Activity Of Candida Antarctica Lipase B Research Articles

Candida antarctica lipase B performance in organic solvent at varying water activities studied by molecular dynamics simulations

Applications of lipases in low-water environments are found across a broad range of industries, including the pharmaceutical and oleochemical sectors. This includes condensation reactions in organic solvents where the enzyme activity has been found to depend strongly on both the solvent and the water activity (aw). Despite several experimental and computational studies, knowledge is largely empirical, and a general predictive approach is much needed. To close this gap, we chose native Candida antarctica lipase B (CALB) and two mutants thereof and used molecular dynamics (MD) simulations to gain a molecular understanding of the effect of aw on the specific activity of CALB in hexane. Based on the simulations, we propose four criteria to understand the performance of CALB in organic media, which is supported by enzyme kinetics experiments. First, the lipase must be stable in the organic solvent, which was the case for native CALB and the two mutants studied here. Secondly, water clusters that form and grow close to the active site must not block the path of substrate molecules into the active site. Thirdly, the lipase’s lid must not cover the active site. Finally, mutations and changes in aw must not disrupt the geometry of the active site. We show that mutating specific residues close to the active site can hinder water cluster formation and growth, making the lipase resistant to changes in aw. Our computational screening criteria could potentially be used to screen in-silico designed variants, so only promising candidates could be pushed forward to characterisation.

Solvent-dependent activity of Candida antarctica lipase B and its correlation with a regioselective mono aza-Michael addition - experimental and molecular dynamics simulation studies

With the aim of gaining understanding of the molecular basis of commercially available Candida antarctica lipase B (CALB) immobilized on polyacrylic resin catalyzed regioselective mono aza-Michael addition of Benzhydrazide to Diethyl maleate we decided to carry out molecular dynamics (MD) simulation studies in parallel with our experimental study. We found a correlation between the activity of CALB and the choice of solvent. Our study showed that solvent affects the performance of the enzyme due to the binding of solvent molecules to the enzyme active site region, and the solvation energy of substrates in the different solvents. We also found that CALB is only active in nonpolar solvent (i.e. Hexane), and therefore we investigated the influence of Hexane on the catalytic activity of CALB for the reaction. The results of this study and related experimental validation from our studies have been discussed here.

Construction of magnetic switchable Pickering interfacial catalysis system and its application in the hydrolysis of crude rice bran oil

SUMMARYThe high refining cost of traditional rice bran oil with high acid value and its large environmental impact have limited its further application. In this study, a lipase‐catalysed hydrolysis reaction of crude rice bran oil at the oil–water interface was performed using a Pickering emulsion system stabilised by modified magnetic microcrystalline cellulose (MMC) particles. The results revealed that the modified MMC particles exhibited a rod‐like structure, and a contact angle of 84.8°. In addition, the stability of the Pickering emulsion can be enhanced when the modified MMC was used as an emulsifier, and at an optimal addition amount of 2.0wt.%. The saturation magnetisation value of the emulsifier decreased from 38.8 to 8.9 emu g−1 after 12 reuses. Furthermore, the hydrolysis rates of crude rice bran oil in the Pickering emulsion system and a two‐phase system at a Candida antarctica lipase B (CALB) content of 2mg mL−1 were 92.8% and 58.8%, respectively. In addition, the relative activity of CALB after 10 reuses was 71.8%. These findings provide a sustainable interface biocatalysis method and provide ideas for the rational development of high acid value oils.

