Candida Antarctica 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.

Biocatalytic synthesis of oxadiazole thioethers and evaluation of their antitumor activity.

A multicomponent enzyme-catalyzed process is suggested for the synthesis of a novel series of 1,3,4-oxadiazole thioether derivatives with yields ranging from 65 to 94%. Novozym 435, the immobilized form of Candida antarctica lipase B (CALB), was found to efficiently catalyze the reaction. The products were evaluated for antitumor activities against two cancer cell lines, HT-29 (human colorectal cancer cell) and HepG2 (human liver cancer cell), by MTT assays. Among them, two compounds exhibited higher antitumor activities, for both cell lines, compared to doxorubicin. In silico molecular docking and computational ADME analysis were performed to propose a mode of action for the anti-cancer activities and to predict drug-likeness, respectively.

Metagenomic discovery of lipases with predicted structural similarity to Candida antarcticalipase B.

Here we employed sequence-based and structure-based screening for prospecting lipases that have structural homolog to Candida antarctica lipase B (CalB). CalB, a widely used biocatalyst, was used as structural template reference because of its enzymatic properties. Structural homolog could aid in the discovery of novel wild-type enzymes with desirable features and serve as a scaffold for further biocatalyst design. The available metagenomic data isolated from various environments was leveraged as a source for bioprospecting. We identified two bacteria lipases that showed high structural similarity to CalB with <40% sequence identity. Partial purification was conducted. In comparison to CalB, the enzymatic characteristics of two potential lipases were examined. A candidate exhibited optimal pH of 8 and temperature of 50°C similar to CalB. The second lipase candidate demonstrated an optimal pH of 8 and a higher optimal temperature of 55°C. Notably, this candidate sustained considerable activity at extreme conditions, maintaining high activity at 70°C or pH 9, contrasting with the diminished activity of CalB under similar conditions. Further comprehensive experimentation is warranted to uncover and exploit these novel enzymatic properties for practical biotechnological purposes.

Chemo- and regio-selective enzymatic lipophilisation of rutin, and physicochemical and antioxidant properties of rutin ester derivatives.

Enzymes are one of the most powerful tools in organic Green Chemistry and enzymatic reactions offer numerous advantages like regio- and enantio-selectivity along with their eco-friendly and sustainable nature. More specifically, lipases can catalyse both ester hydrolysis and formation depending on the nature of the substrate and water content. Herein, the focus is on the development of an enzymatically catalysed lipophilisation of natural compounds using lipases of microbial origin and the investigation of the optimal reaction conditions, aiming ultimately to ameliorate the compounds' properties. The flavonoid disaccharide rutin (quercetin-3-O-rutinoside) was the model compound on which the acylation protocol was built, allowing an efficient procedure to be established, while simultaneously offering the possibility of developing rapid, clear and robust methodologies, using state-of-the-art techniques, for analysis and purification of the synthesized compounds. An optimal 72 h reaction at 55 °C, using Candida antarctica lipase B immobilized on acrylic resin, combined with silicon dioxide as dehydrating agent, followed by product purification, achieved conversion ratios up to 50%. Full characterization and evaluation of the physicochemical and antioxidant properties of the esterified compounds was obtained. The lipophilicity of the rutin esters produced increased with increasing alkyl chain length, yet antioxidant properties were unaffected in comparison with the parent compound. A preparatively useful acylation protocol was established, allowing full investigation into the properties of the acylated compounds. It is also applicable for use on mixtures of compounds as most natural products are found in nature in mixtures and such a development greatly enhances the potential of this method for future commercial applications.

Enzymatically catalyzed furan-based copolyesters containing dilinoleic diol as a building block.

