Biocatalytic polyester synthesis: analysis of the evolution of molecular weight and end group functionality

The enzymatic synthesis of polyesters from activated diesters and diols has been investigated. Differences between enzymatic synthesis and traditional chemical condensation processes are discussed. The disappearance of monomers during the initial phase of reaction indicates that enzyme has a higher specificity for transesterification of ester-terminated oligomers. During the intermediate phase, enzymatic polymerization involves a competition between diol and enzyme-bound water for the nucleophilic attack of the acyl enzyme intermediate. Competition between enzymatic transesterification and hydrolysis at different stages of polymerization in nonaqueous media is responsible for termination of polyesters with acid end-groups and also for limiting the polymer molecular weight. The resulting oligoester consists of chains that are either terminated with - OH groups and/or - COOH groups. We have used Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass spectroscopy (MALDI-TOF) along with colorimetric titration techniques to determine the acidity of enzyme-synthesized polyesters. This paper addresses how the enzymatic polymerization proceeds, and compares our results to the growing literature in this field. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 227-239, 1997.     

PEG molecular weight and lateral diffusion of PEG-ylated lipids in magnetically aligned bicelles

Lateral diffusion coefficients of PEG-ylated lipids with three different molecular weight PEG groups (1000, 2000 and 5000) were measured in magnetically-aligned bicelles using the stimulated echo (STE) pulsed field gradient (PEG) ¹H nuclear magnetic resonance (NMR) method. At concentrations below the PEG “mushroom-to-brush” transition, all three PEG-ylated lipids exhibited unrestricted lateral diffusion, with lateral diffusion coefficients comparable to those of corresponding non-PEG-ylated lipids and independent of PEG molecular weight. At concentrations above this transition, lateral diffusion slowed progressively with increasing concentration of PEG-ylated lipid as a result of surface crowding. As well, the lateral diffusion coefficients exhibited a pronounced decrease with increasing PEG group molecular weight and a diffusion-time dependence indicative of obstructed diffusion. We conclude that, while lateral diffusion of PEG-ylated lipids within lipid bilayers is determined primarily by the hydrophobic anchoring group, when crowding at the lipid bilayer surface becomes significant, the size of the extra-membranous domain, in this case the PEG group, can influence lateral diffusion, leading to decreased diffusivity with increasing size and producing obstructed diffusion at high crowding. These findings imply that similar considerations will pertain to lateral diffusion of membrane proteins with large extra-membranous domains.     

PEG molecular weight and lateral diffusion of PEG-ylated lipids in magnetically aligned bicelles

Lateral diffusion coefficients of PEG-ylated lipids with three different molecular weight PEG groups (1000, 2000 and 5000) were measured in magnetically-aligned bicelles using the stimulated echo (STE) pulsed field gradient (PEG) (1)H nuclear magnetic resonance (NMR) method. At concentrations below the PEG "mushroom-to-brush" transition, all three PEG-ylated lipids exhibited unrestricted lateral diffusion, with lateral diffusion coefficients comparable to those of corresponding non-PEG-ylated lipids and independent of PEG molecular weight. At concentrations above this transition, lateral diffusion slowed progressively with increasing concentration of PEG-ylated lipid as a result of surface crowding. As well, the lateral diffusion coefficients exhibited a pronounced decrease with increasing PEG group molecular weight and a diffusion-time dependence indicative of obstructed diffusion. We conclude that, while lateral diffusion of PEG-ylated lipids within lipid bilayers is determined primarily by the hydrophobic anchoring group, when crowding at the lipid bilayer surface becomes significant, the size of the extra-membranous domain, in this case the PEG group, can influence lateral diffusion, leading to decreased diffusivity with increasing size and producing obstructed diffusion at high crowding. These findings imply that similar considerations will pertain to lateral diffusion of membrane proteins with large extra-membranous domains.     

Co-precipitation of protein and polyester as a method to isolate high molecular weight DNA

DNA isolation is often the limiting step in genetic analysis using PCR and automated fragment analysis due to low quality or purity of DNA, the need to determine and adjust DNA concentrations after isolation etc. Several protocols have been developed which are either safe and provide good quality DNA or hazardous and provide excellent quality DNA. In this brief communication I describe a new and rapid method of DNA isolation which employs the co-precipitation of protein and polyester, in the presence of acetone, to remove contaminating proteins from a lysed-tissue sample, thus leaving high quality pure DNA. The advantages of this method are increased safety over the phenol:chloroform and the chaotrophic salt methods and increased purity over the salting-out method. Since the concentrations of DNA isolated using this method are relatively consistent regardless of the amount of starting tissue (within limits), adjustments of the DNA concentrations before use as templates in PCR's are not necessary.     