How to improve the efficiency of biocatalysis in non-aqueous pure deep eutectic solvents: A case study on the lipase-catalyzed transesterification reaction

Non-aqueous biocatalysis has been expanding the application fields of biocatalysis and is playing an increasingly important role in industrial biocatalysis. Deep eutectic solvents (DESs) have been recognized as green reaction media of non-aqueous biocatalysis. To provide guidance for improving the catalytic performance of enzymes in pure DESs, Candida antarctica lipase B (CALB) catalyzed transesterification reaction was chosen as the model reaction to assess the catalytic efficiency in different series of pure DESs. The catalytic efficiency of CALB was strongly influenced by the properties of DESs. Hydrophobic DESs caused less inactivation of CALB than hydrophilic DESs. The highest yield in hydrophobic DESs reached 56.75% after 12 h. The amount of carboxyl group in hydrophobic DESs, the amount of hydroxyl group in hydrophilic DESs, and the length of carbon chain had positive impacts on the catalytic efficiency of CALB in DESs. Learning from the CALB behaviors in DESs, a preliminary attempt in rational design DESs for higher transesterification efficiency was provided. Except for reaction media, the catalytic activity of CALB in DESs was improved by medium engineering means and enzyme engineering measures, such as adding a trace amount of water, imprinting CALB with pH buffer or inorganic salt, and protecting CALB by lyoprotectants. Synergistic effect of multiple activation methods could also be observed in improving the catalytic efficiency. This study can provide insights into expanding the application of DESs in non-aqueous biocatalysis.

Evaluation of Designed Immobilized Catalytic Systems: Activity Enhancement of Lipase B from Candida antarctica

Immobilized enzymatic catalysts are widely used in the chemical and pharmaceutical industries. As Candida antarctica lipase B (CALB) is one of the more commonly used biocatalysts, we attempted to design an optimal lipase-catalytic system. In order to do that, we investigated the enantioselectivity and lipolytic activity of CALB immobilized on 12 different supports. Immobilization of lipase on IB-D152 allowed us to achieve hyperactivation (178%) in lipolytic activity tests. Moreover, the conversion in enantioselective esterification increased 43-fold, when proceeding with lipase-immobilized on IB-S861. The immobilized form exhibited a constant high catalytic activity in the temperature range of 25 to 55 °C. Additionally, the lipase immobilized on IB-D152 exhibited a higher lipolytic activity in the pH range of 6 to 9 compared with the native form. Interestingly, our investigations showed that IB-S500 and IB-S60S offered a possibility of application in catalysis in both organic and aqueous solvents. A significant link between the reaction media, the substrates, the supports and the lipase was confirmed. In our enzymatic investigations, high-performance liquid chromatography (HPLC) and the titrimetric method, as well as the Bradford method were employed.

Screening of solvents for favoring hydrolytic activity of Candida antarctica Lipase B.

Lipases are a group of enzymes of considerable significance in organic synthesis, among which Candida antarctica lipase B (CALB) is one of the most widely studied enzymes. The activity of the biocatalyst has been intensively characterized in many organic media, but this paper aimed to compare the effect of 20 different solvents on the activity of CALB in the hydrolysis of p-nitrophenyl laurate. Nonpolar, polar aprotic, and polar protic solvents were used for enzyme pretreatment and then entered the composition of mixed solvents reaction medium. An impact of solvents on solvation processes affecting the catalysis steps, protein denaturation, and changes of its conformation was discussed. Moreover the hydrolytic activity of CALB with partition coefficient (logP) of the solvent used was correlated. It was emphasized that the substrate solubility plays an important role in solvent selection. In the presence of hydrophobic solvents, hydration layer becomes more hydrophobic facilitating the substrate access to the enzyme surface. In turn, polar compounds are good solvents for organic substrates facilitating the penetration of the aqueous layer that surrounds the surface of the enzyme. Two variants proved to be favorable for ester hydrolysis reaction: isooctane or polar solvent such as acetone, tert -butyl methyl ether, tert-butanol or acetonitrile.

