutes when working with soybean and palm oil. Comparatively, the results obtained with CalBAcc were better than the ones with Novozym 435 since no full conversion was observed.

Multiresponse Optimization of Inoculum Conditions for the Production of Amylases and Proteases by Aspergillus awamori in Solid-State Fermentation of Babassu Cake

Increasing interest in the production of second-generation ethanol necessitates the low-cost production of enzymes from the cellulolytic complex (endoglucanases, exoglucanases, and β-glucosidases), which act synergistically in cellulose breakdown. The present work aimed to optimise a bioprocess to produce these biocatalysts from the fungus Penicillium funiculosum ATCC11797. A statistical full factorial design (FFD) was employed to determine the optimal conditions for cellulase production. The optimal composition of culture media using Avicel (10 g·L−1) as carbon source was determined to include urea (1.2 g·L−1), yeast extract (1.0 g·L−1), KH2PO4 (6.0 g·L−1), and MgSO4 ·7H2O (1.2 g·L−1). The growth process was performed in batches in a bioreactor. Using a different FFD strategy, the optimised bioreactor operational conditions of an agitation speed of 220 rpm and aeration rate of 0.6 vvm allowed the obtainment of an enzyme pool with activities of 508 U·L−1 for FPase, 9,204 U·L−1 for endoglucanase, and 2,395 U·L−1 for β-glucosidase. The sequential optimisation strategy was effective and afforded increased cellulase production in the order from 3.6 to 9.5 times higher than production using nonoptimised conditions.

Environmental factors modulating the stability and enzymatic activity of the Petrotoga mobilis Esterase (PmEst)

This work aimed at investigating the simultaneous production of amylases and proteases by solid-state fermentation (SSF) of babassu cake using Aspergillus awamori IOC-3914. By means of experimental design techniques and the desirability function, optimum inoculum conditions (C/N ratio of propagation medium, inoculum age, and concentration of inoculum added to SSF medium) for the production of both groups of enzymes were found to be 25.8, 28.4 h, and 9.1 mg g−1, respectively. Significant influence of both initial C/N ratio and inoculum concentration was observed. Optimum amylolytic activities predicted by this multiresponse analysis were validated by independent experiments, thus indicating the efficacy of this approach.