Crystal structures of a novel family IV esterase in free and substrate-bound form.

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme-substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV. DATABASE: Structural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo-PtEst1) and 6Z69 (PtEst1-inhibitor complex).

Hepatic lipase (LIPC) sequencing in individuals with extremely high and low high-density lipoprotein cholesterol levels.

Common variants in the hepatic lipase (LIPC) gene have been shown to be associated with plasma lipid levels; however, the distribution and functional features of rare and regulatory LIPC variants contributing to the extreme lipid phenotypes are not well known. This study was aimed to catalogue LIPC variants by resequencing the entire LIPC gene in 95 non-Hispanic Whites (NHWs) and 95 African blacks (ABs) with extreme HDL-C levels followed by in silico functional analyses. A total of 412 variants, including 43 novel variants were identified; 56 were unique to NHWs and 234 were unique to ABs. Seventy-eight variants in NHWs and 89 variants in ABs were present either in high HDL-C group or low HDL-C group. Two non-synonymous variants (p.S289F, p.T405M), found in NHWs with high HDL-C group were predicted to have damaging effect on LIPC protein by SIFT, MT2 and PP2. We also found several non-coding variants that possibly reside in the circRNA and lncRNA binding sites and may have regulatory potential, as identified in rSNPbase and RegulomeDB databases. Our results shed light on the regulatory nature of rare and non-coding LIPC variants as well as suggest their important contributions in affecting the extreme HDL-C phenotypes.

Carbon nanotube-lipase hybrid nanoflowers with enhanced enzyme activity and enantioselectivity.

Various nanoflowers are synthesized as supports for different methods of enzyme immobilization; however, the activities of these immobilized enzymes are limited because of their confinement in the nanoflowers. In order to increase the performance of nanoflowers, in this study, different protein-phosphate hybrid nanostructures were successfully synthesized and further enhanced by carbon nanotubes (CNTs) under the same conditions. Only Cu3(PO4)2 complex nanostructures exhibited flower-like structures and showed excellent results after enhancement with CNTs in this framework. An esterification reaction between lauric acid and 1-dodecanol was used to test enzyme activity during immobilization, revealing that the Cu3(PO4)2/CNT/protein complex exhibited 68-fold higher activity relative to free lipase and 51-fold higher than that of Cu3(PO4)2/Burkholderia cepacia lipase hybrid nanoflowers in the absence of CNTs. All three hybrid nanostructures showed good performance and exhibited excellent reusability in resolution reactions between 1-phenylethanol and vinyl acetate. Additionally, the substrate enantiomeric excess (ees) reached 98% in only 10 min, and the corresponding Cu3(PO4)2/CNT/protein complex could be recycled eight times without obvious loss of activity. This approach involving nanoflowers enhanced with CNTs will be highly beneficial for decreasing mass-transfer resistance and providing enhanced enzyme loading along with promising potential for industrial application.

Palladium nanoparticles enzyme aggregate (PANEA) as efficient catalyst for Suzuki-Miyaura reaction in aqueous media.

Palladium nanoparticles enzyme aggregate (PANEA) were prepared from Candida antarctica B lipase and palladium salt by precipitation and subsequent in situ Pd nanoparticle formation. This heterogeneous catalyst was successfully used for the Suzuki-Miyaura cross-coupling reaction between bromobenzene with different phenylboronic acid derivatives under mild reaction conditions and using low Pd amount. The nanocatalyst exhibited the highest catalytic activity in a mixture of methanol/water (1:1), obtaining good to excellent product yields from the cross-coupling reaction. A variety of functional groups were accepted and the catalyst was recycled 4 times without activity loss.

Operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis reaction.

