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This research presents the results of the immobilization of Candida Antarctica Lipase B (CALB) on MOF-199 and ZIF-8 and its use in the production of biodiesel through the transesterification reaction using African Palm Oil (APO). The results show that the highest adsorption capacity, the 26.9 mg·g−1 Lipase, was achieved using ZIF-8 at 45 °C and an initial protein concentration of 1.20 mg·mL−1. The results obtained for the adsorption equilibrium studies allow us to infer that CALB was physically adsorbed on ZIF-8 while chemically adsorbed with MOF-199. It was determined that the adsorption between Lipase and the MOFs under study better fit the Sips isotherm model. The results of the kinetic studies show that adsorption kinetics follow the Elovich model for the two synthesized biocatalysts. This research shows that under the experimental conditions in which the studies were carried out, the adsorption processes are a function of the intraparticle and film diffusion models. According to the results, the prepared biocatalysts showed a high efficiency in the transesterification reaction to produce biodiesel, with methanol as a co-solvent medium. In this work, the catalytic studies for the imidazolate, ZIF-8, presented more catalytic activity when used with CALB. This system presented 95% biodiesel conversion, while the biocatalyst formed by MOF-199 and CALB generated a catalytic conversion percentage of 90%. Although both percentages are high, it should be noted that CALB-MOF-199 presented better reusability, which is due to chemical interactions.

Biocatalysis has emerged as a green technology in chemical based catalysis. Immobilization of enzymes onto magnetized nanomaterials enhances downstream processing as it eases their separation from reaction mixture. Synthesis of the nanoparticles was performed in an aqueous solution of FeCl3.6H2O as precursor and NH3 as nucleating agent under microwave irradiation. The maghemite complex was cross-linked with glutaraldehyde to provide conducive environment for enzyme immobilization. Stability of produced immobilized lipases against different conditions were evaluated such as working temperatures from 27 °C to 85 °C and retained more than 64% of its activity at 85 °C. The immobilized lipase complex was able to withstand a wide range of acidic to alkaline environment from pH 5.5 to pH 9.5. The enzyme retains more than 59% of its activity after 14 days of storage and can be recycled for more than 7 cycles with remaining high activity at 93.7%. Developed enzyme was then subjected to microwave irradiation for transesterification of palm oil to produce biodiesel. Highest biodiesel recovery achieved from microwave assisted immobilized lipase catalysed transesterification of palm oil was 70.2%. The physical properties of produced biodiesel was evaluated and fulfilled the ASTM general requirement for fuels.

An efficient, active and stable biocatalyst was prepared by sol–gel CaL-B encapsulation in the presence of a choline–fructose DES, and is able to transform efficiently ten alcohols relevant for various industries.

In the present communication, the simultaneous co-immobilization by covalent binding of lipase A from Candida antarctica (CALA) and lipase B from Candida antarctica (CALB) in glutaraldehyde (GLU) activated chitosan (CHI) was optimized using the Taguchi method. Under optimized conditions (pH 9, 5 mM, 6:1 (protein load/g of support and 1 h), it was possible to reach 80.00 ± 0.01% for the immobilization yield (IY) and 46.01 ± 0.35 U/g for the activity of the derivative (AtD); in this case, load protein and ionic strength were the only statistically significant parameters and, therefore, those that most influenced the immobilization process. Furthermore, at pH 7, CALA-CALB-CHI had a half-life 2–6 times longer than the mixture of CALA and CALB for a temperature range of 50−80 °C. CALA-CALB showed the highest activity at pH 7, whereas CALA-CALB-CHI, except at pH 7, was more active than the soluble lipase mixture in the pH range (5–9), especially at pH 9. CHI, CHI-GLU, and CALA-CALB-CHI were characterized by X-ray powder diffraction (XRPD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscope (SEM), Thermogravimetry (TGA), and Energy Dispersive Spectroscopy (EDS), proving the immobilization of CALA and CALB in chitosan. CALA-CALB-CHI derivative evaluated in the kinetic resolution of halohydrins acetates rac-2-bromo-1-(2-chlorophenyl) ethyl acetate (2a) and rac-2-chloro-1-(2,4-dichlorophenyl) ethyl acetate (2b), to produce the corresponding halohydrins 3a-b, which are intermediates in the synthesis of the drugs chlorprelanine (antiarrhythmic) and luliconazol (antifungal), respectively. (S)-bromohydrin 3a was obtained with 79% enantiomeric excess (ee), whereas (S)-chlorohydrin 3b produced with 98% ee, conversion of 46% and E > 200. Additionally, molecular docking was performed to elucidate the hydrolysis interaction reaction between β-halohydrin acetates and lipases CALA-CALB.