Method for lipase-catalyzed carbonate synthesis via one- and two-step alkoxycarbonylation reactions

Lipase-catalyzed alkoxycarbonylation methods offer potential advantages over the currently practiced industrial scale chemical synthesis of carbonates. We report a method for synthesis of organic carbonates via lipase-catalyzed alkoxycarbonylation between diphenyl carbonate and various alcohols in hexane. This method utilizes precursors that are readily available and does not involve extensive purification of the intermediate. In a two-step process, the two phenyl groups of diphenyl carbonate were substituted by two alcohol nucleophiles. The approach was demonstrated for two-step synthesis of 14 different disubstituted carbonate products. The rates of reaction for the two steps were much slower if the order of nucleophile addition was reversed. Under optimal conditions, complete conversion of diphenyl carbonate occurred within 8-15 h at 50 degrees C, which is a significant improvement from 50-90 h at 24 degrees C. A kinetic model for the alkoxycarbonylation reaction was derived based on the Michaelis-Menten equation, which simplified to first-order kinetics at low and equimolar concentration of substrates.     

Ultrasonic pretreatment for lipase-catalyzed synthesis of 4-methoxy cinnamoyl glycerol

4-Methoxy cinnamoyl glycerol (4MCG) is a very promising UV filters material in personal care products. In order to effectively improve the yield of 4MCG, a systematic study on ultrasonic pretreatment enzymatic esterification for 4MCG products was carried out. An ultrasonic frequency of 35 kHz, ultrasonic power of 150 W and ultrasound irradiation time of 1.5 h was determined to guarantee satisfactory degree of esterification and lipase activity. The optimum production was achieved in organic solvent system at 65 °C with 4MCA to glycerol molar ratio of 1:5, enzyme amount of 15 mg/mL, resulting in a monoester yield of above 66% and 55% after 48 h and 24 h of reaction under ultrasonic pretreatment, respectively. The experimental kinetic data were studied. The reactions were modeled by a system of sequential first-order rate expressions, kinetic parameters were estimated from experimental data fit to the model equations. Results show that the monoester yield in the ultrasonic pretreatment process (24 h) were above 1.5-fold as that in mechanical stirring process without essential damaging to lipase activity. The enzymatic method using ultrasonic pretreatment was obviously superior to the mechanical stirring for enzymatic method and chemical method in terms of conversion rate and the monoester yield. These results are of great significance for applying ultrasonic pretreatment method to prepare 4MCG.     

A comparison of different strategies for lipase-catalyzed synthesis of partial glycerides

Summary Four different approaches for the synthesis of monolaurylglycerol (MLG) by non specificPseudomonas cepacia lipase in a crude and purified form have been studied: a. The direct esterification of glycerol by lauric acid in bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsion systems; b. the transesterification of glycerol by vinyl laurate in the presence or not of any solvent; c. solid-phase glycerolysis of trilaurin; and, d. transesterification of protected glycerol, 1,2-O-isopropylidene glycerol, by vinyl laurate, in the presence or not of any solvent. It was found that in the two latter cases (d and c) the formation of pure MLG was occurred, while in the first two cases (a and b) apart of MLG the formation of DLG was also observed.     

Application of lipase-catalyzed transformations for the synthesis of insect pheromones and related compounds

This review describes efficient means of preparing optically pure insect pheromones and related compounds via lipase-catalyzed enantioselective reaction on a large scale. (1) A new synthesis of the Japanese beetle pheromone, (R,Z)-(−)-5-(1-decenyl)oxacyclopentan-2-one, established by a combination of two lipase-catalyzed transformation was demonstrated. (2) A chemico-enzymatic procedure for the syntheses of both enantiomers of cupreous chafer beetle pheromone, (R,Z)- and (S,Z)-5-(1-octenyl)oxacyclopentan-2-one, was described. (3) An optical resolution of (±)-2,3-epoxy-8-methyl-1-nonanol, the key intermediate of the synthesis of gypsy moth pheromone, was demonstrated. (4) A practical chemico-enzymatic synthesis of (+)-disparlure in large scale was demonstrated. (5) A facile synthesis of carboxyalkyl acrylate, which is special monomers in the synthesis of the new polymers, by two lipase-catalyzed regioselective reactions was described.     