Industrial use of Novozym 435 in synthesis of structured lipids and biodiesel via alcoholysis is limited by mass transfer effects of the glycerides through immobilized enzymes and its low operational stability under operation conditions. To better understand this, differently modified Novozym 435 preparations, differing in their surface nature and in their interactions with reactants, have been compared in the alcoholysis of Camelina sativa oil. The three modifications performed have been carried out under conditions where all exposed groups of the enzyme have been modified. These modifications were: 2,4,6-trinitrobenzensulfonic acid (Novo-TNBS), ethylendiamine (Novo-EDA) and polyethylenimine (Novo-PEI). Changes in their operational performance are analyzed in terms of changes detected by scan electron microscopy in the support morphology. The hydrophobic nature of the TNBS accelerates the reaction rate; t-ButOH co-solvent swells the macroporous acrylic particles of Lewatit VP OC 1600 in all biocatalysts, except in the case of Novo-PEI. This co-solvent only increases the maximal conversions obtained at 24h using the modified biocatalysts. t-ButOH reduces enzyme inactivation by alcohol and water. In a co-solvent system, these four biocatalysts remain fully active after 14 consecutive reaction cycles of 24h, but only Novo-TNBS yields maximal conversion before cycle 5. Some deposits on biocatalyst particles could be appreciated during reuses, and TNBS derivatization diminishes the accumulation of product deposits on the catalyst surface. Most particles of commercial Novozym(®) 435 are broken after operation for 14 reaction cycles. The broken particles are fully active, but they cause problems of blockage in filtration operations and column reactors. The three derivatizations studied make the matrix particles more resistant to rupture.

Protein-Coated Microcrystals from Candida rugosa Lipase: Its Immobilization, Characterization, and Application in Resolution of Racemic Ibuprofen.

In this study, an economical heterogeneous biocatalyst, protein-coated microcrystals (PCMCs), was prepared from a commercial Candida rugosa lipase (CRL) and used for catalyzing esterification of (R, S)-ibuprofen enantiomers with isooctanol in isooctane. The main variables controlling the process (precipitating solvents, pH, saturated K2SO4 solution, and water content) were optimized via single-factorial experiments. Under optimum conditions, the enantiomeric excess of active S(+)-ibuprofen and total conversion rate were 97.34 and 49.83 %, respectively, and the corresponding enzyme (PCMC-CRL) activity attained 387.29 µmol/min/g protein, a 5.78-fold enhancement over the free lipase powder. Additionally, the thermostability, organic-solvent tolerance, and operational stability of PCMC-CRL were greatly improved as compared to the free enzyme. Fourier transform infrared (FTIR) spectroscopy was employed to reveal the correlation between conformation and enzyme activity enhancement. Moreover, the PCMC-CRL retained most of its original activity following use in more than 15 successive batches, suggesting that it exhibits adequate operational stability. These results indicate that PCMC-CRL is of great potential use in industrial applications.

Towards efficient chemical synthesis via engineering enzyme catalysis in biomimetic nanoreactors.

Biocatalysis with immobilized enzymes as catalysts holds enormous promise in developing more efficient and sustainable processes for the synthesis of fine chemicals, chiral pharmaceuticals and biomass feedstocks. Despite the appealing potentials, nowadays the industrial-scale application of biocatalysts is still quite modest in comparison with that of traditional chemical catalysts. A critical issue is that the catalytic performance of enzymes, the sophisticated and vulnerable catalytic machineries, strongly depends on their intracellular working environment; however the working circumstances provided by the support matrix are radically different from those in cells. This often leads to various adverse consequences on enzyme conformation and dynamic properties, consequently decreasing the overall performance of immobilized enzymes with regard to their activity, selectivity and stability. Engineering enzyme catalysis in support nanopores by mimicking the physiological milieu of enzymes in vivo and investigating how the interior microenvironment of nanopores imposes an influence on enzyme behaviors in vitro are of paramount significance to modify and improve the catalytic functions of immobilized enzymes. In this feature article, we have summarized the recent advances in mimicking the working environment and working patterns of intracellular enzymes in nanopores of mesoporous silica-based supports. Especially, we have demonstrated that incorporation of polymers into silica nanopores could be a valuable approach to create the biomimetic microenvironment for enzymes in the immobilized state.