Butyl oleate synthesis by the lipase B from Candida antarctica (CalB) under extreme halophilic conditions was investigated in the present research through the AOT/Water/Isooctane reverse micellar system. The impact of aqueous content (Wo=H2OSurfactant) and NaCl variation on the enzymatic activity of CalB in the butyl oleate reaction in reverse micelles was explored. The results indicated that, based on the increase of NaCl, it is remarkable to achieve higher enzymatic activity up to 444.85 μmolmin at 5 M NaCl and Wo = 10, as the best esterification conditions at pH 7.2 and 30 °C. However, it was clear that butyl oleate synthesis by lipase CalB increased based on the reduction in the average reverse micelle size, where reverse micelle sizes were determined by dynamic light scattering (DLS). This increase in butyl oleate synthesis demonstrated the potential of reverse micelles as systems that enhance mass transport phenomena in heterogeneous biocatalysis. Furthermore, reverse micelles are promising systems for extreme halophilic lipases research.

Proanthocyanin (PA) dimers isolated from Acacia mearnsii bark were enzymatically acylated with palmitic acid as the acyl donor by immobilized Candida antarctica lipase on acrylic resin (Novozym 435). The acylation reaction conditions were optimized by comparing the amount of enzyme, the temperature, the reaction solvents, initial water content, substrate molar ratios and reaction time. The highest acylation conversion of 96.53% was achieved under the follow conditions: PA dimers/palmitic acid at a molar ratio of 1: 10 in tert-amyl alcohol; initial water content of 5% at 60 ℃ for 12 h with 30 g/L enzyme dosage. Introducing palmitic acid into PA dimers significantly improved both the lipophilicity and antioxidant properties. The 1-octanol/water partition coefficient of the PA dimers and their derivatives showed that the lipophilicity of the derivatives were 2.4 times higher than that of the PA dimers. The derivatives exhibited strong antioxidant scavenging capacities, approximately 1.6 times greater than the original dimers. This work is of great significance to expand the application of natural PA dimers in cosmetic and food industries and also lay a foundation for the high value-added utilization of A. mearnsii.

MCM-48 mesoporous support was synthesized with the ionic solid 1-tetradecyl-3-methylimidazolium chloride ([C14MI]Cl) as a structure-directing agent for in situ immobilization of Candida antarctica B (CALB). The MCM-48[C14MI]Cl support showed characteristics of mesoporous material of interest, with a pore size of 20.30 and 73.41 A for the support without and with the enzyme, respectively. The elongation of the carbonic chain of the ionic solid directly influenced the increase in the specific area and pore volume of the material. In addition, the decrease in the specific area and pore volume for support with the enzyme showed the effectiveness of immobilization in situ. It was possible to obtain the ideal levels for the best activities of esterification of the enzyme with optimization of a mathematical model. The optimized variables were 0.31 g of enzyme and 3.35% of ionic solid with a maximum esterification activity of 392.92 U/g and 688% of yield. The support showed residual activity above 50% when stored under refrigeration for 75 days. At 60 and 80 °C, the enzyme immobilized on the support retained more than 80 and 40% of its residual activity, respectively. In addition, the support presented the possibility of reuse for up to 10 cycles with residual activity of approximately 50%. The support synthesized in the present study presents a great industrial opportunity for the immobilization and use of the CALB enzyme.