A more environmentally friendly method for creating sustainable alternatives to traditional aromatic-aliphatic polyesters is a valuable step towards resource-efficiency optimization. A library of furan-based block copolymers was synthesized via temperature-varied two-step polycondensation reaction in diphenyl ether using Candida antarctica lipase B (CAL-B) as a biocatalyst where dimethyl 2,5-furandicarboxylate (DMFDCA), α,ω-aliphatic linear diols (α,ω-ALD), and bio-based dilinoleic diol (DLD) were used as the starting materials. Nuclear magnetic spectroscopy (1H and 13C NMR), Fourier transform spectroscopy (FTIR) and size exclusion chromatography (SEC) were used to analyze the resulting copolymers. Additionally, crystallization behavior and thermal properties were studied using X-ray diffraction (XRD), digital holographic microscopy (DHM), and differential scanning microscopy (DSC). Finally, oxygen transmission rates (OTR) and dynamic mechanical analysis (DMTA) of furan-based copolyesters indicated their potential for medical packaging.

Incrementing MCT Character of Coconut Oil Using Enzyme Catalyzed Interesterification.

The fatty acid composition of coconut oil was modified using enzyme catalyzed interesterification with the aim of obtaining a product more alike to commercial MCT oils. This modification was carried out with the aim to obtain a product with some of the health benefits shown by MCT oils. Initially, lipase B from Candida antarctica immobilized on acrylic resin and lipozyme TL IM were tested as enzyme catalysts for the reaction. The enzyme catalysts have shown similar performance and lipozyme TL IM has been chosen as the catalyst based on its lower cost. The effects of reaction time, oil to methyl octanoate ratio, and enzyme loading on the reaction performance have been investigated with response surface methodology (RSM) utilizing the Box-Behnken approach. The optimized reaction was scaled up to 20 g. The possibility to source the medium chain fatty acid esters from coconut oil fatty acid distillate using a simple procedure was demonstrated and the possibility to use these esters for the interesterification of coconut oil has been demonstrated as well.

Yeast Lipid Produced through Glycerol Conversions and Its Use for Enzymatic Synthesis of Amino Acid-Based Biosurfactants

The aim of the present work was to obtain microbial lipids (single-cell oils and SCOs) from oleaginous yeast cultivated on biodiesel-derived glycerol and subsequently proceed to the enzymatic synthesis of high-value biosurfactant-type molecules in an aqueous medium, with SCOs implicated as acyl donors (ADs). Indeed, the initial screening of five non-conventional oleaginous yeasts revealed that the most important lipid producer was the microorganism Cryptococcus curvatus ATCC 20509. SCO production was optimised according to the nature of the nitrogen source and the initial concentration of glycerol (Glyc0) employed in the medium. Lipids up to 50% w/w in dry cell weight (DCW) (SCOmax = 6.1 g/L) occurred at Glyc0 ≈ 70 g/L (C/N ≈ 80 moles/moles). Thereafter, lipids were recovered and were subsequently used as ADs in the N-acylation reaction catalysed by aminoacylases produced from Streptomyces ambofaciens ATCC 23877 under aqueous conditions, while Candida antarctica lipase B (CALB) was used as a reference enzyme. Aminoacylases revealed excellent activity towards the synthesis of acyl-lysine only when free fatty acids (FAs) were used as the AD, and the rare regioselectivity in the α-amino group, which has a great impact on the preservation of the functional side chains of any amino acids or peptides. Aminoacylases presented higher α-oleoyl-lysine productivity and final titer (8.3 g/L) with hydrolysed SCO than with hydrolysed vegetable oil. The substrate specificity of both enzymes towards the three main FAs found in SCO was studied, and a new parameter was defined, viz., Specificity factor (Sf), which expresses the relative substrate specificity of an enzyme towards a FA present in a FA mixture. The Sf value of aminoacylases was the highest with palmitic acid in all cases tested, ranging from 2.0 to 3.0, while that of CALB was with linoleic acid (0.9-1.5). To the best of our knowledge, this is the first time that a microbial oil has been successfully used as AD for biosurfactant synthesis. This bio-refinery approach illustrates the concept of a state-of-the-art combination of enzyme and microbial technology to produce high-value biosurfactants through environmentally friendly and economically sound processes.

Tuning Selectivity in CalA Lipase: Beyond Tunnel Engineering.

