Enzymatic synthesis of sugar esters in non-aqueous media has advantages over chemical synthesis, particularly regioselectivity. However, conventional non-aqueous media do not solubilise sugars well and can destabilise the enzyme. Natural deep eutectic solvents (NADES) are environmentally friendly alternatives, and reactive NADES (R-NADES), in which sugars act simultaneously as solvent components and substrates, are especially promising. We investigated the acetylation of alpha-d-glucose with vinyl acetate catalysed by Candida antarctica lipase B (CALB), comparing an R-NADES composed of choline chloride and alpha-d-glucose with a solvent-free system, under identical substrate molar ratios (1:1) and reaction time (72 h). The solvent-free system reached 82% conversion, but its volumetric productivity of 6-O-acetyl-alpha-d-glucopyranose was low (0.021 g L-1 h-1) due to limited glucose solubility. The anhydrous R-NADES achieved 44% conversion but exhibited higher volumetric productivity (1.05 g L-1 h-1), reflecting its higher substrate concentration, despite its high viscosity. Water was added at 10-30% (v/v) to reduce viscosity and the highest conversion (91%) and volumetric productivity (2.16 g L-1 h-1) were obtained with 20% water. The R-NADES with 20% water also had a 3.8-fold higher enzyme-normalised volumetric productivity than the solvent-free system. This enhancement results from reduced viscosity and favourable enzyme-solvent interactions. The results demonstrate that water-modified R-NADES significantly intensify enzymatic sugar-ester synthesis, reinforcing their potential as green reaction media for biocatalysis.

Kinetic models are important tools for guiding the design and optimization of lipase-catalyzed processes. These processes follow the Ping Pong bi bi mechanism, for which mechanistic kinetic equations can be derived. However, when there are several competing reactions, fully mechanistic models contain a large number of parameters, making it difficult to obtain reliable estimates, so simplified models are necessary. We present a two-step approach to developing semi-mechanistic models of such processes. The first step involves the estimation of the selectivities of the enzyme, using profiles for the reaction species plotted against the degree of reaction, while the second step involves empirical fitting to the same data, but plotted as a function of time. We demonstrate this two-step approach through four case studies based on the literature data for the lipase-catalyzed esterification of fatty acids with trimethylolpropane to produce biolubricants. The semi-mechanistic models were able to describe the data well. Our approach has the advantage of allowing selectivities to be estimated without confounding effects from phenomena such as enzyme denaturation and inhibition. It therefore provides a promising framework for developing models of enzyme-catalyzed processes that obey Ping Pong bi bi kinetics.

The lipase-catalyzed desymmetrization of diols is important in the production of various chiral biotechnological products. Almost two decades ago, it was proposed that Rhizomucor miehei lipase (RML) suffers from allosteric inhibition during the desymmetrization of a 3-OH-protected pentane-1,3,5-triol derivative by selective acetylation, in a dynamic kinetic resolution aimed at producing only one of the monoacetate enantiomers. The proposal was that the diol itself bound to an allosteric site, provoking a conformational change that inhibited the binding of the monoacetate enantiomers to the active site, with this inhibition only being lifted when the diol was completely consumed. This proposed homotropic allostery has been cited over the last two decades as evidence of allosteric inhibition of lipases. However, we demonstrate that the data obtained during this RML-catalyzed desymmetrization can be equally well explained by a model in which the selectivities of the lipase remain constant during the process. In other words, we show that the data can be described without invoking homotropic allosteric inhibition.