Green polymer chemistry: enzyme catalysis for polymer functionalization.

Enzyme catalyzed reactions are green alternative approaches to functionalize polymers compared to conventional methods. This technique is especially advantageous due to the high selectivity, high efficiency, milder reaction conditions, and recyclability of enzymes. Selected reactions can be conducted under solventless conditions without the application of metal catalysts. Hence this process is becoming more recognized in the arena of biomedical applications, as the toxicity created by solvents and metal catalyst residues can be completely avoided. In this review we will discuss fundamental aspects of chemical reactions biocatalyzed by Candida antarctica lipase B, and their application to create new functionalized polymers, including the regio- and chemoselectivity of the reactions.

The surfactant-induced conformational and activity alterations in Rhizopus niveus lipase.

In this study, we have reported the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and structural stability of Rhizopus niveus lipase. Secondary structural changes were monitored by Far-UV CD which shows that surfactant induces helicity in the Rhizopus niveus lipase protein which was maximum in case of CTAB followed by SDS, CHAPS, and Brij-35. Similarly, tertiary structural changes were monitored by tryptophan fluorescence. We also carried out enzyme kinetics assays which showed that activity was enhanced by 1.5- and 1.1-fold in the presence of CHAPS and Brij-35, respectively. Furthermore, there was a decline in activity by 20 and 30 % in case of SDS and CTAB, respectively. These studies may be helpful in understanding detergent-lipase interaction in greater detail as lipases are used in many industrial processes.

Increasing the activity and enantioselectivity of lipases by sol-gel immobilization: further advancements of practical interest.

The entrapment of lipases in hydrophobic silicate matrices formed by sol-gel mediated hydrolysis of RSi(OCH3)3/Si(OCH3)4 as originally reported in 1996 has been improved over the years by a number of modifications. In the production of second-generation sol-gel lipase immobilizates, a variety of additives during the sol-gel process leads to increased activity and enhanced stereoselectivity in esterifying kinetic resolution. Recent advances in this type of lipase immobilization are reviewed here, in addition to new results regarding the sol-gel entrapment of the lipase from Burkholderia cepacia. It constitutes an excellent heterogeneous biocatalyst in the acylating kinetic resolution of two synthetically and industrially important chiral alcohols, rac-sulcatol and rac-trans-2-methoxycyclohexanol. The observation that the catalyst can be used 10 times in recycling experiments without losing its significant activity or enantioselectivity demonstrates the practical viability of the sol-gel approach.

Crystallographic analysis of ground and space thermostable T1 lipase crystal obtained via counter diffusion method approach.

Three-dimensional structure of thermostable lipase is much sought after nowadays as it is important for industrial application mainly found in the food, detergent, and pharmaceutical sectors. Crystallization utilizing the counter diffusion method in space was performed with the aim to obtain high resolution diffracting crystals with better internal order to improve the accuracy of the structure. Thermostable T1 lipase enzyme has been crystallized in laboratory on earth and also under microgravity condition aboard Progress spacecraft to the ISS in collaboration with JAXA (Japanese Aerospace Exploration Agency). This study is conducted with the aims of improving crystal packing and structure resolution. The diffraction data set for ground grown crystal was collected to 1.3 Å resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.40 Å, b = 80.95 Å, and c = 99.81 Å, whereas the diffraction data set for space grown crystal was collected to 1.1 Å resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.31 Å, b = 80.85 Å, and c = 99.81 Å. The major difference between the two crystal growth systems is the lack of convection and sedimentation in microgravity environment resulted in the growth of much higher quality crystals of T1 lipase.

Conformation and catalytic properties studies of Candida rugosa Lip7 via enantioselective esterification of ibuprofen in organic solvents and ionic liquids.