Engineering studies of Candida (Pseudozyma) antarctica lipase A (CalA) have demonstrated the potential of this enzyme in the selective hydrolysis of fatty acid esters of different chain lengths. CalA has been shown to bind substrates preferentially through an acyl-chain binding tunnel accessed via the hydrolytic active site; it has also been shown that selectivity for substrates of longer or shorter chain length can be tuned, for instance by modulating steric hindrance within the tunnel. Here we demonstrate that, whereas the tunnel region is certainly of paramount importance for substrate recognition, residues in distal regions of the enzyme can also modulate substrate selectivity. To this end, we investigate variants that carry one or more substitutions within the substrate tunnel as well as in distal regions. Combining experimental determination of the substrate selectivity using natural and synthetic substrates with computational characterization of protein dynamics and of tunnels, we deconvolute the effect of key substitutions and demonstrate that epistatic interactions contribute to procuring selectivity toward either long-chain or short/medium-chain fatty acid esters. We demonstrate that various mechanisms contribute to the diverse selectivity profiles, ranging from reshaping tunnel morphology and tunnel stabilization to obstructing the main substrate-binding tunnel, highlighting the dynamic nature of the substrate-binding region. This work provides important insights into the versatility of this robust lipase toward diverse applications.

Stereoselective Synthesis of γ‐(Acyloxy)carboxylic Acids and γ‐Lactones Featuring the Switch of Stereopreference of Candida antarctica Lipase B in Sodium γ‐Hydroxycarboxylate Homologues

AbstractScalable protocols of straightforward synthesis of enantiomeric γ‐(acyloxy)carboxylic acids and γ‐lactones are presented. The key step is lipase‐catalyzed stereoselective acylation of γ‐hydroxycarboxylic acid sodium salt in organic solvent followed by acidification of the product, extraction and acidic relactonization of the unreacted enantiomer. The mixture of γ‐(acyloxy)carboxylic acid and γ‐lactone is separated either by extraction with solution of sodium bicarbonate or by distillation. A switch of enantioinduction of Candida antarctica lipase B along homologous nucleophiles from R configuration of γ‐hydroxyhexanoic acid salt to S configuration of the C7 and longer‐chain homologues has been disclosed. Both enantiomers of γ‐(acyloxy)pentanoic acids; γ‐(acetyloxy)octanoic and ‐nonanoic acids with S configuration; [(1S,5R)‐5‐(chloroacetyloxy)cyclopent‐2‐en‐1‐yl]acetic acid and enantiomeric γ‐lactones derived from them were prepared with e. r. &gt;98.5/1.5. The rates of acylation of C5 to C9 homologous salts differ by three orders of magnitude but remain applicable for preparative synthesis by variation of the enzyme loading and reaction time.

Chemoenzymatic Protocol for the Synthesis of Enantiopure β-Blocker (S)-Bisoprolol

The β-blocker (S)-bisoprolol hemifumarate has been synthesised in 96% enantiomeric excess with 19% total yield in a six-step synthesis. A transesterification reaction of the racemic chlorohydrin 1-chloro-3-(4-((2-isopropoxyethoxy)methyl)phenoxy)propan-2-ol catalysed by lipase B from Candida antarctica resulted in the R-chlorohydrin in high enantiomeric purity. Reaction of this building block with isopropylamine in methanol gave (S)-bisoprolol, and further reaction with fumaric acid gave (S)-bisoprolol fumarate in 96% ee. Specific rotation value confirmed the absolute configuration of the enantiopure drug.

Chemo-Enzymatic Synthesis of Enantiopure β-Antagonist (S)-Betaxolol

The β-blocker (S)-betaxolol has been synthesized in 99% enantiomeric excess (ee) from the commercially available precursor 4-(2-hydroxyethyl)phenol. The racemic chlorohydrin 1-chloro-3-(4-(2-(cyclopropylmethoxy)ethyl)phenoxy)propan-2-ol was esterified with vinyl acetate catalyzed by lipase B from Candida antarctica, which gave the R-chlorhydrin (R)-1-chloro-3-(4-(2-(cyclopropylmethoxy)ethyl)phenoxy)propan-2-ol in 99% ee with 38% yield. The enantiomeric excess of the R-chlorohydrin was retained in an amination reaction with isopropylamine in methanol to yield (S)-betaxolol in 99% ee and with 9% overall yield. We are under way to improve the yield.