The stereoselectivity of lipases during the hydrolysis of triacylglycerols is important in various biotechnological processes, including the partial hydrolysis of triacylglycerols to produce monoacylglycerols and diacylglycerols, which are added to foods as emulsifiers and functional ingredients. The reaction scheme is complex, with twelve hydrolysis reactions and eight acyl migration reactions. The stereoselectivity profile is typically determined for the hydrolysis of triolein, with profiles being plotted against time for the various diolein and monoolein isomers. A recently proposed method for characterizing stereoselectivity fits a dynamic model to these profiles and estimates 28 parameters, not all of which are selectivities, exacerbating problems of correlation between parameters. We propose an improved method in which experimental profiles for the intermediates are plotted against the percentage consumption of triolein. Our complex model has 19 parameters, all of which are selectivities. We refine our model by eliminating parameters based on the Bayesian Information Criterion. We demonstrate our method by applying it to literature data for the hydrolysis of triolein by porcine pancreatic lipase, Chromobacterium viscosum lipase and Pseudomonas fluorescens lipase. The simplified models for these lipases contained 6, 7 and 9 parameters, respectively. A significant advantage of our method is that it eliminates the effects of denaturation and inhibition of the lipase, even if they occur in the system.

Lipases are commonly immobilized covalently on agarose supports to improve their stability and reusability. However, immobilized lipases often have low activity in aqueous environments since they are typically immobilized with the lid domain covering the active site (i.e. in the closed conformation), restricting the access of substrates. The nonionic surfactant Triton X-100 (TX-100) facilitates the opening of the lid and access of the substrate. However, the addition of TX-100 in its free form leads to foaming problems and contamination of the final product, hindering industrial applications. In this work, we functionalize polygalacturonic acid (PGA) with TX-100 and use it as a novel agent for coating the immobilized lipase from Thermomyces lanuginosus (TLL). This coating stabilizes the lid of TLL in the open conformation, improving catalytic performance, avoiding the need to add free surfactants. The specific activities of the coated immobilized lipases for the hydrolysis of p-nitrophenyl propionate (p-NPP) were 3- to 5-fold higher than that of the uncoated lipase, and were similar to that of the free TLL in the presence of free TX-100. These findings show that coating with PGA-TX is an effective strategy for enhancing lipase activity, offering a promising alternative to conventional surfactant-based activation.

Fast pyrolysis of vegetable oils and residues generates bio-oil (BO), a renewable hydrocarbon source with high acidity that limits its direct use in refineries. In this study, BOs were produced from refined soybean oil (RSO) and waste cooking oil (WCO) at 525 degrees C in a continuous bench-scale pyrolysis at 525 degrees C, with a 390 +/- 8 g h-1 feed rate, under steady-state conditions. The resulting bio-oils exhibited high acidity (acid index of 145 and 127 mg KOH g-1, respectively) and elevated olefinic and oxygen contents, making them corrosive and unsuitable for co-refining with petroleum. To reduce acidity, ethyl esterification was performed using lipase B from Candida antarctica (CALB), using a Box-Behnken 33 factorial design. Variables included temperature (40-60 degrees C), bio-oil:ethanol mass ratio (1:1-1:5), and catalyst concentration (3-10% w/w). The acid index was reduced by up to 76%, with optimal conditions (62 degrees C, 1:1 mass ratio, 11% CALB) yielding a final value of 28 mg KOH g-1. Similar reductions were obtained for waste cooking oil bio-oil, confirming robustness across feedstocks. CALB retained over 70% activity after three cycles, demonstrating stability. This enzymatic esterification process shows strong potential for lowering bio-oil acidity, enabling integration into petroleum refineries, diversifying feedstocks, and advancing renewable fuel production.

Research background. Although there are many studies of the bioimprinting of lipases, there is no study comparing the strategies of bioimprinting prior to immobilization (pre-immobilization) and bioimprinting after immobilization (post-immobilization). Likewise, there is no study that compares bioimprinting of lipases immobilized from a pure lipase preparation and lipases immobilized from a crude extract. We therefore investigated these strategies, using the metagenomic lipase LipC12. Experimental approach. We immobilized LipC12 covalently on the commercial support Immobead 150P and treated it with various bioimprinting agents, either pre-immobilization or post-immobilization. We also compared immobilization from a pure LipC12 preparation and immobilization from a crude cell-free extract. Results and conclusions. The best improvements in triolein-hydrolyzing-activity in n-hexane, compared to a non-bioimprinted control, were obtained with post-immobilization bioimprinting, using oleic acid dissolved in t-butanol: a 12-fold improvement for immobilization from a pure LipC12 preparation and an almost 14-fold improvement for immobilization from the crude cell-free extract. This bioimprinting agent also gave a 3.5-fold increase in activity for the synthesis of ethyl oleate in n-hexane, this result being obtained for pre-immobilization bioimprinting and immobilization from the cell-free extract. Novelty and scientific contribution. This study is the first to compare pre-immobilization and post-immobilization bioimprinting strategies, as well as bioimprinting of enzymes immobilized from both pure enzyme preparations and crude cell-free extracts. These results encourage further investigation into bioimprinting strategies.