Influence of PEG induced drought stress on molecular and biochemical constituents and seedling growth of Egyptian barley cultivars

In order to investigate the effects of drought stress on germination components of barley cultivars, a laboratory experiment was conducted in a factorial randomized complete design with four replications. The controlled experiment included ten of Egyptian barley cultivars namely; (Giza 123, 124, 125, 126, 127, 129, 130, 134, 135 and 2000) as first factor. The second factor included 4 levels of drought stress inducer by applying 0, 5, 10 and 20% of polyethylene glycol-6000 (PEG) which is equivalent to four osmotic potential levels including ?0.001, ?0.27, ?0.54 and ?1.09 MPa, respectively. The results showed that, the highest reduction was related to the drought level of 20% PEG among the barley cultivars. The best cultivars in terms of germination traits were Giza 134, Giza 127, and Giza 126 this indicate their tolerance to drought stress and Giza 130, 135, 2000 cultivars was moderately tolerance and remaining is less tolerance. The protein band 27 kDa and 78 kDa showed high intensity after stress in almost all cultivars. Those two protein bands their exciting was very clear in treated barley leaf tissue. It could be related to dehydrine and oxygen evolving enhancer protein 2 (OEE2) which involved in drought stress tolerance response. Cultivars Giza 127, 130 and 134 showed highest tolerance response under drought stress. The antioxidant enzymes PAGE pattern of Peroxidase (POX), Sodium dismutase (SOD) and Ascorbate peroxidase (APX) for Barley cultivars under drought stress revealed a high activities for Giza 126, 127, 134, 136 and 2000 under ?0.5 MPa osmotic stress by PEG in most of their isoforms. Based on similarity coefficient values the highest values were 1.0 with 100% similarly between tolerant cultivars Giza 130 and Giza 127. Similarly between the susceptible cultivars 125 and Giza 129 was 60%.These data confirmed by the growth parameters which we ranked as tolerant to drought stress.     

Influence of dissolved oxygen tension and agitation speed on alginate production and its molecular weight in cultures of Azotobacter vinelandii*

The alginate production by Azotobacter vinelandii, as well as the molecular weight of the polymer, are strongly influenced by the dissolved oxygen tension (DOT) and stirring speed of the culture. Under high DOT (5% of air saturation), the bacteria produced more alginate (4.5 g/l) than that obtained at low (0.5%) oxygen tension (1.0 g/l) in cultures conducted at 300 rpm. On the other hand, under constant DOT (3%), the higher the stirring speed (from 300 to 700 rev./min), the higher the specific growth rate and the alginate production rate. However, low agitation speed (300 rev./min) lead the culture to produce a polymer of high molecular weight (680 000 g/g mol) whereas a low molecular weight (352 000 g/g mol) alginate was isolated from cultures conducted at high (700 rev./min) stirring speed. At 700 rev./min, the MMW increased to a plateau between 1 and 3% DOT and then decreased to a minimum of 0.11 x 10(6) g/g mol at 7%. Microscopic observations revealed the presence of cell aggregates (one order of magnitude larger than individual cells) when the culture was conducted at 300 rev./min. Oxygen gradients occurring within the aggregates could be responsible of this phenomenon. At high agitation rate, the MMW of the alginate dropped towards the end of the culture in all conditions evaluated. Alginase activity was detected, which would be responsible for this phenomenon.     

Low molecular weight RNA of Drosophila cells which is induced by heat shock--I. Synthesis and its effect on protein synthesis

When Drosophila cells were heat shocked, low molecular weight RNA of a novel type was synthesized and accumulated in the cytoplasm. This RNA, which is about 320 nucleotides long, inhibited the translation of Drosophila mRNAs in rabbit reticulocyte lysate. It remains to be clarified whether this RNA is involved in the regulation mechanism of heat shock at the translational level.     