Tailored Particle Catalysts for Multistep One‐pot Chemoenzymatic Cascade in Pickering Emulsions

AbstractChemoenzymatic cascades are an important tool for advanced synthesis in chemistry. However, these cascades are often limited due to the incompatibility issue between two distinct catalysts and reactions. To address this issue, we present a simple multistep one‐pot platform, in which nanoparticle catalysts are prepared to allow chemo‐ and biocatalytic reactions performed sequentially in water and Pickering emulsions. The preparation of particle catalysts is accomplished in just two steps by polymer modifications and [RuCl2(p‐cymene)]2 coordination, while the benefits of using them for chemoenzymatic synthesis are multifaceted. They act not only as asymmetric catalysts for asymmetric transfer hydrogenation from acetophenone to 1‐phenylethanol in water with up to 99 % conversion and 93 % ee, but also as an emulsifier to form stable Pickering emulsions. By the addition of Candida antarctica lipase B into the emulsions, the second‐step reaction of enantioselective acylation was achieved with 38 % conversion and 99 % ee. Therefore, we successfully present a simple method to enable chemoenzymatic cascades by combining particle catalysts and enzymes in water and Pickering emulsions in a sequential fashion, which can be generalized for other cascade syntheses with different chemo‐ and biocatalysts in the future.

Microencapsulation of Lipases Produced by Dripping and Jet Break-Up for Biodiesel Production

A high-performance and scalable lipase immobilization method using a dipping and jet break-up technique was reported for the production of microcapsule biocatalysts with an entrapped cascade of lipase enzyme. The lipase from Candida antarctica (CALb) recombinant Aspergillus oryzae and from the vegetal of Jatropha curcas L. (var. Sevangel) in Morelos State of Mexico were entrapped by mixing with a sodium alginate biopolymer at different concentrations. The obtained microcapsules were hardened in a CaCl2 solution, aiming at developing Ca2+ alginate microbeads with sizes mostly from 220 to 300 μm. The relationship between the process variables with the shape and size of the alginate drops before and after the gelation was established with aid of optical image analysis. The results showed that a critical Ohnesorge number (Oh) &gt; 0.24 was required to form spherical microencapsulated beads. The biodiesel production via esterification/transesterification reaction was performed using the crude Jatropha curcas L. oil as feedstock in a batch reactor using lipase microcapsules as biocatalysts. Under the optimal reaction condition (ethanol-to-oil mass ratio: 10; water content 9.1 wt%, microencapsulated biocatalyst mass: 5.25 g, reaction temperature: 35 °C, pH of reaction mixture 7.5, stirring force 6 g), an approximately 95% fatty acid ethyl esters (FAEE) yield could be obtained. The biodiesel obtained from this work completely satisfied with the related ASTM D6751 and EN14214 standards. The microencapsulation technique reported herein allows the production of lipase microcapsules on a continuous large scale with the characteristics required for sustainable biofuel production and it can be also applied in other fields such as food processing and the pharmaceutical industry.

Chemoenzymatic Synthesis of Optically Active Alcohols Possessing 1,2,3,4-Tetrahydroquinoline Moiety Employing Lipases or Variants of the Acyltransferase from Mycobacterium smegmatis

The enzymatic kinetic resolution (EKR) of racemic alcohols or esters is a broadly recognized methodology for the preparation of these compounds in optically active form. Although EKR approaches have been developed for the enantioselective transesterification of a vast number of secondary alcohols or hydrolysis of their respective esters, to date, there is no report of bio- or chemo-catalytic asymmetric synthesis of non-racemic alcohols possessing 1,2,3,4-tetrahydroquinoline moiety, which are valuable building blocks for the pharmaceutical industry. In this work, the kinetic resolution of a set of racemic 1,2,3,4-tetrahydroquinoline-propan-2-ols was successfully carried out in neat organic solvents (in the case of CAL-B and BCL) or in water (in the case of MsAcT single variants) using immobilized lipases from Candida antarctica type B (CAL-B) and Burkholderia cepacia (BCL) or engineered acyltransferase variants from Mycobacterium smegmatis (MsAcT) as the biocatalysts and vinyl acetate as irreversible acyl donor, yielding enantiomerically enriched (S)-alcohols and the corresponding (R)-acetates with E-values up to 328 and excellent optical purities (&gt;99% ee). In general, higher ee-values were observed in the reactions catalyzed by lipases; however, the rates of the reactions were significantly better in the case of MsAcT-catalyzed enantioselective transesterifications. Interestingly, we have experimentally proved that enantiomerically enriched 1-(7-nitro-3,4-dihydroquinolin-1(2H)-yl)propan-2-ol undergoes spontaneous amplification of optical purity under achiral chromatographic conditions.