Little attention has been given to the kinetics of enzyme-catalyzed modification of flavonoids and flavonoid glycosides, especially the successive addition of multiple functional groups. The only currently available model is not consistent with the principles of enzyme kinetics. We developed a simple kinetic model, using the lipasecatalyzed acetylation of phlorizin in a solvent-free system as a case study. Our model assumes that the acetylation is irreversible, the concentration of the solvent/acetyl-donor remains constant and the lipase inactivates in a first-order process. We estimated the parameters of the model by fitting it to literature data for the lipasecatalyzed acetylation of phlorizin, with triacetin as the acetyl-donor. Our model fitted well to profiles for phlorizin, phlorizin-6"-O-acetate and phlorizin-3",6"-O-diacetate obtained at several initial phlorizin concentrations. The estimated parameters were a specificity constant of 0.380 h- 1%(w/v)- 1 for phlorizin, a selectivity of 0.362 for the second acetylation in relation to the first, saturation constants of 22.3 mmol L- 1 for phlorizin and 76.5 mmol L- 1 for phlorizin-6"-O-acetate, an inhibition constant for phlorizin-3",6"-O-diacetate of 3.5 mmol L- 1 and a first-order denaturation constant of 0.0125 h-1. Our work not only presents a model that describes well the acetylation of phlorizin, it also demonstrates a strategy for modelling and parameter estimation that can be applied to other enzyme-catalyzed modifications of flavonoids and flavonoid glycosides.

This study presents an innovative method for synthesizing β-amino carbonylated compounds, specifically 2-[phenyl(phenylamino)methyl] cyclohexanone, achieving high conversions and diastereomeric ratios. Using trypsin or α-chymotrypsin in both free and immobilized forms on titanate nanotubes (NtsTi), synthesized through alkaline hydrothermal methods, successful immobilization yields were attained. Notably, α-chymotrypsin, when free, displayed a diastereoselective synthesis of the anti -isomer with 97 % conversion and 16 : 84 ( syn  :  anti ) diastereomeric ratio, which slightly decreased upon immobilization on NtsTi. Trypsin, in its free form, exhibited diastereoselective recognition of the syn -isomer, while immobilization on NtsTi (trypsin/NtsTi) led to an inversion of diastereomeric ratio. Both trypsin/NtsTi and α-chymotrypsin/NtsTi demonstrated significant catalytic efficiency over five cycles. In conclusion, NtsTi serves as an effective support for trypsin and α-chymotrypsin immobilization, presenting promising prospects for diastereoselective synthesis and potential industrial applications. Furthermore, it offers promising prospects for the diastereoselective synthesis of 2-[phenyl(phenylamino)methyl] cyclohexanone through multicomponent Mannich reaction and future industrial application.

Layered double hydroxides (LDHs) are brucite-like nanomaterials that have been used to immobilize several enzymes. However, layered hydroxide salts (LHSs), another group of brucite-like nanomaterials, have not yet been used for enzyme immobilization. In this work, we prepared two types of layered hydroxide salts, zinc hydroxide nitrate (ZHN: Zn-5(OH)(8)(NO3)(2)(.)2H(2)O) and zinc hydroxide chloride (ZHC: Zn-5(OH)(8)(Cl2H2O)-H-.) and used them to immobilize Pseudomonas cepacia lipase (LipPS). The best protein loading for both ZHN and ZHC was 162.5 mg g(-1) of LHS, which gave high values of triolein-hydrolyzing activity in organic medium (103 U g(-1) for LipPS-ZHN and 105 U g(-1) for LipPS-ZHC), immobilization efficiencies above 90% and activity retentions above 170%. In the kinetic resolution of (R,S)-1-phenylethanol, LipPS-ZHN gave better results, with 50% conversion being obtained in 2 h and an ee(s) of 99%. With LipPS-ZHC, the conversion at 2 h was 40% and the ee(s), was lower, only 73%. For both immobilized materials, the ee(p) was higher than 99% and E was higher than 200. The immobilized materials were stable after 5 cycles of reuse in successive 2-h kinetic resolutions. These results demonstrate that the layered hydroxide salts ZHN and ZHC have good potential as supports for the immobilization of lipases.