Lipases are widely used enzymes that catalyze hydrolysis and alcoholysis of fatty acid esters. At high concentrations of small alcohols such as methanol or ethanol, many lipases are inhibited by the substrate. The molecular basis of the inhibition of Candida antarctica lipase B (CALB) by methanol was investigated by unbiased molecular dynamics (MD) simulations, and the substrate binding kinetics was analyzed by Markov state models (MSMs). The modeled fluxes of productive methanol binding at concentrations between 50 mM and 5.5 M were in good agreement with the experimental activity profile of CALB, with a peak at 300 mM. The kinetic and structural analysis uncovered the molecular basis of CALB inhibition. Beyond 300 mM, the kinetic bottleneck results from crowding of methanol in the substrate access channel, which is caused by the gradual formation of methanol patches close to Leu140 (helix α5), Leu278, and Ile285 (helix α10) at a distance of 4-5 A from the active site. Our findings demonstrate the usefulness of unbiased MD simulations to study enzyme-substrate interactions at realistic substrate concentrations and the feasibility of scale-bridging by an MSM analysis to derive kinetic information.

The current research work focuses on the conventional bio-catalysed synthesis of amyl levulinate with immobilized Candida antarctica lipase B in the absence of a toxic solvent. The green catalyst and solvent free system offered considerable benefits like mild and gentle condition requirement, lesser-energy demand, easy down-streaming and reusability of the biocatalyst. The process parameters were examined, and the maximum conversion (73.2%) was obtained under the optimum condition of 50℃ temperature, 1:3 molar ratio of levulinic acid to amyl alcohol using 0.8% (w/v) biocatalyst at 250 rpm speed. The immobilized lipase was characterized by SEM, BET, XRD, FTIR and amyl levulinate was characterized by FTIR and GC-MS analysis. The enzyme lipase was effectively reused till 5 cycles for the formation of ester. The thermodynamic parameters (ΔH, ΔG, and ΔS) and energy of activation (Ea) were determined for the assessment of energy requirement. The activation energy was determined to be 48.42 kJ/mol. The research protocol strikes the development of biocatalytic synthetic route of amyl levulinate without using any toxic solvent, which may contribute to green chemistry approach.

Biocatalysts with tailor-made or controllable stereoselectivity are valuable and highly desired in the precision synthesis of chiral compounds. Most lipases display excellent (R)-enantioselectivity for various sec-alcohols. In order to reverse the inherent (R)-enantioselectivity of lipase A from Bacillus subtilis (BSLA), an important amino acid position of BSLA (H76) which can reverse the (R)-enantioselectivity to (S) in the hydrolytic kinetic resolution (KR) of various esters derived from sec-alcohols was discovered, basing on the comparison with the well-known “gourd”-like pocket of Candida antarctica lipase B (CALB). After iterative mutagenesis, the best mutant H76 ​A/N18C was obtained, displaying high (S)-selectivity for the model reaction (45% conv. and 92% ee). Meanwhile, a single-point mutation of N18W was also found which can remarkably improve the inherent (R)-enantioselectivity of BSLA toward sec-alcohol. A series of enantiocomplementary sec-alcohols can be prepared through the KR catalyzed by WT BSLA or mutants, and the highest enantiomeric ratio (E) value is up to >300 for (R)-enantiomer and 178 for (S)-enantiomer, respectively.

Aliphatic polyesters are widely used in biomedical and environmental areas, and their use has grown due to environmental issues. The use of lipases as catalysts for the synthesis of polyesters is an environmentally benign alternative. In this work, the effect of monomer chain length on polyester synthesis was studied with seven diacids and six diols in a bulk system using immobilized Candida antarctica lipase B (Novozym®435). Firstly, the reaction temperature and the thermal stability of Novozym®435 (N435) were evaluated. The half-life of the commercial lipase was 24.8 h at 90 °C, and N435 maintained a residual activity of 38% after 96 h of incubation. The biocatalyst played an essential role in the reactions using monomers with longer alkylene chain length diacids (azelaic and sebacic acids) and diols (1,4-butanediol, 1,6-hexanediol, and 1,8-octanediol), giving a higher reactivity than reactions of shorter chain-length diacids (oxalic, malonic, succinic, glutaric and adipic acids) and diols (ethylene glycol and 1,3-propanediol). Polycondensation reactions carried out with 2,3-butanediol did not present a significative molecular weight. Otherwise, the reaction performed with 1,6-hexanediol resulted in polyesters with weight-average molecular weights (Mw) of 18,346 g mol−1 and 27,121 g mol−1 by reacting with azelaic and sebacic acids, respectively, at 90 °C using 5 wt.% of N435. The thermal properties of polyazelates and polysebactes were analyzed by DSC and TGA, which showed that aliphatic polyesters are practically stable at up to 380 °C, indicating their high thermal stability.