Optimised Protocol for Drying Aqueous Enzyme Solutions in Organic Solvents – Comparison of Free and Immobilised Candida antarctica Lipase B

AbstractHere, we propose an optimised protocol for controlling the initial water activity (aw) in organic reaction mixtures with soluble lipase to ensure reproducible and consistent enzyme activity measurements. Pre‐equilibration above saturated salt solutions was used to set aw of the reaction media, where aw was tracked with a hygrometer. Consistent stirring of the suspension and volume correction to compensate for solvent evaporation were found to be critical. The protocol was tested on Candida antarctica lipase B (CALB) in soluble and immobilised forms in three organic solvents (acetonitrile, methyl tert‐butyl ether, and hexane) at four different water activities ranging from 0.12 to 0.97. Soluble and immobilised CALB had similar aw‐profiles and showed the highest enzyme activity in hexane. The optimised protocol expands the possibility to study enzyme kinetics from immobilised enzymes to soluble enzymes.

Synthesis of organic-inorganic hybrid nanoflowers of lipases from Candida antarctica type B (CALB) and Thermomyces lanuginosus (TLL): Improvement of thermal stability and reusability

Enzyme immobilization is used to improve the application of enzymes, allowing the reuse of biocatalysts and increasing their stability under reaction conditions. Immobilization of enzymes through structures, such as nanoflowers, is an innovative, simple, and low-cost method compared to other techniques. In this context, the main objective of this work is to synthesize hybrid biocatalytic nanostructures, similar to flowers, of lipases from Candida antarctica type B (CALB) and Thermomyces lanuginosus (TLL). The production of nanoflowers occurred by precipitation of lipases with CuCl2 or CuSO4 salts for 72 h. However, challenges and obstacles were faced in obtaining effective and practical nanoflowers, such as nanoflowers’ low thermal stability and reusability. To overcome these challenges, two conditions were tested: nanoflowers cross-linked with glutaraldehyde and nanoflowers and nanoparticles cross-linked with glutaraldehyde. This last biocatalyst prepared by CuSO4 precipitation showed better thermal stability (half-life about 230 and 233 min for CALB and TLL, respectively, under incubation at 60 °C and pH 7). The CALB biocatalyst retained 70 % of its initial activity (2.31 U) after 10 cycles of hydrolysis. Therefore, this work shows not only the problems and barriers of nanoflowers synthesis, but also the possibility of producing more stable and efficient biocatalysts using improved protocols.

Efficient Activity Enhancement of a Lipase from Sporisorium reilianum for the Synthesis of a Moxifloxacin Chiral Intermediate via Rational Design

Lipase-catalyzed stereoselective resolution of cis -(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate ( cis -(±)-1) is an attractive route for the synthesis of ( S,S )-2,8-diazobicyclo[4.3.0]nonane, an important chiral intermediate of the fluoroquinolone antibiotic, moxifloxacin. In our previous study, a lipase from Sporisorium reilianum (SRL) was identified to possess excellent thermostability and pH stability. However, the low enzymatic activity of the SRL is a challenge that must be addressed. A rational design was initially employed for SRL tailoring according to the engineered Candida antarctica lipase B (CALB), resulting in a beneficial variant called SRL-I194N/V195L. Subsequently, two key amino acid residues in loop 6, L145 and L154, which might modulate the lid conformation between open and closed, were identified. A tetra-site variant, SRL-I194N/V195L/L145V/L154G (V13), with a significantly enhanced activity of 87.8 U∙mg −1 was obtained; this value was 2195-fold higher than that of wild-type SRL. Variant V13 was used to prepare optically pure (2 S ,3 R )-dimethyl 1-acetylpiperidine-2,3-dicarboxylate ((2 S ,3 R )-1), resolving 1 mol∙L −1 cis -(±)-1 with a conversion of 49.9% in 2 h and absolute stereoselectivity ( E > 200). Excellent stability with a half-life of 92.5 h was also observed at 50 °C. Overall, the study findings reveal a lipase with high activity toward cis -(±)-1 at an industrial level and may offer a general strategy for enhancing the enzyme activity of other lipases and other classes of enzymes with a lid moiety.