There has been recent interest in using the beta-galactosidase of Bacillus circulans to produce prebiotic oligosaccharides, including galactooligosaccharides (GOS), lactulose and lactosucrose. The types and amounts of the transgalactosylation products produced depend on the selectivities of the enzyme for the various transgalactosylation and hydrolysis reactions that occur. To date, the selectivities of the beta-galactosidase of B. circulans for these reactions have not been adequately characterized. In the current work, we undertake four case studies using literature data for systems in which different product mixtures are produced (i) GOS; (ii) GOS and disaccharides; (iii) GOS and lactulose; and (iv) GOS and lactosucrose. We analyze this data to obtain quantitative estimates of the relevant selectivities. We show that, in the production of GOS, the isoform beta-Gal-A is significantly less selective against hydrolysis reactions than the other beta-galactosidase isoforms. In the production of lactulose, the beta-galactosidase of B. circulans gives high lactulose yields, despite having a 19- to 33-fold preference for producing GOS over lactulose. In the production of lactosucrose, the beta-galactosidase of B. circulans also prefers to produce GOS.

The present work deals with the preparation and characterization of Zn2Al layered double hydroxides (LDHs) intercalated with chloride and dodecyl sulfate ions and their use as supports for the immobilization of Pseudomonas cepacia lipase (LipPS). LipPS was immobilized on LDH by adsorption, in situ entrapment and delamination entrapment methods. The performance of free and immobilized LipPS in the transesterification of (R,S)-1-phenylethanol was compared. The immobilized derivatives prepared by adsorption gave the highest conversions (c = 50%), with an enantiomeric excess of the product (ee(p)) greater than 99% and a high enantiomeric ratio (E > 200). For immobilization by in situ entrapment (coprecipitation), a conversion of 31.5% and E > 200 were obtained. For immobilization by delamination entrapment, a conversion of 36% and E= 155 were obtained. Although the best results were obtained by adsorption, these results show that the delamination entrapment method can also be used to prepare bioinorganic-enzyme materials based on LDH. Our work therefore increases the range of methods that can be used to immobilize lipases for use in the production of optically pure secondary alcohols.

There has been recent interest in using the β-galactosidase of Aspergillus oryzae to produce lactulose and fructosyl-galactooligosaccharides, for use as prebiotics. The success of the enzymatic process depends on the selectivities of the enzyme for the various transgalactosylation and hydrolysis reactions that occur in these systems, but the methods that have been used to date to express these selectivities are not adequate. In the current work, we demonstrate a method for determining the selectivity of the β-galactosidase of A. oryzae in two case studies done with literature data: (1) the production of lactulose from a mixture of lactose and fructose and (2) the production of fructosyl-galactooligosaccharides (fGOS) from lactulose. In the first case study, we demonstrate that the enzyme has a 4- to 5-fold preference for producing GOS over lactulose when an equimolar mixture of fructose to lactose is used, but that the selectivity for producing lactulose increases as the initial fructose to lactose molar ratio increases. In the second case study, we show that the enzyme has about a 1.5-fold preference for producing fGOS4 and fGOS5 over the initial transgalactosylation product, fGOS3. The selectivities that we determine in our work will be important parameters for time-based models used to guide the development and optimization of processes for the production of lactulose and fructosyl-galactooligosaccharides as prebiotics.

