Enantioselective Hydrolysis of d,l‐Menthyl Benzoate to L‐(–)‐Menthol by Recombinant Candida rugosa Lipase LIP1

The stereoselective synthesis of L ‐menthol is an attractive process in the flavor and fragrance industry. One promising way to obtain optically pure menthol is the enantioselective hydrolysis of menthol esters under enzymatic catalysis. We developed an effective and highly enantioselective method for the synthesis of L ‐(−)‐menthol (>99% EE) by hydrolyzing the key industrial starting compound, d, l ‐menthyl benzoate. The enzyme of choice was the lipase from Candida rugosa (CRL). While commercially available preparations of this lipase showed only minor selectivity (E=15), excellent enantiomeric purity (E>100) was achieved using the heterologously expressed isoenzyme LIP1.     

Computer Modeling Explains the Structural Reasons for the Difference in Reactivity of Amine Transaminases Regarding Prochiral Methylketones

Amine transaminases (ATAs) are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the (R)-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the (S)-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee) of both (R)- and (S)-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of <10% (without affecting the enantioselectivity). In contrast, the commercially engineered V. fluvialis variant, ATA-256, still enabled an 87% conversion, yielding a corresponding amine with >99% ee. Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results.     

Lipase‐catalyzed acidolysis and phospholipase D‐catalyzed transphosphatidylation of phosphocholine

Two approaches on enzymatic phospholipid modification were studied: (1) transphosphatidylation of the 1,2‐dilauroyl‐sn‐glycero‐3‐phosphocholine (DLPC) and ethanolamine in biphasic and anhydrous organic solvent systems by phospholipase D (PLD) and (2) incorporation of oleic acid into the sn1‐position of DLPC in organic solvents with different immobilized lipases at controlled water activity. First, DLPC was chemically synthesized from glycerophosphocholine and lauric acid. Next, PLD‐catalyzed head group exchange of DLPC with ethanolamine was studied using an enzyme from Streptomyces antibioticus expressed recombinantly in E. coli. A comparison of the free PLD with the biocatalyst activated by a salt‐activation technique using KCl showed that the salt‐activated enzyme (PLD‐KCl) was 10–12 folds more active based on the amount of protein used. Thus, DLPC was quantitatively converted to 1,2‐dilauroyl‐sn‐glycero‐3‐phosphoethanolamine in an anhydrous solvent system within 12 h at 60 °C. For the acidolysis of DLPC with oleic acid, among the four lipases studied (CAL‐B, Lipozyme TL IM, Lipozyme RM IM and lipase D immobilized on Accurel EP‐100), Lipozyme TL IM showed the highest activity and incorporation of oleic acid. A quantitative incorporation was achieved at 40 °C using a 8‐fold molar excess of oleic acid in n‐hexane at a water activity of 0.11.     

Thermostabilization of an esterase by alignment-guided focussed directed evolution

Site-saturation libraries of the Pseudomonas fluorescens esterase were created targeting three surface positions to increase its thermostability on the basis of the B-factor iterative test principle. All three positions were saturated simultaneously using our recently developed protocol for the design of 'small, but smart' mutant libraries bearing only consensus-like mutations. Hence, the library size could be significantly reduced while ensuring a high hit rate. Variants could be identified that showed significantly improved stability (8° C higher compared with the wild type) without compromising specific activity. Subsequent iterative saturation mutagenesis gave an esterase mutant with a 9° C increased melting point, but unchanged catalytic properties.     

Enzymatic removal of 3‐monochloro‐1,2‐propanediol (3‐MCPD) and its esters from oils

