Continuous in situ water activity control for organic phase biocatalysis in a packed bed hollow fiber reactor

Packed bed hollow fiber membrane reactors were used to carry out organic phase biocatalysis at constant water activity. The performance of the device was tested by carrying out the esterification of dodecanol and decanoic acid in hexane. Lipase from Candida rugosa, immobilized on microporous polypropylene and packed in the shell space of the reactor, was used to catalyze the reaction. In situ water activity control was accomplished by pumping appropriate saturated salt solutions through the microporous hollow fiber polypropylene membranes. Water generated by reaction in the organic phase, pumped continuously through the shell of the reactor, was transferred into the bulk of the aqueous phase under the water activity gradient. The reactor performance was found to be strongly dependent on the controlling water activity. By carefully selecting this control activity it was found possible to obtain complete esterification. The water activity of the organic phase could be maintained very close to that of the saturated salt solution used. The reactor could be operated in the continuous mode for 100 h without any degradation in its performance. (c) 1996 John Wiley & Sons, Inc.     

Whole-cell biocatalysis in organic media

The use of water-immiscible organic solvents in whole-cell biocatalysis has been exploited for biotransformations involving sparingly water-soluble or toxic compounds. These systems can overcome the problem of low productivity levels in conventional media due to poor substrate solubility, integrate bioconversion and product recovery in a single reactor, and shift chemical equilibria enhancing yields and selectivities; nevertheless, the selection of a solvent combining adequate physicochemical properties with biocompatibility is a difficult task. The cell membrane seems to be the primary target of solvent action and the modification of its characteristics the more relevant cellular adaptation mechanism to organic solvent-caused stress. Correlations between the cellular toxicity or the extractive capacities of different solvents and some of their physical properties have been proposed in order to minimize preliminary, solvent-selection experimental work but also to help in the understanding of the molecular mechanisms of toxicity and extraction. The use of whole cells in organic-media biocatalysis provides a way to regenerate cofactors and carry out bioconversions or fermentations requiring multi-step metabolic pathways; some processes already are commercially exploited. Immobilization can further protect cells from solvent toxicity, and has thus been effectively used in organic solvent-based systems. Several examples of extractive fermentations and other whole-cell bioconversions in organic media are presented.     

Salt hydrates for water activity control with biocatalysts in organic media

Addition of solid salt hydrate(s) to the reaction mixture is a convenient method of water activity control. This article discusses the theoretical background to their use, and gives a compilation of data on the water activity values produced by 48 hydrate pairs of possible use in this application.     

A water activity control system for enzymatic reactions in organic media

A water activity control system for enzymatic synthesis in organic media, for litre-scale reactors has been constructed. Water activity, a(w), is a key factor when using enzymes in non-conventional media and the optimum value varies for different enzymes. The control system consists of a water activity sensor in the headspace of a jacketed glass reactor (equipped with narrow steel tubes to introduce air), gas-washing bottles containing blue silica gel (a(w)=0) and water (a(w)=1), a PC to monitor water activity and a programmable logic controller (PLC) to control the water activity. The system was evaluated by adjusting water activity in the medium, with a deviation from the set point of less than +/-0.05. Synthesis of cetyl palmitate, under controlled water activity and catalysed by two different lipase preparations, namely, Novozym 435 (immobilised Candida antarctica lipase B) and immobilised Candida rugosa lipase, were also performed. Novozym 435 catalyses reactions very well at extremely low water activity while C. rugosa lipase shows low activity for a(w)<0.5.     

Measurement of pH changes in an inaccessible aqueous phase during biocatalysis in organic media

Summary The pH of the aqueous phase trapped within biocatalyst particles in organic media may be measured using very hydrophobic esters of fluorescein. These remain completely in a pentan-3-one phase, but they ionise there in response to the pH of an equilibrated aqueous phase. They show that the catalyst pH may be significantly shifted from the pre-adjusted value by partitioning of an acidic reactant (N-formyl-tyrosine during chymotrypsin-catalysed esterification) or product (acetic acid during lipase-catalysed ester hydrolysis).     

On-line water removal during enzymatic asymmetric esterification in organic media

Summary Enantioselective esterification of 2-bromopropionic acid with n-butanol using Candida cylindracea lipase was carried out in n-pentane at various initial water contents. Reaction rate as well as enantioselectivity decreased at high water content. A heteroazeotropic distillation method was applicable to remove the excess water continuously and to work at the optimum reaction conditions.     

