1,3‐Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates

The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL‐CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA’ observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 μmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL‐SOY‐CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL‐SOY‐CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12‐fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2‐fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL‐SOY‐CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier‐free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910–920, 2018     

Production of L-phenylalanine from phenylpyruvate using resting cells of Escherichia coli

Summary We describe the production of L-phenylalanine from phenylpyruvate using resting cells of a genetically modified strain of Escherichia coli. Fermentations were carried out by continuously raising the feeding rate of D-glucose. We reached a biomass of 10 g/l dry weight and an aminotransferase activity of 14000 U/l The maximum phenylalanine concentration achieved was 173 mmol/l with a phenylpyruvate molar conversion yield of 95%.Dedicated to Prof. Dr. Heinz Harnisch on the occasion of his 60th birthday     

Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush-border-membrane vesicles from hamster jejunum.

The effects of bile salts on Na+-coupled accumulation of D-glucose and L-alanine by brush-border-membrane vesicles isolated from hamster jejunum were investigated. The approximate percentage inhibition of Na+-coupled D-glucose accumulation produced by various bile salts at a concentration of 1 mM were: deoxycholate and chenodeoxycholate, 60%; glycine and taurine conjugates of deoxycholate and chenodeoxycholate, 40--50%; lithocholate, 45%; cholate and its glycine and taurine conjugates, less than 10%. Inhibition of Na+-coupled accumulation of D-glucose was rapid, reversible and not due to dissolution of the vesicles. Na+-coupled accumulation of L-alanine was also inhibited by deoxycholate. Deoxycholate but not cholate enhanced (1) the rate of Na+ influx, (2) the rate of influx of D-glucose and L-alanine in the absence of a Na+ gradient and (3) the rate of efflux of D-glucose and L-alanine from vesicles preloaded with this sugar or amino acid. Deoxycholate-stimulated efflux of D-glucose was not blocked by phlorizin, which completely prevented efflux in the absence of this bile salt. These results suggest that selected bile salts inhibit Na+-coupled accumulation of D-glucose and L-alanine by enhancing the rate of dissipation of the Na+ gradient required for substrate accumulation. In addition, bile salts may also decrease D-glucose and L-alanine accumulation by increasing the rate of efflux of these substrates across the brush-border plasma membrane.     

Kidney brush-border membrane transporters: differential sensitivity to diethyl pyrocarbonate

The effects of the histidine modifier, diethyl pyrocarbonate (DEPC), on brush-border membrane transport systems were studied in rat kidney. DEPC caused a strong inhibition of sodium-dependent phosphate and D-glucose uptake. Phosphate uptake remained linear up to 10 s in control and DEPC-treated membrane vesicles. The D-glucose carrier was more sensitive than the phosphate carrier with half-times of inhibition being 4 and 7 min, respectively. Sodium-independent phosphate and D-glucose uptake remained unaffected by DEPC. Intravesicular volume and two enzyme activities endogenous to the luminal membrane (alkaline phosphatase and aminopeptidase M) remained unaffected by DEPC. Increasing the preincubation pH from 5 to 9 increased phosphate transport inhibition caused by DEPC from 73 to 88% in the presence of DEPC. Hydroxylamine was able to completely reverse phosphate uptake inhibition by DEPC (100%), but only partially reversed the D-glucose uptake inhibition (16%). Sodium or substrate (D-glucose or phosphate) in the preincubation media were unable to protect their respective carriers from DEPC. Sodium-dependent transport of L-glutamine, L-phenylalanine, L-leucine, L-alanine, L-glycine, beta-alanine and L-proline were inhibited at different levels ranging from 70 to 90%. Three transport processes were found insensitive to DEPC modification: L-glutamate, L-lysine and D-fructose. None of the amino acid transporters was protected against DEPC by sodium and/or their respective substrates. Sodium influx was inhibited by DEPC (47%) in the absence of any substrate. Our results show a differential sensitivity of sodium-dependent transporters to DEPC and suggest an important role for histidine residues in the molecular mechanisms of these transporters. More experiments are in progress to further characterize the residue(s) involved in these transport inhibitions by DEPC.     