How Candida antarctica lipase B can be activated in natural deep eutectic solvents: experimental and molecular dynamics studies

AbstractBACKGROUNDNatural deep eutectic solvents (NADESs), a class of green solvents which completely accords to 12 principles of green chemistry, have proven to have great potential applications in enzymatic reactions. Despite strong interest, the role of NADESs in these processes, and the molecular interaction between enzymes and NADESs, still remain ambiguous. In the present study, the stability and activity of Candida Antarctica lipase B (CALB) were studied, and the mechanism by which CALB was activated in NADESs was explored systematically from both molecular‐ and macroscopic‐scale perspectives.RESULTSThe results suggested that the activity of CALB in all NADESs was significantly higher than that of the control (ethanol). Moreover, the stability of CALB increased to 115.48 ± 1.36% and 108.54 ± 1.26% in betaine‐glycerin (B‐Gly) and choline chloride‐glycerol (C‐Gly), but decreased to 91.69 ± 3.26% and 92.31 ± 3.36% in betaine‐xylitol (B‐X) and choline chloride‐xylitol (C‐X), respectively. The results of circular dichroism (CD), fluorescence spectroscopy and molecular dynamics studies (MD) indicated that there was no significant change in the secondary structure of CALB. Furthermore, the results of MD provide some information supporting that CALB was stabilized by the hydrogen(H)‐bonding interaction between surface amino residues of CALB and NADESs and was activated via the H‐bonding interaction between substrate and NADESs in the acyl‐binding pocket.CONCLUSIONThe mechanism by which CALB was activated and stabilized via the H‐bonding interactions in NADESs was revealed in the present study. This provides a scientific basis from which to further explore the potential application of NADESs in enzymatic reactions in food engineering and health‐related fields. © 2019 Society of Chemical Industry

A Correlation between the Activity of Candida antarctica Lipase B and Differences in Binding Free Energies of Organic Solvent and Substrate

The ability of enzymes to operate in organic solvent is now widely accepted and is the basis for extensive research in enzymology. The challenge is to select the solvent media that allows the modulation of enzyme activity. For a rational selection of a solvent, it is necessary to understand the effect of organic solvent molecules on enzyme structure and the enzymatic reaction on a molecular level. To gain such insight, we combined experimental kinetics studies with full atomic molecular dynamics simulations and found a correlation between the activity of Candida antarctica lipase B (CALB) [for the esterification reaction between butyric acid and ethanol at a fixed water activity] and the binding of the solvent/substrate molecules in the active site region of CALB. We have investigated the influence of four organic solvents—hexane (HEX), methyl tertiary butyl ether (MTBE), acetonitrile (ACN), and tertiary butanol (TBU)—on the catalytic activity of CALB for the esterification reaction. The solvents have bee...

Activity modulation of core and shell in nanozeolite@enzyme bi-functional catalyst for dynamic kinetic resolution

A core–shell nanozeolite@enzyme bi-functional catalyst is prepared by using nanozeolite β as acidic core and immobilized Candida antarctica lipase B (CALB) as enzyme shell for the purpose of dynamic kinetic resolution (DKR), and polydiallyldimethylammonium chloride (PDDA) is used as interlayer to compart core and shell. The activities of core and shell in bi-functional catalyst are modulated to achieve the matching between racemization and kinetic resolution (KR) rates in DKR, i.e., a slow racemization rate on core while a fast KR rate on shell. Nanozeolite β with intermediate SiO2/Al2O3 ratio provides proper acid amount for racemization step. A relatively thick layer of PDDA not only improves the activity of CALB by its coverage for surface acidic sites but also limits the accessibility and diffusion of substrate towards the acidic core. The CALB shell with larger immobilized amount and higher enzyme activity offers enhanced driving force of DKR process, leading to higher conversion, selectivity and yield. The preparation and activity modulation of core–shell catalyst provide an ideal method to improve the catalytic performance of bi-functional catalyst.