The present study reports the effects of three commercial immobilized lipases namely Novozyme 435 from Candida antarctica lipase B (CALB), Lipozyme TL IM from Thermomyces lanuginosus and Lipozyme RM IM from Rhizomucor miehei on the production of trimethylolpropane (TMP) ester from high oleic palm methyl ester (HO-PME) and TMP. The TMP ester is a promising base oil for biolubricants that are easily biodegradable and non-toxic to humans and the environment. Enzymatic catalysts are insensitive to free fatty acid (FFA) content, hence able to mitigate the side reactions and consequently reduce product separation cost. The potential of these enzymes to produce TMP ester in a solvent-free medium was screened at various reaction time (8, 23, 30 and 48 h), operating pressure (0.1, 0.3 and 1.0 mbar) and enzyme dosage (1, 3, 5 and 10% w/w). The reaction was conducted at a constant temperature of 70 °C and a molar ratio of 3.9:1 (HO-PME: TMP). Novozyme 435 produced the highest yield of TMP ester of 95.68 ± 3.60% under the following conditions: 23 h reaction time, 0.1 mbar operating pressure and 5% w/w of enzyme dosage. The key lubrication properties of the produced TMP ester are viscosity index (208 ± 2), pour point (− 30 ± − 2 °C), cloud point (− 15 ± − 2 °C), onset thermal degradation temperature (427.8 °C), and oxidation stability, RPVOT (42 ± 4 min). The properties of the TMP ester produced from the enzymatic transesterification are comparable to other vegetable oil-based biolubricants produced by chemical transesterification.

The production of biodiesel from oily waste has been reported as a promising alternative for the use of biomass in energy. However, the high content of free fatty acids makes traditional chemical catalysis processes difficult. This work aimed to evaluate the enzymatic production of biodiesel from oily residues derived from sanitation and its optimization for large-scale applications. We evaluated the production of biodiesel from a University Restaurant grease trap using lipases as catalysts (Candida antarctica, Thermomyces lanuginosa, Mucor miehei). The experiments were performed by using a factorial design of type 32 to evaluate the best conditions of temperature (30, 40 and 50 °C) and enzyme concentration (5, 7.5 and 10% w/w) for each different enzyme. When the enzyme obtained from C. antarctica was used, the high yield was obtained in all studied conditions and none of the variables was statistically significant. As for the enzymes from the T. lanuginosa and the M. miehei significant effect of the temperature was reported, the higher yield was obtained with lower temperatures (30–40 °C). The concentration of enzymes only was statistically significant with enzyme from M. miehei, which had better yield with 10% w/w fat. Still, the lowest yields of biodiesel were obtained when using the enzyme from M. miehei. The enzymatic production of biodiesel from oily residues can be an interesting strategy for reducing production costs and an alternative for the sustainable treatment of oily residues from sanitation.