Modelling and experimental studies on lipase-catalyzed isoamyl acetate synthesis in a microreactor

A lipase-catalyzed synthesis of isoamyl acetate was studied in a continuously operated pressure-driven microreactor. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. By adjusting flow rates of both phases, a parallel laminar flow with liquid–liquid boundary in the middle of the microchannel could be reestablished and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. A three-dimensional mathematical model was developed, considering the velocity profile developed for steady-state conditions between two immiscible fluids. The model contained convection, diffusion, and enzyme reaction terms, where esterification rate was described with a Ping-Pong Bi-Bi mechanism and inhibition by both substrates. Experimental data, which were in good agreement with model simulations, have demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations, which is much faster as in any literature reported so far. According to model simulations, obtained by non-equidistant finite differences numerical solutions of complex non-linear equations system, further microreactor design and process optimization are feasible.     

High yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry

Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 °C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications.     

Design and synthesis of low molecular weight compounds with complement inhibition activity

An attempt was made to synthesize a series of non-cytotoxic low molecular weight compounds of varying substitutions and functionalities having pharmacophore activity like carbonyl compounds, carboxylic acid and bioisosteres like tetrazole and phenyl acrylic acid. The in vitro assay of these analogues for the inhibition of complement activity revealed significant inhibitory activity for varying substituents and, particularly, for bioisosteres, that is, tetrazole and phenyl acrylic acid derivatives.     

Effect of alcohol chain length on the optimum conditions for lipase-catalyzed synthesis of adipate esters

Immobilized Candida antarctica lipase B, Novozym® 435, was used in the esterification of adipic acid and alcohols with different chain lengths (C₁–C₁₈). Optimum conditions for the synthesis of adipate esters were obtained using response surface methodology (RSM) with respect to important reaction parameters including time, temperature, substrate molar ratio and amount of enzyme. Alcohol chain length specificity of the enzyme in the synthesis of adipate esters was also determined. Minimum reaction time (215 min) for achieving maximum ester yield was obtained for butyl alcohol. Methanol required an increased time (358 min) and enzyme amount (10.2%, w/w) for attaining maximum yield. The maximum required temperature and time of 65°C and 523 min, respectively, were obtained for the synthesis of dioctadecyl adipate. The results demonstrate that alcohol chain length is a determining parameter in optimization of the lipase-catalyzed synthesis of adipate esters. Reactions under optimized conditions yielded a high percentage of esterification (>97%). The optimum conditions can be used to scale up the process.     

Optimization and kinetic study of immobilized lipase-catalyzed synthesis of ethyl lactate

Abstract

Enzymatic synthesis of ethyl lactate catalyzed by immobilized lipase has been investigated. The reaction variables (including the molar ratio of ethanol to acid, total substrate amount, temperature, reaction time and rotation speed) were selected in accordance with the Plackett–Burman design and were further optimized via response surface methodology. The molar ratio of ethanol to acid, total substrate amount and reaction time were screened out as significant variables for the optimization study. A 20-run, full-factorial, central composite design was used to construct the statistical model and the optimal conditions obtained were as follows: molar ratio of ethanol to acid of 8.3:1, total substrate amount of 0.4 g, reaction time of 26.87 h with temperature of 55°C and rotation speed of 150 rpm. Under the optimal conditions, the yield of ethyl lactate was up to 24.32%; close to the 25.13% obtained using the commercial lipase, Novozym 435. Due to the low cost and simple immobilization process, the lipase prepared in the present work could have great potential in enzymatic applications. Additionally, a kinetic model with inhibition by both ethanol and lactic acid following a ping-pong bi-bi mechanism was proposed.     

Low molecular weight polyethylenimine-graft-Tween 85 for effective gene delivery: synthesis and in vitro characteristics

The development of safe and efficient gene delivery systems is still a challenge for successful gene therapy. In this work, low molecular weight polyethylenimine (PEI 2K) was modified by Tween 85, which bears three oleate chains. Tween 85 modified PEI 2K (TP) could condense DNA efficiently, and TP/DNA complexes (TPCs) showed high resistance to salt-induced aggregation and enzymatic degradation. In addition, TP did not show the obvious cytotoxicity. The introduction of Tween 85 led to a significant increase in the cellular uptake of complexes with higher transfection efficiency, which was strongly inhibited by the addition of free Tween 85 in MCF-7/ADR cells, but not in MCF-7 cells. These results indicated that TP could be a potentially safe and effective copolymer for gene delivery, and TPCs could be taken up mainly by Tween 85-mediated endocytosis in MCF-7/ADR cells.