Optimization of Enzymatic Transesterification of Acid Oil for Biodiesel Production Using a Low-Cost Lipase: The Effect of Transesterification Conditions and the Synergy of Lipases with Different Regioselectivity.

This study describes the enzymatic production of second-generation biodiesel using low-quality acid oil as a substrate. Biolipasa-R, a commercially available and low-cost lipase, was employed for enzymatic transesterification. Response surface methodology was applied to optimize the enzymatic transesterification process. The optimal conditions for biodiesel production, which comprised 42% lipase concentration (per weight of oil), 32% water content (per weight of oil), a methanol to oil molar ratio of 3:1, pH 7.0 and reaction temperature 30°C, resulted in the highest fatty acid methyl ester (FAME) content (71.3%). Subsequently, the synergistic effect of two lipases with different regioselectivities under the optimum transesterification conditions was studied, aiming at the enhancement of process efficiency. The transesterification efficiency of immobilized Biolipasa-R was determined and compared to that of Biolipasa-R in its free form. The results revealed a good performance on FAME content (66.5%), while the recycling of immobilized lipase resulted in a decrease in transesterification efficiency after three consecutive uses.

Enzymatic Photometric Assays for the Selective Detection of Halides.

Halides are substrates and products of a number of biotechnologically important enzymes like dehalogenases, halide methyltransferases, and halogenases. Therefore, the determination of halide concentrations in samples is important. The classical methods based on mercuric thiocyanate are very dangerous, produce hazardous waste, and do not discriminate between chloride, bromide, and iodide. In this chapter, we describe a detailed protocol for the determination of halide concentrations based on the haloperoxidase-catalyzed oxidation of halides. The resulting hypohalous acids are detected using commercially available colorimetric or fluorimetric probes. The biocatalytic nature of the assays allows them to be implemented in one-pot cascade reactions with halide-generating enzymes. Since chloride is ubiquitous in biological systems, we also describe convenient photometric assays for the selective detection of bromide and iodide in the presence of chloride, obviating the need for laborious dialyses to obtain halide-free enzymes and reagents.

Recovery of Hydroxytyrosol from Olive Mill Wastewater Using the Promiscuous Hydrolase/Acyltransferase PestE.

Olive mill wastewater (OMWW) is produced annually during olive oil extraction and contains most of the health-promoting 3-hydroxytyrosol of the olive fruit. To facilitate its recovery, enzymatic transesterification of hydroxytyrosol (HT) was directly performed in an aqueous system in the presence of ethyl acetate, yielding a 3-hydroxytyrosol acetate rich extract. For this, the promiscuous acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was engineered by rational design. The best mutant for the acetylation of hydroxytyrosol (PestE_I208A_L209F_N288A) was immobilized on EziG2 beads, resulting in hydroxytyrosol conversions between 82 and 89 % in one hour, for at least ten reaction cycles in a buffered hydroxytyrosol solution. Due to inhibition by other phenols in OMWW the conversions of hydroxytyrosol from this source were between 51 and 62 %. In a preparative scale reaction, 13.8 mg (57 %) of 3-hydroxytyrosol acetate was extracted from 60 mL OMWW.

Rational engineering of Luminiphilus syltensis (R)-selective amine transaminase for the acceptance of bulky substrates.

Despite the plethora of information on (S)-selective amine transaminases, the (R)-selective ones are still not well-studied; only a few structures are known to date, and their substrate scope is limited, apart from a few stellar works in the field. Herein, the structure of Luminiphilus syltensis (R)-selective amine transaminase is elucidated to facilitate engineering towards variants active on bulkier substrates. The V37A variant exhibited increased activity towards 1-phenylpropylamine and to activity against 1-butylamine. In contrast, the S248 and T249 positions, located on the ß-turn in the P-pocket, seem crucial for maintaining the activity of the enzyme.

From Natural Methylation to Versatile Alkylations Using Halide Methyltransferases.