Whole cell biocatalysis in nonconventional media

Summary In this paper biocatalytic reactions carried out by whole cells in nonconventional media are reviewed. Similar relationships are observed between solvent hydrophobicity and catalytic activity in reactions carried out by isolated enzymes and whole cells. In addition to the effect of organic solvent on biocatalyst stability, microbial cells are susceptible to damaging effects caused by the organic phase. In general, more hydrophobic solvents manifest lower toxicity towards the cells. Whole cell biocatalysts require more water than isolated enzymes and two-phase systems have been most widely used to study whole cell biocatalysis. Immobilization makes cell biocatalysts more resistant to organic solvents and helps achieve homogeneous biocatalyst dispersion. Cell entrapment methods have been widely used with organic solvent systems and mixtures of natural and/or synthetic polymers allow adjustment of the hydrophobicity-hydrophilicity balance of the support matrix. Some examples of stereoselective catalysis using microbial cells in organic solvent media are presented.     

非常规介质中细胞生化反应研究进展

对完整细胞在非常规介质中的生物催化反应进行了回顾,分别总结了产物为醇,甾体,有机酸,生物大分子及其它各类反应的研究进展。并从溶剂和细胞两种角度对主要的研究方法进行了阐述 。     

Optimization of organic solvent in multiphase biocatalysis

The microbial epoxidation of propene and 1-butene was used to study some fundamental aspects of two-liquid-phase biocatalytic conversions. Introduction of a water-immiscible organic solvent phase in a free-cell suspension gave rise to a series of undesired phenomena, e.g., inactivation by the solvent, clotting of biomass, and aggregation of cells at the liquid-liquid interface. Immobilization of the cells in hydrophilic gels, e.g., calcium alginate, prevented direct cell-organic solvent contact and the related clotting and aggregation of biomass. However, the gel entrapment did not seem to provide additional protection against the organic solvent. The influence of various organic solvents on the retention of immobilized-cell activity was related to solvent properties like the polarity (as expressed by the Hildebrand solubility parameter) and the molecular size (as expressed by the molecular weight or molar volume). High activity retention was favored by a low polarity in combination with a high molecular weight. The solubility parameter also proved useful to describe the capacity of various organic solvents for oxygen and alkene oxides. This facilitated the optimization of the solvent polarity.     

Protein-protein interaction in low water organic media

Trypsin either modified with polyethylene glycol or as a suspended powder was used to catalyze digestion of protein substrates in benzene in order to get insight into protein-protein interactions in water-immiscible organic media. Depending on whether suspended or soluble trypsin was used, catalysis was found to proceed differently. In the first case, the amount of water in the reaction mixture (up to 1% v/v) appeared to be critical, and adsorption of water from the reaction medium by the protein substrate allowed it to behave as a hydrophilic support material comparable to that involved in immobilized enzymes. In the latter case, the presence of an additional nucleophile was a prerequisite for catalysis to proceed, and thus both water and nucleophile concentrations had some influence on trypsin activity. Phe-NH(2) was the most potent nucleophile for proteolysis catalyzed by polyethylene glycol-modified trypsin in organic media containing 1-2% water (v/v). The organic solvent-soluble enzyme was found to bind reversibly to the protein substrate as a function of both extent of hydration of the reaction medium and time of incubation. The overall results strongly suggested that modified trypsin catalyzed peptide bond hydrolysis at the protein substrate-organic solvent interface. Peptide mapping of bovine insulin digest by reversed-phase high-performance liquid chromatography definitely showed that enzyme-catalyzed proteolysis did occur in organic solvents with a concomitant and significant transpeptidation reaction.     

Chemical modification of alpha-chymotrypsin for obtaining high transesterification activity in low water organic media

Alpha-chymotrypsin was made more hydrophilic by modifying 11 (out of 16) ε-amino groups with pyromellitic dianhydride. The hydrophilic preparation was precipitated with n-propanol. This preparation gave significantly higher initial rates at the optimum a_w (127.51 nmol mg^?1 min^?1 in n-octane and 21.30 nmol mg^?1 min^?1 in acetonitrile at a_w=0.33) compared with the lyophilized preparation (53.50 nmol mg^?1 min^?1 in n-octane and 0.26 nmol mg^?1 min^?1 in acetonitrile at a_w=0.97). FT-IR showed that the precipitate of modified alpha-chymotrypsin has a higher content of alpha-helices and beta-sheets compared to the lyophilized powder.     

