Evaluation of green solvents: Oil extraction from oleaginous yeast Lipomyces starkeyi using cyclopentyl methyl ether (CPME)

Cyclopentyl methyl ether (CPME) was evaluated for extracting oil or triacylglycerol (TAG) from wet cells of the oleaginous yeast Lipomyces starkeyi. CPME is a greener alternative to chloroform as a potential solvent for oil recovery. A monophasic system of CPME and biphasic system of CPME:water (1:0.7) performed poorly having the lowest TAG extraction efficiency and TAG selectivity compared to other monophasic systems of hexane and chloroform and the biphasic Bligh and Dyer method (chloroform:methanol:water). Biphasic systems of CPME:water:alcohol (methanol/ethanol/1‐propanol) were tested and methanol achieved the best oil extraction efficiency compared to ethanol and 1‐propanol. Different biphasic systems of CPME:methanol:water were tested, the best TAG extraction efficiency and TAG selectivity achieved was 9.9 mg/mL and 64.6%, respectively, using a starting ratio of 1:1.7:0.6 and a final ratio of 1:1:0.8 (CPME:methanol:water). Similar results were achieved for the Bligh and Dyer method (TAG extraction efficiency of 10.2 mg/mL and TAG selectivity of 66.0%) indicating that the biphasic CPME system was comparable. The fatty acid profile remained constant across all the solvent systems tested indicating that choice of solvent was not specific for any certain fatty acid. This study was able to demonstrate that CPME could be used as an alternative solvent for the extraction of oil from the wet biomass of oleaginous yeast. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1096–1103, 2017     

Bioconversion of Phosphatidylserine by Phospholipase D from Streptomyces racemochromogenes in a Microaqueous Water-Immiscible Organic Solvent

Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) in a biphasic system was limited by the hydrolysis reaction. A biphasic system can produce a large amount of water. To solve this problem, a microaqueous water-immiscible organic solvent was used for the first time in the bioconversion of phosphatidylserine (PS). The transphosphatidylation among 40 µmol PC, 800 µmol L-serine, and 0.17 U/mL PLD in 2.133 mL of butyl acetate with 6.25% water (V/V) was conducted at a trans-phosphatidylation rate of 88% (mol/mol), and no hydrolytic reaction was observed. Compared to commonly used biphasic systems, this system shows a similar transphosphatidylation rate, whereas the undesirable hydrolysis of phospholipids was completely suppressed.     

A comparison of lipase-catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.     

Immobilization of Thermoanaerobium brockii alcohol dehydrogenase on SBA-15

The activity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH) adsorbed on mesoporous silica SBA-15 was compared with that of the free enzyme in water and in biphasic system (water phase up to 50% v/v water). TBADH was active at a water concentration ≥10% v/v. In the reduction reaction of sulcatone to sulcatol carried out in biphasic systems, the yield obtained with SBA-15-adsorbed TBADH was up to 5.5-fold higher than that with the free enzyme, which suggests a higher stability of the immobilized enzyme toward the organic solvent. The nature of the organic solvent substantially influenced the degree of conversion that, for example, was 7.4% in toluene and 31.6% in petroleum ether.     

Catalysis by amine oxidases in nonaqueous media

The synthetic potential of amine oxidases was examined in different reaction systems, ranging from aqueous solutions to organic solvents with low water content. Substantial conversion was achieved in biphasic systems, which eliminated the product inhibition observed in the aqueous system. The conversion was particularly high in the more hydrophobic solvents. The use of low water systems was studied using amine oxidase immobilized on celite and pre-equilibrated in a salt hydrate environment to reach a constant water activity. Addition of water in the solvent was shown to be unnecessary, with significant conversion being attained through the water supplied by pre-equilibration of the immobilized enzyme at a_w=0.55. The use of organic solvent-containing reaction systems thus presents a convenient method for oxidising poorly water-soluble amines using amine oxidases.     

Operational concept for the improved synthesis of (R)-3,3'-furoin and related hydrophobic compounds with benzaldehyde lyase

Biphasic reaction systems for enzyme catalysis are an elegant way to overcome limited solubility and stability of reactants and facilitate continuous processes. However, many synthetically useful enzymes are not stable in biphasic systems of water and organic solvent. The entrapment in polymer beads of polyvinyl alcohol has been shown to enable the stable operation of enzymes unstable in conventional biphasic reaction systems. We report the extension of this concept to continuous operation in a fluidised bed reactor. The enzyme benzaldehyde lyase was used for the continuous synthesis of enantiopure (R)-3,3'-furoin. The results show enhanced stability with half-life times under operation conditions of more than 100 h, as well as superior enzyme utilisation in terms of productivity. Furthermore, racemisation and oxidation of the product could be successfully prevented under the non-aqueous and inert reaction conditions.     