Halide methyltransferases (HMTs) enable the enzymatic synthesis of S-adenosyl-l-methionine (SAM) from S-adenosyl-l-homocysteine (SAH) and methyl iodide. Characterisation of a range of naturally occurring HMTs and subsequent protein engineering led to HMT variants capable of synthesising ethyl, propyl, and allyl analogues of SAM. Notably, HMTs do not depend on chemical synthesis of methionine analogues, as required by methionine adenosyltransferases (MATs). However, at the moment MATs have a much broader substrate scope than the HMTs. Herein we provide an overview of the discovery and engineering of promiscuous HMTs and how these strategies will pave the way towards a toolbox of HMT variants for versatile chemo- and regioselective biocatalytic alkylations.

Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs.

Biocatalytic alkylations are important reactions to obtain chemo-, regio- and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide-based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one-pot cascades with O-methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4-dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.

Activity and specificity studies of the new thermostable esterase EstDZ2.

In this paper, we study the activity and specificity of EstDZ2, a new thermostable carboxyl esterase of unknown function, which was isolated from a metagenome library from a Russian hot spring. The biocatalytic reaction employing EstDZ2 proved to be an efficient method for the hydrolysis of aryl p-, o- or m-substituted esters of butyric acid and esters of secondary alcohols. Docking studies revealed structural features of the enzyme that led to activity differences among the different substrates.

Cutinases as stereoselective catalysts: Specific activity and enantioselectivity of cutinases and lipases for menthol and its analogs.

The specific activity and enantioselectivity of immobilized cutinases from Aspergillus oryzae (AoC) and Humicola insolens (HiC) were compared with those of lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML) and Lipase B from Candida antarctica (CALB) for menthol and its analogs that include isopulegol, trans-2-tert-butylcyclohexanol (2TBC), and dihydrocarveol (DHC). Common features of these alcohols are two bulky substituents: a cyclohexyl ring and an alkyl substituent. Dissimilarities are that the alkyl group reside at different positions or have dissimilar structures. The aim was to develop an understanding at a molecular level of similarities and differences in the catalytic behavior of the selected cutinases and lipases as a function of substrate structural elements. The experimental results reflect the (-)-enantioselectivity for AoC, HiC, TLL, and RML, while CALB is only active on DHC with (+)-enantioselectivity. In most cases, AoC has the highest activity while HiC is significantly more active than other enzymes on 2TBC. The E values of AoC, HiC, TLL, and RML for menthol are 27.8, 16.5, 155, and 125, respectively. HiC has a higher activity (>10-fold) on (-)-2TBC than AoC while they exhibit similar activities on menthol. Docking results reveal that the bulky group adjacent to the hydroxyl group determines the enantioselectivity of AoC, HiC, TLL, and RML. Amino acid residues that dominate the enantioselectivity of these enzymes are AoC's Phe195 aromatic ring; HiC's hydrophobic Leu 174 and Ile 169 groups; TLL's ring structures of Trp89, His258 and Tyr21; and Trp88 for RML. Results of this study highlight that cutinases can provide important advantages relative to lipases for enantioselective transformation, most notably with bulky and sterically hindered substrates.

The challenge of using isopropylamine as an amine donor in transaminase catalysed reactions.

Amine transaminases (ATAs) propose an appealing alternative to transition metal catalysts as they can provide chiral amines of high purity from pro-chiral compounds by asymmetric synthesis. Industrial interest on ATAs arises from the fact that chiral amines are present in a wide spectrum of pharmaceutical and other high value-added chiral compounds and building blocks. Despite their potential as useful synthetic tools, several drawbacks such as challenges associated with the thermodynamic equilibrium can still impede their utilization. Several methods have been developed to displace the equilibrium, such as the use of alanine as an amine donor and the subsequent removal of pyruvate with a two-enzyme system, or the use of o-xylylene diamine. To date, the preferred amine donor remains isopropylamine (IPA), as the produced acetone can be removed easily under low pressure or slight heating, without complicating the process with other enzymes. Despite its small size, IPA is not widely accepted from wild-type ATAs, and this fact compromises its wide applicability. Herein, we index the reported biocatalytic aminations with IPA, comparing the sequences, while we discuss significant parameters of the process, such as the effect of temperature and pH, as well as the protein engineering and process development advances in the field. This information is expected to provide an insight for potential designs of tailor-made ATAs and IPA processes.