Effects of substrate pretreatment and water activity on lipase-catalyzed cellulose acetylation in organic media

Lipase-catalyzed acetylation of cellulose solubilized in the dimethyl sulfoxide/paraformaldehyde organic solvent system was conducted with lipase A12 from Aspergillus niger. The accompanying side cellulase activity of the A. niger lipase partly accounted for the enhanced acetylation mediated by the enzyme, via facilitating the partial degradation of cellulose substrate as evidenced by high-performance size exclusion chromatograph analysis. The enzymatic cellulose acetylation was improved by substrate pretreatment with cellulase or ultrasound by 18 and 14%, respectively, as a result of the reduced substrate molecular size. Additionally, the ultrasound-pretreated cellulose as the starting substrate was beneficial for the cellulose solution preparation due to the increased accessible surface of cellulose as evidenced by its increased sedimentation volume and SEM micrographs. The effect of thermodynamic water activity (aw) on lipase catalytic activity in organic media was also investigated. The maximum acetylation extent (nearly 11 wt %) occurred at aw = 0.52, which was improved by 51% relative to the enzymatic reaction with no control of water activity. The much larger extent to which the lipase-catalyzed cellulose acetylation was enhanced by water activity optimization than by substrate pretreatment further supported the predominant role played by the major lipase activity of the A. niger lipase over its side cellulase activity in catalyzing cellulose ester synthesis in organic media.     

Extraction of hemoglobin with calixarenes and biocatalysis in organic media of the complex with pseudoactivity of peroxidase

The present work involves the use of p-tert-butylcalix[4,6,8]arene carboxylic acid derivatives (^tButyl[4,6,8]CH₂COOH) for selective extraction of hemoglobin. All three calixarenes extracted hemoglobin into the organic phase, exhibiting extraction parameters higher than 0.90. Evaluation of the solvent accessible positively charged amino acid side chains of hemoglobin (PDB entry 1XZ2) revealed that there are 8 arginine, 44 lysine and 30 histidine residues on the protein surface which may be involved in the interactions with the calixarene molecules. The hemoglobin–^tButyl[6]CH₂COOH complex had pseudoperoxidase activity which catalysed the oxidation of syringaldazine in the presence of hydrogen peroxide in organic medium containing chloroform. The effect of pH, protein and substrate concentrations on biocatalysis was investigated using the hemoglobin–^tButyl[6]CH₂COOH complex. This complex exhibited the highest specific activity of 9.92 × 10^?2 U mg protein^?1 at an initial pH of 7.5 in organic medium. Apparent kinetic parameters (V′_max, K′_m, k′_cat and k′_cat/K′_m) for the pseudoperoxidase activity were determined in organic media for different pH values from a Michaelis–Menten plot. Furthermore, the stability of the protein–calixarene complex was investigated for different initial pH values and half-life (t_1/2) values were obtained in the range of 1.96 and 2.64 days. Hemoglobin–calixarene complex present in organic medium was recovered in fresh aqueous solutions at alkaline pH, with a recovery of pseudoperoxidase activity of over 100%. These results strongly suggest that the use of calixarene derivatives is an alternative technique for protein extraction and solubilisation in organic media for biocatalysis.     

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Summary The V_max and K_M of various forms of lipase from Pseudomonas cepacia (powder, adsorbed onto Celite or covalently linked to polyethylene glycol) were determined in organic solvents preequilibrated to water activities (a _w) from <0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that K_M for the nucleophile increased with increasing a _w for all three lipase forms. V_max increased with increasing a _w for polyethylene glycol-lipase, whereas there was an optimum at intermediate a _w values (0.11 – 0.38) for lipase powder and Celite-immobilized lipase.     

Rules for optimization of biocatalysis in organic solvents

General rules for the optimization of different biocatalytic systems in various types of media containing organic solvents are derived by combining data from the literature, and the logarithm of the partition coefficient, log P, as a quantitative measure of solvent polarity. (1) Biocatalysis in organic solvents is low in polar solvents having a log P < 2, is moderate in solvents having a log P between 2 and 4, and is high in a polar solvents having a log P > 4. It was found that this correlation between polarity and activity parallels the ability of organic solvents to distort the essential water layer that stabilizes the biocatalysts. (2) Further optimization of biocatalysis in organic solvents is achieved when the polarity of the microenvironment of the biocatalyst (log P(i)) and the continuous organic phase (log P(cph)) is tuned to the polarities of both the substrate (log P(s)) and the product (log P(p)) according to the following rules: |log P(i) - log P(s)| and |log P(cph) - log P(p)| should be minimal and |log P(cph) - log P(s)| and |log P(i) - log P(p)| should be maximal, with the exception that in the case of substrate inhibition log P(i), should be optimized with respect to log P(s) In addition to these simple optimization rules, the future developments of biocatalysis in organic solvents are discussed.     

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.