Environmentally friendly processes are nowadays a trending topic to get highly desired chemical compounds and, in this sense, the use of enzyme-catalyzed routes is becoming a promising alternative to traditional synthetic methods. In the present paper, a hybrid quantum mechanics/molecular mechanics (QM/MM) computational study on the epoxidation of alkenes catalyzed by the Ser105Ala variant of the promiscuous Candida antarctica lipase B (CALB) is presented in an attempt to search for alternative paths to get useful intermediates in industries. The catalyzed reaction, described at the atomistic level with a model of the full solvated in a box of water molecules, is compared with the alternative epoxidation of alkenes by peroxy acids in chloroform. Free-energy profiles obtained at the density functional theory (DFT)/MM level show how Ser105Ala CALB is capable of epoxide short alkenes in a two-step process with free-energy barriers, in agreement with available experimental data, that are significantly lower than those of the single-step reaction in solution. The possible (R)-enantioselectivity dictated by the binding step, explored by means of alchemical QM/MM free-energy perturbation (FEP) methods, and the preference for the (S)-enantiomer derived from the free-energy landscape of the chemical steps would cancel out, thus predicting the lack of enantioselectivity experimentally observed. In general, our results provide general information on the molecular mechanism employed by a highly promiscuous enzyme, with potential applications in biotechnology.

A long-standing goal in the field of biotechnology is to develop and understand design rules for the stabilization of enzymes upon immobilization to materials. While immobilization has sometimes been successful as a strategy to stabilize enzymes, the design of synthetic materials that stabilize enzymes remains largely empirical. We sought to overcome this challenge by investigating the mechanistic basis for the stabilization of immobilized lipases on random copolymer brush surfaces comprised of poly(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA), which represent novel heterogeneous supports for immobilized enzymes. Using several related but structurally diverse lipases, including Bacillus subtilis lipase A (LipA), Rhizomucor miehei lipase, Candida rugosa lipase, and Candida antarctica lipase B (CALB), we showed that the stability of each lipase at elevated temperatures was strongly dependent on the fraction of PEGMA in the brush layer. This dependence was explained by developing and applying a new algorithm to quantify protein surface hydrophobicity, which involved using unsupervised cluster analysis to identify clusters of hydrophobic atoms. Characterization of the lipases showed that the optimal brush composition correlated with the free energy of solvation per enzyme surface area, which ranged from -17.1 kJ/mol·nm2 for LipA to -11.8 kJ/mol·nm2 for CALB. Additionally, using this algorithm, we found that hydrophobic patches consisting of aliphatic residues had a higher free energy than patches consisting of aromatic residues. By providing the basis for rationally tuning the interface between enzymes and materials, this understanding will transform the use of materials to reliably ruggedize enzymes under extreme conditions.

The synthesis of both enantiomers of a key intermediate in the synthesis of halofuginone was accomplished by a Candida antarctica lipase B (CAL-B)-catalyzed kinetic resolution of the corresponding racemate. When the resolution was carried out in the versatile solvent cyclopentyl methyl ether (CPME) using p-chlorophenylbutyrate (PCPB) as the acylating reagent, the highest enantiomeric ratio (E) values were measured, and highly enantioenriched (95% ee) compounds could be obtained in a single iteration. As an example, one of the two enantiomers was used as a starting material to prepare (+)-halofuginone in a three-step procedure.

The biomimetic mineralization of zeolitic imidazolate framework-8 (ZIF-8) has been reported as a strategy for enzyme immobilization, enabling the heterogenization and protection of biomacromolecules. Here, we report the preparation of different Candida antarctica lipase B biocomposites (CALB@ZIF-8) formed by altering the concentrations of Zn2+ and 2-methylimidazole (2-mIM). The influence of synthetic conditions on the catalytic activity of the lipase CALB was examined by hydrolysis and transesterification assays in aqueous and organic media, respectively. We demonstrated that for both reactions, activity was retained for the biocomposites formed at low Zn2+/2-mIM ratios but notably almost entirely lost when the ligand concentration used to form the biocomposites was increased. Additionally, phosphate buffer could regenerate the activity of larger particles by degrading the crystal surfaces and releasing encapsulated CALB into solution. Transesterification reactions using CALB@ZIF-8 biocomposites were undertaken in 100% hexane, giving rise to enhanced CALB activity relative to the free enzyme. These observations highlight the fundamental importance of synthetic protocols and operating parameters for developing enzyme@MOF biocomposites with improved activity in challenging conditions.