Angiotensin converting enzyme inhibitory activity of amino acid esters of carbohydrates

L-alanyl-D-glucose, L-valyl-D-glucose, L-phenylalanyl-D-glucose and L-phenylalanyl-lactose esters were synthesized enzymatically using two lipases viz., Rhizomucor miehei lipase (RML) and porcine pancreas lipase (PPL) and tested for their potential as inhibitors of angiotensin converting enzyme (ACE) in vitro. The esters exhibited concentration related ACE inhibitory activity. The potency of the various esters measured in terms of IC50 values were as follows: L-phenylalanyl-D-glucose, IC50-0.121 mM (mixture of five diastereomeric esters: 6-O-24.1%; 3-O-23.3%; 2-O-19.2%; 2,6-di-O-16.6% and 3,6-di-O-16.8% from the total yield of 92.4%); L-phenylalanyl-lactose, IC50-0.229 mM (mixture of three diastereomeric esters: 6-O-42.1%; 6'-O-30.9%; and 6,6'-di-O-27.0% from the total yield of 50.58%); alanyl-D-glucose, IC50-0.23 mM (mixture of five diastereomeric esters: 6-O-46.7%; 3-O-11.5%; 2-O-19.9%; 2,6-di-O-6.6% and 3,6-di-O-15.3% from the total yield of 26.5%) and L-valyl-D-glucose, IC50-0.396 mM (mixture of five diastereomeric esters: 6-O-32.4%; 3-O-26.5%; 2-O-26.4%; 2,6-di-O-8.8% and 3,6-di-O-5.9% from the total yield of 68.2%). These in vitro data suggest a potential therapeutic role for the aminoesters of carbohydrates as inhibibitors of ACE.     

Application of central composite rotatable design to lipase catalysed synthesis of m-cresyl acetate

Esterification of m-cresol with acetic acid using porcine pancreas lipase (PPL) was investigated by response surface methodology (RSM). A central composite rotatable design (CCRD) involving 32 experiments of five variables at five levels was employed to analyse the esterification behaviour. The effect of five variables studied namely, m-cresol concentrations (0.005–0.025 mol), enzyme/substrate ratios (0.18–1.22 activity units/millimol, AU/mmol), incubation periods (6–54 h), pH (4–8) and buffer volumes (0–0.2 ml) was useful in arriving at an optimum ester yield. The methodology projected conditions for higher yields up to 6.0 mmol. Validation experiments carried out under these predicated conditions showed good correspondence between experimental and predicted yields. CCRD treatment clearly showed the inhibitory nature of m-cresol in the esterification process. The reaction required the presence of buffer for better conversions and a minimum amount of 0.1 ml buffer was found necessary for this reaction. Buffer pH values around 6.0 and below appeared to favour better esterification than those at pH values in the range 6.0–8.0. However an optimum condition for maximum yield was: incubation period: 54 h; buffer volume: 0.2 ml; pH: 8; E/S ratio: 0.83 AU/mmol; m-cresol: 0.02 mol; predicted yield: 6.0 mmol; experimental yield: 6.4 mmol.     

Control of propranolol intake by direct chromatographic detection of alpha-naphthoxylactic acid in urine

A rapid chromatographic procedure with a C18 column, a mobile phase of 0.15 M sodium dodecyl sulfate (SDS)-10% (v/v) 1-propanol at pH 3 (0.01 M phosphate buffer), and fluorimetric detection, is reported for the control of propranolol (PPL) intake in urine samples, which are injected directly without any other treatment than filtration. The peak of PPL was only observed in samples taken a few hours after ingestion of the drug due to its extensive conjugation and metabolisation. The detection of several unconjugated PPL metabolites was therefore considered: desisopropylpropranolol (DIP), propranolol glycol (PPG), alpha-naphthoxylactic acid (NLT) and alpha-naphthoxyacetic acid (NAC). NLT showed the best characteristics: it eluted at a much shorter retention time than PPL, its concentration in urine samples was greater and it did not present any interference from endogeneous compounds in urine, common drugs or drugs administered in combination with PPL. The limit of quantification, measured as the concentration of analyte providing a relative standard deviation of 20%, was 24 ng/ml, and the day-to-day imprecision was below 4% for concentrations above 200 ng/ml. The procedure allows the routine control of PPL at therapeutic urine levels. Urinary excretion studies showed that the detection of NLT is possible at least up to 20-30 h after oral administration.     

Salt-assisted acid hydrolysis of starch to D-glucose under microwave irradiation

The effect of inorganic salts on the hydrolysis of starch in a microwave field was investigated and it was found that some inorganic salts can effectively accelerate the acid hydrolysis of starch. The yield of D-glucose reached 111 wt% (equal to the theoretical yield).     