Selection of Xenobiotic-Degrading Microorganisms in a Biphasic Aqueous-Organic System

Microbial selection on mixtures of chlorinated and nonchlorinated compounds that are poorly soluble in water and/or toxic to growing microbial cells was examined in both biphasic aqueous-organic and monophasic aqueous systems. A biphasic system in which silicone oil was used as the organic phase permitted the acceleration of acclimation, leading to rapid selection and to an increase in xenobiotic compound degradation. In contrast, acclimation, selection, and degradation were very slow in the monophasic aqueous system. The variation in microbial growth rate with the degree of dispersion (i.e., dispersion at different silicone oil concentrations and agitation rates), and cell adhesion to the silicone oil indicate that the performance of the biphasic aqueous-organic system is dependent on the interfacial area between the two phases and that microbial activity is important at this interface. Therefore, the biphasic water-silicone oil system could be used for microbial selection in the presence of xenobiotic compounds that are toxic and have low water solubility.     

Evaluation of parallel milliliter-scale stirred-tank bioreactors for the study of biphasic whole-cell biocatalysis with ionic liquids

As clear structure-activity relationships are still rare for ionic liquids, preliminary experiments are necessary for the process development of biphasic whole-cell processes involving these solvents. To reduce the time investment and the material costs, the process development of such biphasic reaction systems would profit from a small-scale high-throughput platform. Exemplarily, the reduction of 2-octanone to (R)-2-octanol by a recombinant Escherichia coli in a biphasic ionic liquid/water system was studied in a miniaturized stirred-tank bioreactor system allowing the parallel operation of up to 48 reactors at the mL-scale. The results were compared to those obtained in a 20-fold larger stirred-tank reactor. The maximum local energy dissipation was evaluated at the larger scale and compared to the data available for the small-scale reactors, to verify if similar mass transfer could be obtained at both scales. Thereafter, the reaction kinetics and final conversions reached in different reactions setups were analysed. The results were in good agreement between both scales for varying ionic liquids and for ionic liquid volume fractions up to 40%. The parallel bioreactor system can thus be used for the process development of the majority of biphasic reaction systems involving ionic liquids, reducing the time and resource investment during the process development of this type of applications.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

1H- and 2H-NMR relaxation in serum albumin solutions

The proton and deuteron relaxation times T1 and T2 were investigated in water and heavy water solutions of fatless human serum albumin. The temperature, concentration and Larmor precession frequency dependences can be well described by the conception of fast exchange in a simple biphasic model of water molecules rotation in the first hydration layer with slight anisotropy of motion. In the protonated systems the intermolecular dipole-dipole relaxation mechanism must be taken into account.     

Enzymes in preparative organic synthesis: Coincidence of the pH optimum for catalyst effectiveness with the pH optimum for the catalyzed reaction equilibrium

The study concerned the pH profile of the apparent equilibrium constant for synthesis of N-benzoyl-L -phenylalanine ethyl ester from the respective acid and ethanol in the biphasic system chloroform + 5% (v/v) water. The substitution of water (as a reaction medium) for the biphasic aqueous–organic system shifted the pH profile toward neutral pH values. As a result the pH range thermodynamically conducive to synthesis of the final product in the biphasic system coincided with the pH optimum of the catalytic activity of the enzyme used (α-chymotrypsin). This approach should, in principle, be considered as general: first, per se it is independent of a catalyst (enzyme) nature; second, the biphasic method helps the shift ionic equilibria involving not only organic acids, but also bases. A physical mechanism of the ionic equilibrium shift is the same is both cases, namely, a preferable extraction from water into an organic phase of one generally nonionic (more hydrophobic) form of the reagent.     