Cloning, expression and characterization of cold active esterase (EstN7) from Bacillus cohnii strain N1: A novel member of family IV.

Esterases and lipases from extremophiles have attracted great attention due to their unique characteristics and wide applications. In the present study, an open reading frame (ORF) encoding a novel cold active esterase (EstN7) from Bacillus cohnii strain N1 was cloned and expressed in Escherichia coli. The full-length esterase gene encoding a protein of 320 amino acids with estimated molecular weight of 37.0 kDa. Amino acid sequence analysis revealed that the EstN7 belongs to family IV lipases with a characteristic penta-peptide motif (GXSXG), the catalytic triad Ser, Asp, His and the conserved HGGG motif of the family IV. The recombinant enzyme was purified to apparent homogeneity using nickel-affinity chromatography with a purification fold of 5 and recovery 94.5%. The specific activity of the purified enzyme was 336.89 U/mg. The recombinant EstN7 showed optimal activity at 5 °C moreover, EstN7 displayed full robust stability in the presence of wide range of organic solvents. The purified enzyme had Km and Vmax of 45 ±â€¯0.019 µM and 1113 µmol min-1 mg-1, respectively on p-NP-acetate. These promising characteristics of the recombinant EstN7 would underpin its possible usage with high potential in the synthesis of fragile compounds in pharmaceutical industries.

S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic.

S-adenosyl-L-homocysteine (SAH) hydrolases (SAHases) are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD+) as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB) are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD+ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC) and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor), nor the holo-enzyme (i.e., in the NAD+-bound state) were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors.

Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity.

Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity.

A coupled photometric assay for characterization of S-adenosyl-l-homocysteine hydrolases in the physiological hydrolytic direction.

S-Adenosyl-l-homocysteine hydrolases (SAHases) are important metabolic enzymes and their dysregulation is associated with some severe diseases. In vivo they catalyze the hydrolysis of S-adenosyl-l-homocysteine (SAH), the by-product of methylation reactions in various organisms. SAH is a potent inhibitor of methyltransferases, thus its removal from the equilibrium is an important requirement for methylation reactions. SAH hydrolysis is also the first step in the cellular regeneration process of the methyl donor S-adenosyl-l-methionine (SAM). However, in vitro the equilibrium lies towards the synthetic direction. To enable characterization of SAHases in the physiologically relevant direction, we have developed a coupled photometric assay that shifts the equilibrium towards hydrolysis by removing the product adenosine, using a high affinity adenosine kinase (AK). This converts adenosine to AMP and thereby forms equimolar amounts of ADP, which is phosphorylated by a pyruvate kinase (PK), in turn releasing pyruvate. The readout of the assay is the consumption of NADH during the lactate dehydrogenase (LDH) catalyzed reduction of pyruvate to lactic acid. The applicability of the assay is showcased for the determination of the kinetic constants of an SAHase from Bradyrhizobium elkanii (KM,SAH 41±5µM, vmax,SAH 25±1µM/min with 0.13mg/mL enzyme). This assay is a valuable tool for in vitro characterization of SAHases with biotechnological potential, and for monitoring SAHase activity in diagnostics.

Amine Transaminase Engineering for Spatially Bulky Substrate Acceptance.

Amine transaminase (ATA) catalyzing stereoselective amination of prochiral ketones is an attractive alternative to transition metal catalysis. As wild-type ATAs do not accept sterically hindered ketones, efforts to widen the substrate scope to more challenging targets are of general interest. We recently designed ATAs to accept aromatic and thus planar bulky amines, with a sequence-based motif that supports the identification of novel enzymes. However, these variants were not active against 2,2-dimethyl-1-phenyl-propan-1-one, which carries a bulky tert-butyl substituent adjacent to the carbonyl function. Here, we report a solution for this type of substrate. The evolved ATAs perform asymmetric synthesis of the respective R amine with high conversions by using either alanine or isopropylamine as amine donor.

Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases.

Pyridoxal-5'-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.

Protein-engineering of an amine transaminase for the stereoselective synthesis of a pharmaceutically relevant bicyclic amine.