Vitamin E-loaded polymeric nanoparticles from biocompatible adipate-based copolymer obtained using the nanoprecipitation method

An enzymatic synthesis of new aliphatic biopolyesters - poly(butylene adipate)-co-(dilinol adipate) (PBA-DLA) with the use of monomers obtained from natural sources was carried out. Lipase B from the strain Candida antarctica (CALB) was used as a biocatalyst. The chemical structure of the PBA-DLA copolyester was confirmed by 1H NMR. In vitro biological studies did not show its cytotoxic effect on mouse fibroblast cells. PBA-DLA nanoparticles obtained in a single-stage nanoprecipitation process were used to encapsulate hydrophobic α-tocopherol (α-TP), the main component of vitamin E, obtaining an encapsulation efficiency of 48−74% (EE%) depending on the concentration of α-TP (2.5 ; 5; 10 mg/ml), which was confirmed by dynamic light scattering (DLS) analysis, ultraviolet spectroscopy (UV-VIS) and 1H NMR.

Synthesis of Ibuprofen Monoglyceride Using Novozym®435: Biocatalyst Activation and Stabilization in Multiphasic Systems

This work was focused on the enzymatic esterification of glycerol and ibuprofen at high concentrations in two triphasic systems composed of toluene+ibuprofene (apolar) and glycerol or glycerol–water (polar) liquid phases, and a solid phase with the industrial immobilized lipase B from Candida antarctica named Novozym®435 (N435) acting as the biocatalyst. Based on a preliminary study, the concentration of the enzyme was set at 30 g·L−1 and the stirring speed at 720 r.p.m to reduce external mass transfer limitations. To obtain more information on the reaction system, it was conducted at a wide range of temperatures (50 to 80 °C) and initial concentrations of ibuprofen (20–100 g·L−1, that is, 97 to 483 mM). Under these experimental conditions, the external mass transfer, according to the Mears criterion (Me = 1.47–3.33·10−4 &lt;&lt; 0.15), was fast, presenting no limitation to the system productivity, regardless of the presence of water and from 50 to 80 °C. Considering that the enzyme is immobilized in a porous ion-exchange resin, limitations due to internal mass transfer can exist, depending on the values of the effectiveness factor (η). It varied from 0.14 to 0.23 at 50 to 80 °C and 0.32–1 mm particle diameter range in the absence of water, and in the same ranges, from 0.40 to 0.66 in the presence of 7.4% w/w water in the glycerol phase. Thus, it is evident that some limitation occurs due to mass transfer inside the pores, while the presence of water in the polar phase increases the productivity 3–4 fold. During the kinetic study, several kinetic models were proposed for both triphasic reacting systems, with and without first-order biocatalyst deactivation, and their fit to all relevant experimental data led to the observation that the best kinetic model was a reversible hyperbolic model with first-order deactivation in the anhydrous reaction system and a similar model, but without deactivation, for the system with added water at zero time. This fact is in sharp contrast to the use of N435 in a water-glycerol monophasic system, where progressive dissolution of ibuprofen in the reacting media, together with a notable enzyme deactivation, is observed.

Immobilization of Lipase B from Candida antarctica in Octyl-Vinyl Sulfone Agarose: Effect of the Enzyme-Support Interactions on Enzyme Activity, Specificity, Structure and Inactivation Pathway.

Lipase B from Candida antarctica was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and p-nitrophenol butyrate (pNPB) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (pNPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different.