In this study, the synthesis of biopolyols derived from castor oil and glycerol was performed by enzymatic glycerolysis in a solvent-free system applying the lipases: Novozym® 435 (N435), from Candida antarctica fraction B, immobilized on macroporous anionic resin, and Eversa® Transform 2.0, a soluble formulation from Thermomyces lanuginosus. The biopolyols presented different conversion into mono- and diacylglycerol (MAG and DAG) owing to regioselectivity of the lipases. The resulting biopolyols were employed for the synthesis of polyurethane foams using different amounts of chemical blowing agent (water). The NCO source employed was polymeric methylene diphenyl diisocyanate (pMDI). The foams were compared, and the results showed that the PU foams obtained by using the biopolyol from lipase N435 presented uniform pore size and more desirable mechanical characteristics. Although this has arisen, the results obtained by using the lipase Eversa® Transform 2.0 showed the possibility of applying a low-cost enzyme to obtain biopolyols and foams, and there may be a possibility of competition with chemical catalysts.

We report a simple enzymatic procedure for the synthesis of short-chained flavor esters by direct esterification of natural acids with short-chain primary alcohols mediated by lipase B from Candida antarctica entrapped in a tailored sol–gel matrix in the presence of three additives, using vacuum for water removal. Maximal immobilization yields (100%) were obtained for all three biocatalysts, while the enzyme loading and the synthetic activity depend on the used additive (6.7, 7.6, and 7.7 μg enzyme/mg biocatalyst for β-cyclodextrin, polyvinyl alcohol, and glycerol, respectively, and 76–110% recovered activity as compared with free lipase). The process was optimized using the reaction of hexan-1-ol with butyric acid as the model with significant conversion improvements (from 94%) reducing the alcohol/enzyme ratio (from 100:1 to 25:1 weight ratio) for all novel biocatalysts. Other short-chain flavor esters were prepared with excellent yields (>90%) under the previously established optimal conditions. Atom economy (90.53), E-factor (17.22), atom efficiency (88.36), mass intensity (18.58), and reaction mass efficiency (60.07) as relevant sustainability metrics were calculated for the preparative scale model reaction with glycerol as the additive. Based on our results, this green and sustainable new approach can be used for the synthesis of many flavor esters.

A new series of nonionic gemini amphiphiles have been synthesized in a multi-step chemoenzymatic approach by using a novel A2 B2 -type central core consisting of conjugating glycerol and propargyl bromide on 5-hydroxy isophthalic acid. A pair of hydrophilic monomethoxy poly(ethylene glycol) (mPEG) and hydrophobic linear alkyl chains (C12 /C15 ) were then added to the core to obtain amphiphilic architectures. The aggregation tendency in aqueous media was studied by dynamic light scattering, fluorescence spectroscopy and cryogenic transmission electron microscopy. The nanotransport potential of the amphiphiles was studied for model hydrophobic guests, that is, the dye Nile Red and the drug Nimodipine by using UV/Vis and fluorescence spectroscopy. Evaluation of the viability of amphiphile-treated A549 cells showed them to be well tolerated up to the concentrations studied. Being ester based, these amphiphiles exhibit stimuli-responsive sensitivity towards esterases, and a rupture of amphiphilic architecture was observed in the presence of immobilized Candida antarctica lipase (Novozym 435), thus facilitating release of the encapsulated guest from the aggregate.

The very slow degradation of biodegradable polymers in the marine environment is due to the lack of dedicated degradation enzymes in open seas. As a result, introducing monomers that have a fast hydrolysis process is required to accelerate seawater degradation. Poly(butylene succinate-co-glycolate) (PBSGA) copolyesters with glycolic acid (GA) units ranging from 5 to 40% were synthesized by our newly developed polymerizing method based on oligo(glycolic acid). The results of ¹H-NMR and GPC revealed that short GA segments were evenly distributed between BS segments, obtaining random copolyesters with a weight-average molecular weight over 6.24 * 10⁴ g/mol. The copolymerized GA units hinder its crystallization capability and increase hydrophilicity of the PBSGAs, which still displayed mechanical properties comparable or even better than most biodegradable polymers. Fast degradation in seawater and enzymatic environments (Candida antarctica lipase B enzymes) is proved experimentally. The quick decomposition in seawater was originated from accelerated hydrolysis. For instance, the weight loss of PBSGA40 (compositions of GA units) exceeded 22% after 49 days. Possible degradation mechanisms were proposed based on Fukui function analysis and frontier molecular orbital calculation. Additionally, the energy barrier for hydrolysis was calculated by the density functional theory method, indicating that the hydrolysis of the polymer chain became more and more easy with the increase in GA units. At last, the addition of GA units only had a mild effect on the shelf life of the PBSGAs.