Application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones represents an environmentally benign and economically attractive alternative to transition metal catalyzed asymmetric synthesis. However, the restrictive substrate scope has limited the conversion typically to non-sterically demanding scaffolds. Recently, we reported on the identification and design of fold class I ATAs that effect a highly selective asymmetric synthesis of a set of chiral aromatic bulky amines from the corresponding ketone precursors in high yield. However, for the specific amine synthetic approach extension targeted here, the selective formation of an exo- vs. endo-isomer, these biocatalysts required additional refinement. The chosen substrate (exo-3-amino-8-aza-bicyclo[3.2.1]oct-8-yl-phenyl-methanone), apart from its pharmacological relevance, is a demanding target for ATAs as the bridged bicyclic ring provides substantial steric challenges. Protein engineering combining rational design and directed evolution enabled the identification of an ATA variant which catalyzes the specific synthesis of the target exo-amine with >99.5% selectivity.

Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines.

The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.

Graphene oxide derivatives with variable alkyl chain length and terminal functional groups as supports for stabilization of cytochrome c.

In this study we report the ability of reduced and non-reduced graphene oxide-based nanomaterials (GONs), modified with variable alkyl chain length and terminal functional groups, to act as effective scaffolds for the immobilization of cytochrome c (cyt c) using different immobilization procedures. The GONs/cyt c conjugates are characterized by a combination of techniques, namely atomic force microscopy, X-ray photoelectron and FT-IR spectroscopies as well as thermo-gravimetric and differential thermal analysis. The effect of the structure of functional groups and the surface chemistry of GONs on the immobilization efficiency, the peroxidase activity and the stability of the cyt c was investigated and correlated with conformational changes on the protein molecule upon immobilization. The enhanced thermal stability (up to 2-fold) and increased tolerance (up to 25-fold) against denaturing agents observed for immobilized cyt c, indicates that these functionalized GONs are suitable as nanoscaffolds for the development of robust nanobiocatalysts.

Highly efficient and easy protease-mediated protein purification.

As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH). While GFP and AiiA were already known to be protease resistant, the thermostable enzymes CAL-A, AspFum, and AlaDH were selected due to the documented correlation between thermostability and protease resistance. As proof of principle for PMPP, recombinant GFP remained unaffected whereas most Escherichia coli (E. coli) host proteins were degraded by trypsin. PMPP was highly advantageous compared to the widely used heat-mediated purification of commercial CAL-A. The resistance of AspFum towards trypsin was improved by rational protein design introducing point mutation R20Q. Trypsin also served as economical and efficient substitute for site-specific endopeptidases for the removal of a His-tag fused to AiiA. Moreover, proteolysis of host enzymes with interfering properties led to a strongly improved sensitivity and accuracy of the NADH assay in E. coli cell lysate for AlaDH activity measurements. Thus, PMPP is an attractive alternative to common protein purification methods and facilitates also enzyme characterization in cell lysate.

Structure of product-bound SMG1 lipase: active site gating implications.

Monoacylglycerol and diacylglycerol lipases are industrially interesting enzymes, due to the health benefits that arise from the consumption of diglycerides compared to the traditional triglyceride oils. Most lipases possess an α-helix (lid) directly over the catalytic pocket which regulates the activity of the enzyme. Generally, lipases exist in active and inactive conformations, depending on the positioning of this lid subdomain. However, lipase SMG1, a monoacylglycerol and diacylglycerol specific lipase, has an atypical activation mechanism. In the present study we were able to prove by crystallography, in silico analysis and activity tests that only two positions, residues 102 and 278, are responsible for a gating mechanism that regulates the active and inactive states of the lipase, and that no significant structural changes take place during activation except for oxyanion hole formation. The elucidation of the gating effect provided data enabling the rational design of improved lipases with 6-fold increase in the hydrolytic activity toward diacylglycerols, just by providing additional substrate stabilization with a single mutation (F278N or F278T). Due to the conservation of F278 among the monoacylglycerol and diacylglycerol lipases in the Rhizomucor miehei lipase-like family, the gating mechanism described herein might represent a general mechanism applicable to other monoacylglycerol and diacylglycerol lipases as well. Database: Structural data are available in the Protein Data Bank under the accession numbers 4ZRE (F278D mutant) and 4ZRD (F278N mutant).