Methylosinus trichosporium OB3b whole-cell methane monooxygenase activity in a biphasic matrix

 Reconstituted whole-cell preparations of lyophilized Methylosinus trichosporium OB3b were used to demonstrate soluble methane monooxygenase activity in a two-phase (biphasic) matrix consisting of a buffered aqueous phase and 2,2,4-trimethylpentane (isooctane). The rate of conversion of gaseous propylene to propylene oxide, a non-metabolized liquid, was used as the primary measure of enzyme activity. Appreciable soluble methane monooxygenase activity was detected when the volume of the aqueous phase represented at least 1% of the total volume, although the initial rate of product formation did increase as the volume of the aqueous phase increased. In comparison to the aqueous system, the specific rate and yields in the biphasic system were much less sensitive to increases in the concentrations of formate and protein (the methane monooxygenase). However, there was some evidence that the enzyme system was more stable in the biphasic matrix, since the rate of propylene oxide formation remained linear for an extended period of time. V _(app.) in the biphasic system decreased by a factor of 0.6 relative to the same parameter in the aqueous system. Conversely, K _m(app.) for propylene was 1.6 times greater in the biphasic system. Hence, the apparent catalytic efficiency in the aqueous system was four times that in the biphasic system, as indicated by a decrease in the corresponding ratios of V _(app.) to K _m(app.). Received: 21 July 1995/Received last revision: 1 February 1996/Accepted: 5 December 1996     

A new approach to preparative enzymatic synthesis. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 9, Pages 1351-1361

A new approach to preparative organic synthesis in aqueous-organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system "water-water-immiscible organic solvent." Thereby the enzyme is localized in the aqueous phase-this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations "water-water-miscible organic solvent," in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L-tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L-tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log P_octanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between ? 1.1 and 8.8 were examined. Organic solvents with log P_octanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL^?1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.     

Effects of water on equilibria catalysed by hydrolytic enzymes in biphasic reaction systems

In the literature on hydrolase-catalysed synthetic reactions in aqueous-organic biphasic systems, it has been stated that low water concentrations contribute to favourable shifts in equilibrium. It is argued here that this is not a sufficient condition for mass action effects of water. A simple method of treating such equilibria is suggested, using the thermodynamic activity of water. Only when its activity is significantly reduced below 1 can water contribute to a shift of equilibrium in favour of synthetic products. Such a reduction is not necessarily obtained by creating a biphasic system, however low the water content, but requires that the aqueous phase becomes a very concentrated solution/dispersion of hydrophilic species.     

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study

Background

Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.

Methods and Findings

The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001).

Conclusions

The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.     

Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

ABSTRACT: BACKGROUND: Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. RESULTS: For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems.Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system.Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. CONCLUSIONS: Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.     

Peptide synthesis enzymatically catalyzed in a biphasic system: Water–water–immiscible organic solvent

Synthesis of a peptide bond is suggested to be enzymatically catalysed in a biphasic system “water–water-immiscible organic solvent”. The pH dependence of the apparent equilibrium constant is studied for synthesis of N-acetyl-L -tryptophanyl-L -leucine amide from N-Acet-Trp andL -Leu-NH₂. The reaction was performed in the biphasic system ethyl acetate plus water [from 2 to 2% (v/v)] in the presence of α-chymotrypsin. The suggested approach is preparative value: with the stoichiometric ratio of the reagents, [N-Acet-L -Trp] = [L -Leu-NH₂] = 2 × 10^?3M, the yield is practically 100% (in water, with other conditions being the same, the yield is not over 01.%).     

Dynamic interactions between enzyme activity and the microstructured environment

A new approach for the study of an enzyme's relationship with its own reaction medium has been developed. One technique of micellar enzymology is the use of pseudohomogeneous systems composed of surfactant/water/organic solvent. In such systems, the physicochemical properties and textures of the medium depend on the relative ratios of the different components. Enzymes are catalytically active in such systems and up to the present have been studied in different microenvironments, such as micelles, microemulsions and lyotropic liquid crystals. Our purpose was to develop a system in which the enzyme could, by its activity, modify one of the components in such a way that the relative ratios among them changed sufficiently to produce a transition from one phase domain to another. The three components, water (or glucose in water), octanol and octyl-beta-D-glucoside, form a classical ternary water/oil/surfactant system. The relevant phase diagram shows different macroheterogeneous phases and microstructured domains. The enzyme beta-D-glucosidase hydrolyses octyl-beta-D-glucoside to form glucose and octanol. The enzyme was found to change the relative ratios of water (or glucose in water), octanol and octyl-beta-D-glucoside in such a manner that the physicochemical structure of the medium was modified. At the beginning of the reaction beta-D-glucosidase was present in a micellar solution of octyl-beta-D-glucoside in water. As the enzymatic reaction proceeded, the medium became biphasic. One of the two phases was the micellar solution of octyl beta-D-glucoside in water, while the other phase was either a microemulsion or a liquid crystalline phase. In addition the enzyme, through its catalytic activity, was able to modify the physiocochemical properties of the reaction medium.