A concentration-dependent effect of methanol on Candida antarctica lipase B in aqueous phase

Abstract The influence of methanol on enzyme is of great importance in enzymatic synthesis. The influence of methanol on the activity of Candida antarctica lipase B (CALB) upon hydrolysis of tributyrin was investigated by enzyme kinetics analysis. Dynamic light scattering (DLS) and circular dichroism (CD) was used to measure the variation of hydrodynamic diameter and secondary structure change of CALB at different methanol concentration, respectively. A concentration-dependent influence of methanol on CALB was observed. The activity of CALB slightly increased with less than 1.2 mol l −1 of methanol, followed by sustained decrease at higher methanol concentrations. DLS result indicated that the negative effect of methanol on CALB was supposed to follow different mechanisms due to the significant variation of particle size of CALB at different methanol concentrations. Both reversible competitive inhibition based on enzymatic kinetics analysis and inactivation based on CD spectroscopy study were supposed to contribute to the negative effect of methanol on CALB with methanol concentration in the range of 1.2 mol l −1 to 6 mol l −1 . And after arriving at percolation threshold concentration of 6 mol l −1 , methanol was mainly performed as aggregate and precipitate agent for CALB, which resulted in low effective enzyme concentration in the solution.

The relationship between enhanced enzyme activity and structural dynamics in ionic liquids: a combined computational and experimental study

Candida antarctica lipase B (CALB) is an efficient biocatalyst for hydrolysis, esterification, and polymerization reactions. In order to understand how to control enzyme activity and stability we performed a combined experimental and molecular dynamics simulation study of CALB in organic solvents and ionic liquids (ILs). Our results demonstrate that the conformational changes of the active site cavity are directly related to enzyme activity and decrease in the following order: [Bmim][TfO] > tert-butanol > [Bmim][Cl]. The entrance to the cavity is modulated by two isoleucines, ILE-189 and ILE-285, one of which is located on the α-10 helix. The α-10 helix can substantially change its conformation due to specific interactions with solvent molecules. This change is acutely evident in [Bmim][Cl] where interactions of LYS-290 with chlorine anions caused a conformational switch between α-helix and turn. Disruption of the α-10 helix structure results in a narrow cavity entrance and, thus, reduced the activity of CALB in [Bmim][Cl]. Finally, our results show that the electrostatic energy between solvents in this study and CALB is correlated with the structural changes leading to differences in enzyme activity.

Functional Motions of Candida antarctica Lipase B: A Survey through Open-Close Conformations

Candida antarctica lipase B (CALB) belongs to psychrophilic lipases which hydrolyze carboxyl ester bonds at low temperatures. There have been some features reported about cold-activity of the enzyme through experimental methods, whereas there is no detailed information on its mechanism of action at molecular level. Herein, a comparative molecular dynamics simulation and essential dynamics analysis have been carried out at three temperatures (5, 35 and 50°C) to trace the dominant factors in the psychrophilic properties of CALB under cold condition. The results clearly describe the effect of temperature on CALB with meaningful differences in the flexibility of the lid region (α5 helix), covering residues 141–147. Open- closed conformations have been obtained from different sets of long-term simulations (60 ns) at 5°C gave two reproducible distinct forms of CALB. The starting open conformation became closed immediately at 35 and 50°C during 60 ns of simulation, while a sequential open-closed form was observed at 5°C. These structural alterations were resulted from α5 helical movements, where the closed conformation of active site cleft was formed by displacement of both helix and its side chains. Analysis of normal mode showed concerted motions that are involved in the movement of both α5 and α10 helices. It is suggested that the functional motions needed for lypolytic activity of CALB is constructed from short-range movement of α5, accompanied by long-range movement of the domains connected to the lid region.

Analysis of the Conformational Stability and Activity of Candida antarctica Lipase B in Organic Solvents: INSIGHT FROM MOLECULAR DYNAMICS AND QUANTUM MECHANICS/SIMULATIONS

The conformational stability and activity of Candida antarctica lipase B (CALB) in the polar and nonpolar organic solvents were investigated by molecular dynamics and quantum mechanics/molecular mechanics simulations. The conformation change of CALB in the polar and nonpolar solvents was examined in two aspects: the overall conformation change of CALB and the conformation change of the active site. The simulation results show that the overall conformation of CALB is stable in the organic solvents. In the nonpolar solvents, the conformation of the active site keeps stable, whereas in the polar solvents, the solvent molecules reach into the active site and interact intensively with the active site. This interaction destroys the hydrogen bonding between Ser(105) and His(224). In the solvents, the activation energy of CALB and that of the active site region were further simulated by quantum mechanics/molecular mechanics simulation. The results indicate that the conformation change in the region of active sites is the main factor that influences the activity of CALB.