Application of multi-component reaction for covalent immobilization of two lipases on aldehyde-functionalized magnetic nanoparticles; production of biodiesel from waste cooking oil

Lipase from Rhizomucor miehei (RML) and Thermomyces lanuginosa lipase (TLL) were immobilized on silica core-shell magnetic nanoparticles (Fe₃O₄@SiO₂) produced by coating Fe₃O₄ core with silica shell. The nanoparticles were functionalized with aldehyde groups followed by immobilization of RML and TLL by using a multi-component reaction in an extremely mild condition. Rapid immobilization of both enzymes (1.5−12 h) with high immobilization yields (81–100%) was observed. The maximum loading capacity of the support was determined to be 81 mg for RML and 97 mg for TLL. The thermal stability of the immobilized derivatives of RML and TLL were greatly improved by retaining 54 and 97 % of their initial activities at 65 °C, respectively. The immobilized preparations were used to produce biodiesel by transesterification of waste cooking oil. In an optimization study, Response Surface Methodology (RSM) and a central composite rotatable design (CCRD) were used to study the effect of amount of biocatalyst, temperature, reaction time, water adsorbent (wt.%) and ratio of t-butanol to oil (wt.%) on the yield of biodiesel production. Biodiesel production yield by immobilized TLL reached 93.1 % under optimal conditions while the maximum yield for RML was 57.5 %. Both immobilized derivatives showed high reusability after 5 cycles of the reaction.     

Investigation of the use of agricultural byproducts for fungal protein production

Cellulose and nutrient salts as well as potato pulp and potato protein liquor (PPL), were used as substrates for the cultivation of Chaetomium cellulolyticum in batch and repeated-batch operations. Using cellulose as the substrate a linear relationship existed between the rates of cell mass formation and acid production. The repeated-batch process was controlled by NaOH consumption using a simple computer model. When the production of cell mass and acid stopped because of a lack of substrate cellulose was fed into the reactor. This occurred within 10 min at which point no NaOH-feed was needed to maintain a constant pH. Repeated-batch operations yielded higher cell concentrations and productivities than batch operations. The relationship between the NaOH and H₂SO₄ consumed, and the fungal mass concentration was complex in cultivation media containing potato pulp and PPL, because various substrates were consumed by the fungus simultaneously and successively. Therefore, for repeated-batch cultivation a constant time interval was used. Repeated-batch cultivation of the fungus on potato pulp and PPL did not yield higher cell concentrations and productivities than did batch cultivation. With the optimal pulp-to-PPL ratio a maximum specific growth rate of 0.61 h¹ was obtained. These investigations indicate, that potato pulp and PPL are well suited to fungal protein production by Chaetomium cellulolyticum for fodder supplement.     

Rhizomucor miehei lipase immobilized on reinforced chitosan–chitin nanowhiskers support for synthesis of eugenyl benzoate

An alternative environmentally benign support was prepared from chitosan–chitin nanowhiskers (CS/CNWs) for covalent immobilization of Rhizomucor miehei lipase (RML) to increase the operational stability and recyclability of RML in synthesizing eugenyl benzoate. The CS/CNWs support and RML-CS/CNWs were characterized using X-ray diffraction, fluorescent microscopy, and Fourier transform infrared spectroscopy. Efficiency of the RML-CS/CNWs was compared to the free RML to synthesize eugenyl benzoate for parameters: reaction temperature, stirring rate, reusability, and thermal stability. Under optimal experimental conditions (50°C, 250?rpm, catalyst loading 3?mg/mL), a twofold increase in yield of eugenyl benzoate was observed for RML-CS/CNWs as compared to free RML, with the former achieving maximum yield of the ester at 62.1% after 5?hr. Results demonstrated that the strategy adopted to prepare RML-CS/CNWs was useful, producing an improved and prospectively greener biocatalyst that supported a sustainable process to prepare eugenyl benzoate. Moreover, RML-CS/CNWs are biodegradable and perform esterification reactions under ambient conditions as compared to the less eco-friendly conventional acid catalyst. This research provides a facile and promising approach for improving activity of RML in which the resultant RML-CS/CNWs demonstrated good operational stability for up to eight successive esterification cycles to synthesize eugenyl benzoate.     