Lipase B from Candida antarctica (CAL-B) was immobilised on epoxy-activated polymer support functionalised with polyethyleneimine (PEI) via preparation of enzyme aggregates by precipitation with po...

Candida antarctica lipase B was immobilized on a series of silicone nanofilament-coated matrices of different porosities. In addition to creating a more open surface, SNF’s hydrophobicity allows for a simple immobilization pathway via adsorption. In order to study the impact of the nanostructure, the performance was compared with control samples lacking SNFs. For all materials, the surface was characterized with BET measurements, and the immobilized enzyme was measured as well as the catalytic activity. Enzyme loads ranged between 3.85% w / w and 2.53% w / w and decreased with the decreasing surface area of the carrier material from 200 m2/g to 0.04 m2/g, while the activity per enzyme increases from 824 U to 2040 U. The data suggest that the coating seals off inner surfaces, forcing the enzyme to be immobilized at more accessible positions allowing for higher activity per enzyme. Optimization of the immobilization conditions allowed us to create a thinner enzyme layer which further improved the activity per enzyme to 3129 U. While this activity is comparable to the commercial Novozyme 435 with 3073 U, the SNF-based system performs the catalysis in a thin surface layer of around 13 μm. A favorite area of application is, for example, the creation of enzyme-based detection systems, where the high activity per surface area of up to 89622 U · mg/m2 would lead to high signal strength.

Candida antarctica lipase B (CALB) and Thermomyces lanuginose lipase (TLL) were co-immobilized on epoxy functionalized silica gel via an isocyanide-based multicomponent reaction. The immobilization process was carried out in water (pH 7) at 25 °C, rapidly (3 h) resulting in high immobilization yields (100%) with a loading of 10 mg enzyme/g support. The immobilized preparations were used to produce biodiesel by transesterification of palm oil. In an optimization study, response surface methodology (RSM) and central composite rotatable design (CCRD) methods were used to study the effect of five independent factors including temperature, methanol to oil ratio, t-butanol concentration and CALB:TLL ratio on the yield of biodiesel production. The optimum combinations for the reaction were CALB:TLL ratio (2.1:1), t-butanol (45 wt%), temperature (47 °C), methanol: oil ratio (2.3). This resulted in a FAME yield of 94%, very close to the predicted value of 98%.

Lipases, also known as triacylglycerol hydrolases (E.C.No. 3.1.1.3), are considered as leading biocatalysts in the lipid modification business. With properties like ease of availability, capability to work in heterogeneous media, stability in organic solvents, property of catalyzing at the lipid-water interface and even in nonaqueous conditions, have made them a versatile choice for applications in the food, flavor, detergent, pharmaceutical, leather, textile, cosmetic, and paper industries [1]. The increasing alertness toward sustainable technologies, lesser waste generation and solvent usage and minimization of energy input has brought light toward the production and usage of recombinant/improved lipases. For example, Novozym 435, a broadly used recombinant lipase isolated from Candida antarctica, dominates the lipase industry and has even created a supplier bias in the market. This shows that there is a desperate need for novel, low-cost lipases with better properties. For this, mining of existing extremophilic genomes seems more rewarding. But considering the diversity of industrial requirements such as types of solvents used or carrier systems employed for enzyme immobilization, tailor-designed enzymes are an unrealized pressing priority. Therefore, protein engineering strategies in collaboration with the discovery of new lipases can serve as a vital tool to obtain tailor-made enzymes with specific characteristics.