Influence of alcohol treatments on the activity of lipases immobilized on methyl-modified silica aerogels

The effects of alcohol treatment on the activity and loading amount of Candida rugosa lipase (CRL), Candida Antarctica lipase B (CALB) and Porcine Pancreas lipase (PPL) immobilized on methyl-modified silica aerogels were investigated, and the fluorescent analysis was used to explore the change of lipase hydrophobicity in aqueous solution caused by alcohols. It is found that alcohol types and the stages at which alcohol was added significantly influenced the performance of immobilized lipases through changing the hydrophobicity of the molecules. For CRL and PPL, five kinds of alcohol were added in the adsorption process, and n-butanol and isopropanol improved the apparent activity of CRL and PPL up to 2.5 times and 2 times those of the untreated ones, respectively; however, for CALB, it is better to activate the immobilized CALB after the adsorption process, and the apparent activity of CALB increased up to 2.76 times through n-butanol treatment.

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the alpha-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins alpha-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with alpha-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with alpha-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using alpha-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with alpha-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.

Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems

Candida antarctica lipase B (CALB) carrying a point mutation, N74S, resulting in a non-glycosylated protein was actively expressed in Pichia pastoris yielding 44 mg/L which was similar to that of the glycosylated CALB wild type expressed in P. pastoris. Hence, the major obstacle in the Escherichia coli expression of CALB is not the lack of glycosylation. To understand and improve the expression of CALB in E. coli, a comprehensive investigation of four different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3) and Origami 2(DE3) as well as co-expression with chaperones groES and groEL in Origami B(DE3), all using the pET-22b(+) vector and the T7 lac promoter. Furthermore the E. coli expression was carried out at three different temperatures (16, 25 and 37 °C) to optimise the expression. Periplasmic expression resulted in highest amount of active CALB of the four systems, yielding a maximum of 5.2 mg/L culture at 16 °C, which is an improvement to previous reports. The specific activity of CALB towards tributyrin in E. coli was found to be the same for periplasmic and cytosolic expression. Active site titration showed that the CALB mutant N74S had a lower specific activity in comparison to wild type CALB regardless of expression host. The expected protein identity was confirmed by LC–ESI-MS analysis in E. coli, whereas in P. pastoris produced CALB carried four additional amino acids from an incomplete protein processing.

Candida antarctica Lipase B Chemically Immobilized on Epoxy-Activated Micro- and Nanobeads: Catalysts for Polyester Synthesis

Candida antarctica Lipase B (CALB) was covalently immobilized onto epoxy-activated macroporous poly(methyl methacrylate) Amberzyme beads (235 microm particle size, 220 A pore size) and nanoparticles (nanoPSG, diameter 68 nm) with a poly(glycidyl methacrylate) outer region. Amberzyme beads allowed CALB loading up to 0.16 g of enzyme per gram of support. IR microspectroscopy generated images of Amberzyme-CALB beads showed CALB is localized within a 50 microm thick loading front. IR microspectroscopy images, recorded prior to and after treatment of Amberzyme-CALB with DMSO/aqueous Triton X-100, are similar, confirming that CALB is largely chemically linked to Amberzyme. The activity of CALB immobilized on Amberzyme, Lewatit (i.e., Novozym 435 catalyst), and nanoPSG was assessed for lactone ring-opening and step-condensation polymerizations. For example, the percent conversion of -caprolactone using the same amount of enzyme catalyzed by Amberzym-CALB, Novozym 435, and nanoPSG-CALB for 20 min was 7.0, 16, and 65%, respectively. Differences in CALB reactivity were discussed based on resin physical parameters and availability of active sites determined by active site titrations. Regardless of the matrix used and chemical versus physical immobilization, -CL ring-opening polymerizations occur by a chain growth mechanism without chain termination. To test Amberzyme-CALB stability, the catalyst was reused over three reaction cycles for -CL ring-opening polymerization (70 degrees C, 70 min reactions) and glycerol/1,8-octanediol/adipic acid polycondensation reactions (90 degrees C, 64 h). Amberzyme-CALB was found to have far better stability for reuse relative to Novozym 435 for the polycondensation reaction.