Lipase catalyzed synthesis of organic acid esters of lactic acid in non-aqueous media

Lipases from Rhizomucor miehei (Lipozyme IM20) and porcine pancreas (PPL) were employed as catalysts for the esterification reaction between the hydroxyl group of lactic acid and the carboxyl group of organic acids. Reactions were carried out at both shake-flask and bench-scale levels. Various parameters, such as solvent, temperature, substrate and enzyme concentrations, effect of buffer volume, buffer pH and water volume, were investigated for optimization of yields. While ethylmethyl ketone (EMK) was found to be the best solvent for shake-flask reactions, chloroform gave higher yields at bench-scale level. Detailed studies were carried out with respect to the synthesis of palmitoyl and stearoyl lactic acids. At shake-flask level, maximum yields of 37.5 and 40% were observed in case of palmitoyl and stearoyl lactic acids, respectively, with Lipozyme IM20; at bench-scale level, the maximum yields were 85.1 and 99% respectively, when PPL was employed. Of all the organic acids employed (C(2)--C(18)), only lauric, palmitic and stearic acids gave yields above 50%. At bench-scale level, PPL could be reused for up to three cycles with yields above 40%. Esters prepared were found to conform to Food Chemical Codex (FCC) specifications in terms of acid value, ester value, sodium and lactic acid contents.     

A study on the enzyme catalysed enantioselective hydrolysis of methyl 2-methyl-4-oxopentanoate,a precursor of chiral γ-butyrolactones

Porcine pancreas lipase (PPL) resolution of the α-methyl group of racemic methyl 2-methyl-4-oxopentanoate, a valuable synthetic precursor of fragrances and marine natural products, was enhanced by salt modulation of the enzymatic hydrolysis. For the enantioselective hydrolysis of the title ester, PPL was selected from a series of esterases and lipases, and its enantioselectivity was evaluated by changing the reaction medium parameters. The use of 1.6?mol L^–1 sodium sulfate in phosphate buffer (pH 7.2) improved the enantioselectivity allowing the formation of methyl (2R)-(+)-2-methyl-4-oxopentanoate and (2S)-(–)-2-methyl-4-oxopentanoic acid with an enantiomeric excess of >99% and 71%, respectively. The study showed that a modulation of PPL enantioselectivity could be achieved by using kosmotropic salts in the reaction media. The present method consists of a practical and low-cost option to improve enzymatic kinetic resolution reactions.     

Interface-mediated inactivation of pancreatic lipase by a water-reactive compound: 2-sulfobenzoic cyclic anhydride

2-Sulfobenzoic cyclic anhydride (SBA) rapidly and selectively inactivates porcine pancreatic lipase (PPL) only when added during the hydrolysis of an emulsified ester such as tributyrin or dodecyl acetate. The present data suggest that the inactivation of PPL occurs preferentially at the oil/water interface and not in the aqueous phase, since colipase and bile salt were found to adversely affect the inhibition process. Moreover, it is shown that at a molar ratio of SBA to pure PPL of 1, 40% of the lipase activity was already irreversibly lost. Complete inactivation was observed at SBA to pure PPL molar ratios of 120. A 60% inactivation occurred when 0.5 mol of 3H-labeled SBA was attached per mole of PPL. The SBA-inactivated PPL competes for binding to the dodecyl acetate/water interface as efficiently as the native enzyme. Larger SBA concentrations are required when crude lipase preparations are used as well as with pure PPL in the presence of bile salts and colipase. Lipases were found to have variable sensitivities to SBA inactivation, depending on their origin. In the presence of bile salts and tributyrin at pH 6.0, human gastric lipase activity was not affected by the presence of a 10(6) molar excess of SBA.     

Synthesis of tetrapeptide Bz-RGDS-NH2 by a combination of chemical and enzymatic methods

The tetrapeptide Bz-Arg-Gly-Asp-Ser-NH(2) (Bz-RGDS-NH(2)) was successfully synthesized by a combination of chemical and enzymatic methods in this study. Firstly, the precursor tripeptide Gly-Asp-Ser-NH(2) (GDS-NH(2)) was synthesized by a novel chemical method in four steps including chloroacetylation of l-aspartic acid, synthesis of chloroacetyl l-aspartic acid anhydride, the synthesis of ClCH(2)COAsp-SerOMe and ammonolysis of ClCH(2)COAsp-SerOMe. Secondly, lipase (PPL) was used to catalyze the formation of Bz-RGDS-NH(2) in aqueous water-miscible organic cosolvent systems using Bz-Arg-OEt as the acyl donor and GDS-NH(2) as the nucleophile. The optimum conditions were Bz-Arg-OEt 50 mM; GDS-NH(2) 400 mM; 10 degrees C, 0.1M phosphate buffer, pH 7.5; 60% DMF or 58% DMSO, PPL: 10 mg ml(-1) with the maximum yields of the tetrapeptide of 73.6% for DMF and 70.4% for DMSO, respectively. The secondary hydrolysis of the tetrapeptide product did not take place due to the absence of amidase activity of lipase.     