Enantioselective esterification of ibuprofen was conducted to evaluate the enzyme activity and ees of lipase from Candida rugosa (CRL7) in ten conventional organic solvents and three ionic liquids. Different alcohols were tested for selecting the most suitable acyl acceptor due to the fact that the structure of alcohols (branch and length of carbon chains; location of -OH functional group) could affect the enzyme activity and ees. The results of alcohol and solvent selection revealed that 1-isooctanol and isooctane were the best substrate and reaction medium, respectively, because of the highest enzyme activity and ees. Compared with the control, conformational studies via FT-IR indicate that the variations of CRL7's secondary structure elements are probably responsible for the differences of enzyme activity and ees in the organic solvents and ionic liquids. Moreover, the effects of reaction parameters, such as molar ratio, water content, temperature, and reaction time, in the selected reaction medium, were also examined.

Highly efficient biosynthesis of sucrose-6-acetate with cross-linked aggregates of Lipozyme TL 100 L.

As a short chain monoester, sucrose-6-acetate (S-6-a) is a key intermediate in the preparation of an eminent sweetener (sucralose). To replace the traditional multi-step chemical route for sucralose biosynthesis, enzymatic synthesis of S-6-a was investigated, using cross-linked enzyme aggregates (CLEAs) of Lipozyme TL 100 L. The optimal CLEA preparation conditions was obtained as follows: using 33.3% (v/v) PEG600 co-precipitated with additive of D-sorbierite, then cross-linking with 1.5% (v/v) glutaraldehyde at 0 °C for 4 h. As a result, the immobilized Lipozyme had high specific bioactivity (34.64 U/g) of transesterification in non-aqueous media. With these immobilized enzymes, the optimum transesterification conditions were investigated systematically, including CLEA loading, the mole ratio of vinyl acetate versus sucrose, temperature and reaction time, etc. The results showed that the highest concentration and yield of S-6-a was 49.8 g/L and 87.46%, respectively. Further experiments showed that the resulting CLEAs also had much higher operational stability than the commercial Lipozyme TLIM. The present work has paved a new path for the large-scale bioproduction of S-6-a with immobilized lipase in the future.

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum.

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E((0.3))), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3U/mg protein, which is 0.46 times less than that of the free lipase (35.6U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 degrees C respectively. Kinetic parameters V(max) and K(m) were also determined for the immobilized lipase. It was observed that there is an increase of the K(m) value (7.54mM) and a decrease of the V(max) value (145.0U/mg-protein) comparing with that of the free lipase.

Improvement of catalytic properties of lipase from Arthrobacter sp. by encapsulation in hydrophobic sol-gel materials.

In this work, lipase from Arthrobacter sp. was immobilized by sol-gel encapsulation to improve its catalytic properties. Various silanizing agents including vinyl-trimethoxy silane, octyl-trimethoxy silane, gamma-(methacryloxypropyl)-trimethoxy silane (MAPTMS) and tetraethoxysilane (TEOS) were chosen as the precursors. Among them, MAPTMS was for the first time utilized to encapsulate lipases, and the prepared enzyme by copolymerization of MAPTMS and TEOS exhibited the highest activity in both the hydrolysis of p-nitrophenyl palmitate and the asymmetric acylation of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one. The effects of various immobilization parameters were investigated. Under the optimum conditions of MAPTMS/TEOS=1/1 (mol/mol), water/silane molar ratio (R value)=20 and lipase loading=0.01 g/mL sol, the total activity of the immobilized enzyme reached up to 13.6-fold of the free form. Moreover, the encapsulated lipase exhibited higher thermal stability than the free form and retained 54% of the original activity after uses for 60 d. Enantioselectivity of enzyme was also improved with an E value of 150 after encapsulation from 85 for the free form.

Development of magnetically separable immobilized lipase by using cellulose derivatives and their application in enantioselective esterification of ibuprofen.