The effect of ionic liquid media on activity, selectivity and stability of Candida antarctica lipase B in transesterification reactions

Nineteen different 1,3-dialkylimidazolium-based ionic liquids (ILs) were used as reaction media for the synthesis of butyl butyrate by transesterification from vinyl butyrate and 1-butanol catalyzed by Candida antarctica lipase B (CaLB). The reaction was also carried out in hexane as a reference solvent. In all the water-immiscible ILs assayed, the enzymatic activity and selectivity were higher than that obtained in hexane. However, in water-miscible ILs, the activity was lower than in the reference solvent, although they showed >99.99% selectivity. Two solvent properties, hydrophobicity and nucleophilicity, were considered key parameters for analyzing the behavior of CaLB in ILs. In the case of ILs based on the same anion, the synthetic activity was gradually enhanced by increasing cation hydrophobicity. Furthermore, the activity of CaLB was greater in ILs containing anions of lower nucleophilicity. Stability studies indicate that CaLB exhibited greater stability in water-immiscible ILs than in water-miscible ILs.

Structural evolution and stability of sol–gel biocatalysts

Immobilisation strategies for catalytic enzymes are important as they allow recovery and reuse of the biocatalysts. In this work, sol–gel matrices have been used to immobilise Candida antarctica lipase B (CALB), a commonly used industrial enzyme. The sol–gel bioencapsulate is produced through fluoride-catalysed hydrolysis of mixtures of tetramethylorthosilicate (TMOS) and methyltrimethoxysilane (MTMS) in the presence of CALB, yielding materials with controlled pore sizes and surface chemistries. Sol–gel matrices prolong the catalytic life and enhance the activity of CALB, although the molecular basis for this effect has yet to be elucidated due to the limitations of analytical techniques applied to date. Small angle neutron scattering (SANS) allows such multi-component systems to be characterised through contrast matching. In the sol–gel bioencapsulate system at the contrast match point for silica, residual scattering intensity is due to the CALB and density fluctuations in the matrix. A SANS contrast variation series found the match point for the silica matrix, both with and without enzyme present, to be around 35%. The model presented here proposes a mechanism for the interaction between CALB and the surrounding sol–gel matrix, and the observed improvement in enzyme activity and matrix strength. Essentially, the inclusion of CALB modulates silicate speciation during evolution of the inorganic network, leading to associated variations in SANS contrast. The SANS protocol developed here may be applied more generally to other encapsulated enzyme systems.

The amidase activity of Candida antarctica lipase B is dependent on specific structural features of the substrates

The hydrolytic activity of Candida antarctica lipase B ( CAL-B) was studied using 15 amides with different linear saturated acyl residues and substituents in the aromatic amine. A strong dependence of the hydrolysis rate on the length of the acyl residue and the substituents groups in the aromatic ring of the amides was demonstrated, with the highest hydrolytic initial reaction rates found for the C 10 acyl derivates and benzylamides. The C 10 benzylamide, an amide without substituents in the aromatic ring was hydrolyzed almost as fast as capsaicin and five times faster that the corresponding C 10 vanillyl derivative. Therefore, a benzylamide bearing the non-linear unsaturated acyl residue of capsaicin (8-methyl-6-nonanenoic acid) was synthesized. This substrate was hydrolyzed four times faster than capsaicin. Although it has been widely claimed that lipases rarely display amidase activity, with this contribution we demonstrate that the amidase activity of CAL-B is dependent on the structural features of the substrate.