A posterior probability of linkage-based re-analysis of schizophrenia data yields evidence of linkage to chromosomes 1 and 17

OBJECTIVE: Linkage analysis using 22 Canadian pedigrees identified a promising schizophrenia candidate region on 1q23 with a maximum 2-point HLOD under a recessive model of 5.8 [Brzustowicz et al. 2000]. In the current study, we revisited this data set using a Bayesian linkage analysis technique, namely the posterior probability of linkage (PPL). METHODS: The PPL has been developed as an alternative to traditional linkage analysis. It differs from both LOD scores and 'non-parametric' methods in that it directly measures the probability of linkage given the data, and incorporates prior genomic information. RESULTS: As expected, PPL results for 1q23 supported the previously observed linkage, with an estimated multipoint PPL of 99.7%. However, the PPL supported two further results: a second peak on chromosome 1 at 1p13 with a multipoint with PPL of 70% and a chromosome 17 marker (D17S784 at 17q25) with a multipoint PPL of 44%. CONCLUSIONS: The PPL-based analysis presented has the advantage over other likelihood-based linkage methods in that it avoids maximization and produces a less complex view of the strength of evidence for linkage.     

Hydrolysis of triacetin catalyzed by immobilized lipases: effect of the immobilization protocol and experimental conditions on diacetin yield

The effect of the immobilization protocol and some experimental conditions (pH value and presence of acetonitrile) on the regioselective hydrolysis of triacetin to diacetin catalyzed by lipases has been studied. Lipase B from Candida antarctica (CALB) and lipase from Rhizomucor miehei (RML) were immobilized on Sepabeads (commercial available macroporous acrylic supports) activated with glutaraldehyde (covalent immobilization) or octadecyl groups (adsorption via interfacial activation). All the biocatalysts accumulated diacetin. Covalently immobilized RML was more active towards rac-methyl mandelate than the adsorbed RML. However, this covalent RML preparation presented the lowest activity towards triacetin. For this reason, this preparation was discarded as biocatalyst for this reaction. At pH 7, acyl migration occurred giving a mixture of 1,2 and 1,3 diacetin, but at pH 5.5, only 1,2 diacetin was produced. Yields were improved at acidic pH values and in the presence of 20% acetonitrile (to over 95%). RML immobilized on octadecyl Sepabeads was proposed as optimal preparation, mainly due to its higher specific activity. Each enzyme preparation presented very different properties. Moreover, changes in the reaction conditions affected the various immobilized enzymes in a different way.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Transesterification of phenylalanine by means of chymotrypsin in a continuous fixed bed reactor.

An enzymic transesterification was carried out in a continuously operated fixed bed reactor. The reaction system consisted of immobilized alpha-chymotrypsin (E.C. 3.4.21.1) catalysing the transfer of the L-phenylalanine radical from the racemic propyl ester to 1,4-butanediol, yielding L-phenylalanine 4-hydroxybutyl ester. The desired reaction was accompanied by alcoholysis due to the presence of 1-propanol liberated during the reaction and by hydrolysis of both the propyl and the hydroxybutyl ester. The problem of shifting pH during the reaction due to ester hydrolysis was overcome by adjusting the initial pH of the substrate feed solution appropriately in order to obtain a sufficiently high buffer capacity provided by the free amino group of the esters. Thus, it was possible to work with shifting pH, an obvious disadvantage for operating reactors of low backmixing for this kind of reaction system. The overall reaction scheme was characterized by the appearance of a maximum ester yield as a function of the operating time in case of batch reactors. Surprisingly, the yield was found to become constant as a function of space-time for continuous operation due to a steeper pH drop. The maximum productivity achieved with respect to the hydroxybutyl ester was about 65 mol d-1 l-1 referred to the catalyst volume.     

The selective enzymatic synthesis of lipophilic esters of swainsonine.

The potent and specific inhibitor of Golgi alpha-mannosidase II, swainsonine (SW) has been isolated in high yield from Swainsona procumbens and derivatised by regiospecific enzymatic reactions. In this study the regioselectivity of three commercially available enzymes, subtilisin Carlsberg, porcine pancreatic lipase (PPL) and Candida cylindracea lipase was determined for the acylation of swainsonine in predominantly anhydrous organic medium. The use of subtilisin in pyridine facilitated the single step synthesis of 2-O-butyryl-SW in a 23% yield, whilst catalysis by PPL in tetrahydrofuran gave 2-O-butyryl-SW (6%) and 1,2-di-O-butyryl-SW (31%).