3‐Monochloro‐1,2‐propanediol (3‐MCPD) is a contaminant in processed food well known for about 30 years. More recently, this compound has observed attendance due to its occurrence as fatty acid esters in edible oils and products derived from them. In this study, the first enzymatic approach to remove 3‐MCPD and its esters from aqueous and biphasic systems by converting it into glycerol is described. First, 3‐MCPD was converted in an aqueous system by an enzyme cascade consisting of a halohydrin dehalogenase from Arthrobacter sp. AD2 and an epoxide hydrolase from Agrobacterium radiobacter AD1 with complete conversion to glycerol. Next, it could also be shown, that the corresponding oleic acid monoester of 3‐monochloropropanediol‐1‐monooleic‐ester (3‐MCPD‐ester) was converted in a biphasic system in the presence of an edible oil by Candida antarctica lipase A to yield free 3‐MCPD and the corresponding fatty acid. Hence, also 3‐MCPD‐esters can be converted by an enzyme cascade into the harmless product glycerol. Practical applications: Since several reports have been recently published on the contamination of foods with 3‐MCPD and its fatty acid esters, there is a great demand to remove these compounds and an urgency to find useful methods for this. In this contribution, we present an easy enzymatic way to remove 3‐MCPD and its esters from the reaction media (i.e., plant oil) by converting it to the nontoxic glycerol. The method requires neither high temperature nor organic solvents.     

Lipase‐catalyzed transesterification to remove saturated MAG from biodiesel

Saturated MAG (SMG) are known to be present in FAME intended to be used as biodiesel. These SMG can strongly affect the properties of biofuels such as the cloud point (CP), and they have been implicated in engine failure due to filter plugging. It is shown here that lipase G from Penicillium camemberti can be efficiently used for the transesterification of SMG to fatty acid methyl ester and glycerol even in the presence of the bulk biodiesel. Thus, in samples of commercial biodiesel to which glycerol monostearate (GMS) and glycerol monopalmitate (GMP) had been added, their levels were enzymatically reduced from 2% (w/v) to 0.22% (w/v) for GMP and 0.14% (w/v) for GMS as confirmed by GC‐MS analysis. Practical applications: SMG present in biodiesel can have a pronounced negative effect on the CP, and/or filterability and in‐field performance of the fuel. The lipase‐catalyzed transesterification shown in this paper enables a significant reduction in the amount of SMG, leading to superior biodiesel quality.     

Protein engineering of α/β-hydrolase fold enzymes

The superfamily of α/β-hydrolase fold enzymes is one of the largest known protein families, including a broad range of synthetically useful enzymes such as lipases, esterases, amidases, hydroxynitrile lyases, epoxide hydrolases and dehalogenases. This minireview covers methods developed for efficient protein engineering of these enzymes. Special emphasis is placed on the alteration of enzyme properties such as substrate range, thermostability and enantioselectivity for their application in biocatalysis. In addition, concepts for the investigation of the evolutionary relationship between the different members of this protein superfamily are covered, together with successful examples.     

Lipase-catalyzed synthesis of monoacylglycerides by glycerolysis of campher tree seed oil and cocoa-butter

The lipase-catalyzed glycerolysis of Campher tree seed oil and Cocoa-butter in a solid-phase system was studied. Lipases from Pseudomonas cepacia (PCL) and Chromobacterium viscosum (CVL) were considered as suitable for the synthesis of monoacylglycerols (MAG). The glycerolysis of Campher tree seed oil was performed initially at 25°C followed by cooling to 7°C. This resulted in 86% (PCL) and 90% (CVL) MAG. Maximum concentration of MAG from Cocoa-butter was 89% with both lipases, when the reaction was performed at 25°C. Isolation of MAG was performed by extraction with chloroform at 4°C followed by purification via silica gel chromatography.     

Endocrine reactions, circulatory and resuscitation behavior in ketamine-midazolam anesthesia. A comparative study of ketamine racemate vs. (S)-ketamine in knee surgery