Kinetic resolution of racemates with lipases is the preferred method for producing bioactive compounds. One strategy for obtaining highly enantioselective lipases that are stable in the organic media that are often used in these reactions is to improve existing lipases by protein engineering. In this work, we engineered the lipase LipC12, which has good stability in organic media, but only moderate enantioselectivity. Molecular docking with LipC12 identified V261 as a key position influencing, first, enantioselectivity in the transesterification of (RS)-1-phenylethanol and, second, activity in the hydrolysis of p-nitrophenyl octanoate. Variants were then obtained by site-directed mutagenesis, expressed in Escherichia coli, and their performance in these reactions was evaluated. Enzymes immobilized on Immobead 150 were used in the transesterification while free enzyme was used in the hydrolysis reaction. It was not possible to increase the hydrolytic activity and enantioselectivity simultaneously: some variants had increased enantioselectivity but lower hydrolytic activity, and others had increased hydrolytic activity but lower enantioselectivity. The best result for enantioselectivity was obtained for LipC12(V261Q), with an increase of the E-value (for (R)-1-phenylethanol) from 46 to 110, however, its hydrolytic activity decreased 4-fold in comparison to LipC12(wt). The highest hydrolytic activity was obtained for LipC12(V261F), with a value almost 6-fold higher than that of LipC12(wt). This variant also had an inverted enantiopreference (i.e. for (S)-1-phenylethanol), but with a very low E-value of only 4.

This work aimed to produce porous poly‐hydroxybutyrate (PHB) pellets in order to evaluate the pellets as a support for immobilization of the metagenomic lipase, LipG9. Four types of pelletized PHB particles with different morphological characteristics were obtained using the double emulsion and solvent evaporation technique (DESE). The micropores of these PHB pellets had similar average diameters (about 3 nm), but the pellets had different specific surface areas: 11.7 m 2 g −1 for the PHB powder, 8.4 m 2 g −1 for the control pellets ( Ø < 0.5 mm, produced without the pore forming agent), 10.0 m 2 g −1 for the small pellets ( Ø < 0.5 mm), 9.5 m 2 g −1 for the medium pellets (0.5 < Ø < 0.8 mm) and 8.4 m 2 g −1 for the large pellets ( Ø > 1.4 mm). Purified LipG9 was immobilized by adsorption on these pellets, and the results were compared with those obtained with PHB powder. The highest immobilization yield (83%) was obtained for the medium PHB pellets, followed by large (76%) and small (55%) PHB pellets. The activity of LipG9 immobilized on the pellets, for the synthesis of ethyl oleate in n ‐hexane, was highest for the medium pellets (22 U g −1 ). The immobilization yield was high for PHB powder (99%) but the esterification activity was slightly lower (20 U g −1 ). These results show that pelletized PHB beads can be used for the immobilization of lipases, with the advantage that pelletized PHB will perform better than PHB powder in large‐scale enzyme bioreactors.

Deep eutectic solvents (DESs) have been shown to increase the activity of lipases in biocatalytic processes, especially when added at intermediate concentrations. We evaluated the effect of DESs on the hydrolysis of pnitrophenyl palmitate (p-NPP) by a metagenomic lipase, LipC12. The hydrogen bond acceptor was choline chloride (ChCl), while sorbitol (Sor), xylitol (Xyl), Glycerol (Gly), ethylene glycol (EG), triethylene glycol (TEG) and urea (U) were tested as hydrogen bond donors. The best results were obtained at 25% (v v-1) DES, with the increases in hydrolytic activity, compared to activity in buffer, being 18-fold for ChCl:Sor (2:1), and 10- to 12fold for ChCl:Gly (1:2), ChCl:EG (1:2) and ChCl:Xyl (2:1). Dynamic Light Scattering analysis suggests that protein disaggregation played a key role in this activation: the hydrodynamic radius of LipC12 was 157 nm in buffer, but ranged from about 1 to 7 nm in DESs at different concentrations. The addition of 100 mM NaCl to the DEScontaining media (at 25% v v-1 DES) slightly improved LipC12 activity. Addition of the hydrogen-bond donor alone also increased the p-NPP hydrolyzing activity with 30% (w v-1) sorbitol or xylitol giving 4.5-fold higher activities, compared to activity in buffer. Our fold-improvements in activity are the highest yet reported for the pNPP-hydrolyzing activity of lipases and suggest that more studies are required, especially with other biocatalytic reactions such as esterification and transesterification.