Highly active, stable, and magnetically separable immobilized enzymes were developed using carboxymethyl cellulose (CMC) and diethylaminoethyl cellulose DEAE-C; hereafter designated "DEAE" as supporting materials. Iron oxide nanoparticles penetrated the micropores of the supporting materials, rendering them magnetically separable. Lipase (LP) was immobilized on the surface of the supporting materials by using cross-linked enzyme aggregation (CLEA) by glutaraldehyde. The activity of enzyme aggregates coated on DEAE was approximately 2 times higher than that of enzyme aggregates coated on CMC. This is explained by the fact that enzyme aggregates with amine residues are more efficient than those with carboxyl residues. After a 96-h enantioselective ibuprofen esterification reaction, 6% ibuprofen propyl ester was produced from the racemic mixture of ibuprofen by using DEAE-LP, and 2.8% using CMC-LP.

Studies on crystallization and cross-linking of lipase for biocatalysis.

The development of robust biocatalysts with increased stability and activity is a major challenge to industry. A major breakthrough in this field was the development of cross-linked enzyme crystals with high specificity and stability. A method is described to produce micro crystals of CLEC lipase, which is thermostable and solvent stable. Lipase from Burkholderia cepacia was crystallized using ammonium sulfate and cross-linked with glutaraldehyde to produce catalytically active enzyme. The maximum yield of CLEC was obtained with 70% ammonium sulfate and cross-linked with 5% (v/v) glutaraldehyde. SEM studies showed small hexagonal-shaped crystals of 2-5 microm size. CLEC lipase had improved thermal and reuse stability. It is versatile, having good activity in both polar and nonpolar organic solvents. CLEC lipase was coated using beta cyclodextrin for improving the storage and reuse stability. CLEC was successfully used for esterification of Ibuprofen and synthesis of ethyl butyrate.

Covalent attachment of Candida rugosa lipase on chemically modified hybrid matrix of polysiloxane-polyvinyl alcohol with different activating compounds.

Candida rugosa lipase was immobilized by covalent binding on hybrid matrix of polysiloxane-polyvinyl alcohol chemically modified with different activating agents as glutaraldehyde, sodium metaperiodate and carbonyldiimidazole. The experimental results suggested that functional activating agents render different interactions between enzyme and support, producing consequently alterations in the optimal reaction conditions. Properties of the immobilized systems were assessed and their performance on hydrolytic and synthetic reactions were evaluated and compared with the free enzyme. In hydrolytic reactions using p-nitrophenyl palmitate as substrate all immobilized systems showed higher thermal stability and optima pH and temperature values in relation to the free lipase. Among the activating compounds, carbonyldiimidazole resulted in a total recovery of activity on the support and the highest thermal stability. For the butyl butyrate synthesis, the best performance (molar conversion of 95% and volumetric productivity of 2.33 g L(-1)h(-1)) was attained with the lipase immobilized on POS-PVA activated with sodium metaperiodate. The properties of the support and immobilized derivatives were also evaluated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopies and chemical composition (FTIR).

A high performance lipase preparation: characterization and atomic force microscopy.

The goal of obtaining enzyme forms which show greater stability, higher catalytic efficiency, and reusability has been pursued since last several decades. Some novel biocatalyst designs have been evolved and protein coated micro-crystals (PCMCs) is one of them. Pseudomonas cepacia lipase coated micro-crystals were prepared by simultaneous precipitation of mixture of aqueous lipase solution and salts such as potassium sulphate by organic solvents. This resulted in lipase coated micro-crystals. The structures of micro-crystals were studied by atomic force microscopy (AFM). The AFM picture confirmed the enzyme coating over the potassium sulphate crystals. These PCMCs are in the size range of 500-1000 nm. These enzyme coated micro-crystals showed enhanced transesterification rates. Also, the PCMC were stable at 60 degrees C whereas the free enzyme lost all its activity. The enzyme coated micro-crystals prepared by 50 mg Pseudomonas cepacia lipase gave 96% conversion in 90 min whereas free enzyme gave 8% conversion. Even PCMCs prepared from 3.12 mg lipase gave 90% conversion in 10 h at 60 degrees C where as free lipase was inactive at 60 degrees C.

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18-crown-6 on lipase activity, enantioselectivity, and conformation.

Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.