Clinically used ketamine is a racemic mixture of two isomers, (S)- and (R)-ketamine, in equal amounts. Previous investigations showed the anaesthetic potency of (S)-ketamine to be three times higher than that of (R)-ketamine. The aim of this study was to compare the effects of (S)-ketamine/midazolam and racemic ketamine/midazolam on endocrine and cardiovascular parameters, recovery, and side effects in unpremedicated patients during knee surgery. METHODS: 41 patients scheduled for elective knee surgery were investigated in a prospective, double-blind, and randomised design. For induction of intravenous anesthesia, patients received 0.1 mg/kg midazolam, 0.003 mg/kg atropine, 1 mg/kg (S)-ketamine or 2 mg/kg racemic ketamine, respectively. For tracheal intubation, 1 mg vecuronium and 1.5 mg/kg suxamethonium were injected. After intubation and relaxation with a total dose of 0.1 mg/kg vecuronium, a continuous infusion of 0.5 mg/kg/h (S)- or 1 mg/kg/h racemic ketamine was administered throughout the surgery. In addition, 0.05 mg/kg/h midazolam was infused continuously in both groups throughout surgery. Ventilation was performed with N2O/O2 (FiO2 0.3). Blood samples were taken using a central venous line five times before induction as well as during and after surgery for analysis of adrenaline, noradrenaline (by high-pressure liquid chromatography with electrochemical detection), anti-diuretic hormone (ADH), adrenocorticotropic hormone (ACTH), and cortisol (by radioimmunoassay). In addition, systolic and diastolic arterial pressure (SAP, DAP), heart rate (HR), and arterial oxygen saturation were measured. The time intervals between the end of ketamine and midazolam infusion and the return of consciousness and orientation were recorded. The incidence and quality of dreams and other side effects were reported by the patients. RESULTS: Biometric data of the groups were comparable. Plasma adrenaline and noradrenaline did not change significantly during anaesthesia. ADH increased significantly (p < 0.05) after skin incision in both groups.     

Surgical relevance of diagnostic imaging of abdominal tumors--decision making in pancreatic tumor

1. An association has been reported between QT interval abnormalities and cardiovascular autonomic neuropathy in diabetic patients. The QT interval abnormalities reflect local inhomogeneities of ventricular recovery time and may be related to an imbalance in cardiac sympathetic innervation. Sympathetic innervation of the heart can be visualized and quantified by single-photon emission-computed tomography with m-[123I]iodobenzylguanidine. In this study we evaluated cardiac sympathetic integrity by m-[123I]iodobenzylguanidine imaging and the relationship between both QT interval prolongation and QT dispersion from standard 12-lead ECG variables and m-[123I]iodobenzylguanidine uptake in insulin-dependent diabetic patients. 2. Three patient groups were studied, comprising six healthy control subjects, nine diabetic patients without cardiovascular autonomic neuropathy (CAN-) and 12 diabetic patients with cardiovascular neuropathy (CAN+). Resting 12-lead ECG was recorded for measurement of maximal QT interval and QT dispersion. The QT interval was heart rate corrected using Bazett's formula (QTc) and the Karjalainen approach (QTk). Quantitative measurement (in counts/min per g) and visual defect pattern of m-[123I]iodobenzylguanidine uptake were performed using m-[123I]iodobenzylguanidine single-photo emission-computed tomography. 3. Global myocardial m-[123I]iodobenzylguanidine uptake was significantly reduced in both diabetic patient groups compared with control subjects. The visual defect score of m-[123I]iodobenzylguanidine uptake was significantly higher in CAN+ diabetic patients than in control subjects and in CAN- patients. This score was not significantly different between control subjects and CAN- patients. QTc interval and QT dispersion were significantly increased in CAN+ diabetic patients as compared with control subjects (QTc: 432 +/- 15 ms versus 404 +/- 19 ms, P < 0.05; QT dispersion: 42 +/- 10 versus 28 +/- 8 ms, P < 0.05). QT dispersion was also significantly longer in CAN- diabetic patients than in control subjects (41 +/- 9 ms versus 28 +/- 8 ms, P < 0.05). QTc interval was significantly related to global myocardial m-[123I]iodobenzylguanidine uptake and defect score in diabetic patients (r = -0.648, P < 0.01, and r = 0.527, P < 0.05, respectively). There was no correlation between QT dispersion and both m-[123I]iodobenzylguanidine uptake measures. 4. In conclusion, these findings suggest that m-[123I]iodobenzylguanidine imaging is a valuable tool for the detection of early alterations in myocardial sympathetic innervation in long-term diabetic patients without cardiovascular autonomic neuropathy. Insulin-dependent diabetic patients with cardiovascular autonomic neuropathy have a delayed cardiac repolarization and increased variability of ventricular refractoriness. The cardiac sympathetic nervous system seems to be one of the determinants of QT interval lengthening, but does not appear to be involved in dispersion of ventricular recovery time. It is assumed that QT dispersion is based on more complex electrophysiological mechanisms which remain to be elucidated.