This chapter is written as a basic introduction for readers who are familiar with experimental aspects of the enzymatic hydrolysis of polysaccharides and are interested in modeling their systems, with the aim of using their models to guide process design and optimization. It begins by pointing out that models can address the action of enzymes at different scales, with dynamic macroscale models being the appropriate models for guiding process design and optimization. This is followed by a consideration of features of substrates and enzymes that will affect decisions made during model development. It then discusses the trade-off between simplicity of the model and its ability to give accurate predictions under a range of different conditions. Next, the key features of two different types of models are presented, namely, deterministic models and stochastic models. Finally, the use of models as tools in the estimation of specificity constants is described.

Reactions for the lipase-catalyzed kinetic resolution of chiral diols or the desymmetrization of achiral meso-diols involve two alternative routes, each of which contains two steps. During such reactions, up to four different nucleophiles compete to attack the acyl-enzyme intermediate of the catalytic cycle. The selectivities of the lipase for these different nucleophiles determines the success of the desymmetrization or resolution process and are important parameters for time-based mathematical models of these processes. Current methods for estimating these selectivities are not sufficiently accurate. In the current work, we develop a new approach to determining selectivities in lipase-catalyzed desymmetrization or resolution reactions with diols. The method is based on a model in which the only parameters are selectivities. We demonstrate the application of the method using literature data for three case studies of increasing complexity: (i) the two-step acetylation of phlorizin; (ii) the kinetic resolution of racemic 1,3-butanediol and (iii) the two-step desymmetrization of a meso-diol to produce a monoacetylated diol that is a key intermediate in the synthesis of biotin. We demonstrate the advantages of our estimation method over previously proposed estimation methods. The selectivities estimated by our method will be important parameters in models describing the desymmetrization and resolution of diols.

Packed-bed bioreactors are often used for aerobic solid-state fermentation, since the forced aeration supplies O2 and removes metabolic heat from the bed. Motivated by the potential for applications in biorefineries, we review studies conducted on packed-bed bioreactors over the last decade, evaluating the insights these studies provide into how large-scale packed beds should be designed and operated. Many studies have used low superficial air velocities and suffer from preferential airflow, such that parts of the bed are not properly aerated. Moreover, some studies have proposed ineffective strategies, such as reversing the direction of the airflow or introducing air through perforated pipes within the bed. Additionally, many studies have used narrow water-jacketed packed-bed bioreactors, but these bioreactors do not reflect heat removal in wide large-scale packed beds, in which heat removal through the side walls makes a minor contribution. Finally, we conclude that, although some attention has been given to characterizing the porosities, water sorption isotherms and volumetric heat and mass transfer coefficients of substrate beds, this work needs to be extended to cover a wider range of solid substrates, and work needs to be done to characterize how these bed properties change due to microbial growth.

Interest has increased in using enzyme-catalyzed esterification to produce fatty acid esters of trimethylolpropane for use as biolubricants. Mathematical models of this process can be used to guide the design and scale-up of production processes. The selectivities of the enzyme for the various trimethylolpropane species (unesterified, mono-esterified, di-esterified and tri-esterified) are important parameters of such models. Previous attempts to estimate selectivities in the esterification of trimethylolpropane have not produced accurate estimates. In the current work, we show that the fingerprinting method can be used to obtain accurate estimates. We apply the method to various data sets from the literature, using Bayesian techniques for model fitting. We show that, depending on the reaction conditions, different versions of the model are necessary: (i) A model that treats the reactions as irreversible; (ii) an “irreversible model” that recognizes mass transfer limitations, but treats them using a pseudoprocessive model; and (iii) a model that treats the first reaction as reversible and is appropriate for systems in which water is added at the beginning of the reaction.