Draft:Microaqueous Biosystem

• Comment: The concept of a microaqueous biosystem seems to be entirely the vision of T. Yamane, and I can't find any references to the term by any other author. This suggests a neologism that never took off; see WP:NEO. Also, the text of the article needs a lot of work. All of the issues in the multiple-issues template are serious, and there are long uncited passages and excessive detail for a general audience. This reads like the first draft of a review paper, not an encyclopedia entry. Please revise by trimming content, streamlining text, and especially adding references to the subject that come from other authors, if any can be found. WeirdNAnnoyed (talk) 12:56, 6 April 2025 (UTC)

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A Microaqueous (or micro-aqueous) system is generally defined as the chemical reaction system and a microaqueous (or micro-aqueous) biosystem is generally defined as biochemical reaction system, both of which have only trace amounts of water. In organic chemistry the reactions are usually performed in nonaqueous or anhydrous organic solvents, whereas the biochemical reactions are usually carried out in aqueous media, i.e. in large excess amount of water. In the strictly scientific sense, "nonaqueous" or "anhydrous" system means that it has no water at all. Between these two extreme states, there is a state where it contains small amount of water so that it is named microaqueous system. In this article the microaqueous biosystem is discussed focusing on the enzymatic and/or microbial reactions where the water content is very small, but it plays an important role as compared with conventional biochemical and/or microbial reactions. Because enzymes and biological cells (mainly microbial whole cells) are lumped together as biochemical catalyst, they are often called biocatalysts in total. The concept of "microaqueous biosystem" was proposed in 1988

Merits of the microaqueous system and those of the solvent-free subsystem

The enzymatic and microbial reactions are usually carried out in the excessive water. However, there are cases where performance (the yields and/or the productivities, etc.) can be dramatically increased by the reduction of the water content.

Following are the merits of performing the reactions in microaqueous biosystems:

• One can raise up the solubility of lipophilic (i.e. water-insoluble) substrate.
• One can sift the thermodynamic equilibrium to the synthesis from hydrolysis.
• One can decrease the side reactions depending on water.
• One can change the specificity (i.e. selectivity) of the enzyme.
• The Immobilization of the enzyme is not always necessary (The enzyme is insoluble in organic solvent so that one can separate it simply by the filtration or centrifugation).
• When the immobilization of the enzyme is required, it is realized merely by its adsorption on the surface of particles.
• It is easy to separate the product from an organic solvent having low boiling point.
• The stability of the enzyme is increased by some organic solvents.
• The reaction system is not contaminated by microorganisms.

The last merit is quite important for industrial bioprocess. The microaqueous biosystem is categorized into two subsystems:

• The solvent system
• The solvent-free system (neat system)

The subsystem implies the biosystem being composed of bulk liquid substrates in which only a biocatalyst is suspended or contained. The examples are glycerolizes of vegetable fat.
The merits of the subsystem are as follows:

• Very high volumetric performance (Finally the bioreactor contains only product and biocatalyst)
• No activity loss of the biocatalyst by the organic solvent
• Unnecessary of explosion-proof equipment and/or factory
• Safety in term of human health

This last merit guarantees the health of both the workers of manufacture and consumers of the products.

Biochemical reactions in microaqueous biosystems

Biocatalytic reactions have been actively studied as an interdisciplinary domain between enzyme engineering and organic chemistry for the syntheses or conversions of lipids carbohydrates, peptides, chiral compounds, etc. Types of reactions are as follows:

• oxidation
• reduction
• esters
• amides and glycosides syntheses by synthetic or rearrangement reactions as reverse reactions of hydrolyses
• C-O and C-N bond formations by addition or replacement reactions
• C-C bond formation
• polymerization reactions, etc.

These reactions may be simple in the field of organic chemistry so that the advantage of using enzymes is their superiority of its specificity (reaction specificity, substrate specificity, stereo-specificity, functional group specificity, regiospecificity, etc.). These specificities are realized without introducing protective group, which results in simpler process. Since the enzymatic reactions can be carried out under mild conditions, they are suitable especially for the production of labile substances. The following table shows the reaction types catalyzed by lipase, which is the most frequently used enzyme in microaqueous systems.

Table 1. Classification of Reactions catalyzed by lipases

	Reaction formula
(1) hydrolysis of ester	R1COOR2 + H2O → R1COOH + R2OH
(2) synthesis of ester	R1COOH + R2OH → R1COOR2 + H2O
(3) transesterification	(3.1) alcoholysis 　R1COOR2 + R3OH → R1COOR3 + R2OH (If alcohol is methanol, ethanol, or glycerol, it is called methanolysis, ethanolysis, or glycerolysis, respectively.) (3.2) acidolysis 　R1COOR2 + R4COOH → R4COOR2 + R1COOH (3.3) interesterification 　R1COOR2 + R3COOR4 → R1COOR4 + R3COOR2 (3.4) aminolysis 　R1COOR2 + R3NH2 → R1CONHR3 + R2OH

Immobilization and application of lipases in organic media are comprehensively reviewed in the reference published in 2013.

States of water molecule in microaqueous biosystem

It should be noted that the biochemical reactions in the organic solvents or solvent-free subsystem are affected strongly by water content in terms of reaction rate, yields or selectivity, operational stability, etc. Strictly speaking, the opposite of aqueous system is anhydrous or no-aqueous system where there exists absolutely no water at all, however, biocatalytic reactions do not occur without water because the enzymes are proteins and there must be small or large amounts of water molecules around and/or inside of the protein molecules to assure their fluctuation or vibrations that are essential for their catalytic actions.

Free water has effects on both the ester synthesis and transesterification by lipase.In general, in the region of very low water content, the reaction is limited by the hydration of protein (hydration controlled). In the higher free water content, byproduct is formed due to hydrolysis (the side reaction). The effect of the free water content in the bulk organic solvent on the transesterification reaction is shown in the figure.

File:Microaqueous biosystem, transesterification

Effect of free water concentration on transesterification reaction by enzyme (lipase) (General profile)

The activity of the enzyme is affected by the water bounded directly on/in the enzyme protein, and the water associated with the substrate or the product is the fee water dissolved in the bulk organic solvent. The bound water on the protein (hydrated water) is in equilibrium with the free water in the bulk solvent (Figure, a).
The profile of the equilibrium is quite different between the water-miscible solvents and water-immiscible solvents.
For the water-miscible solvents, the equilibrium isotherm exhibits Langmuir adsorption isotherm curve having a plateau at greater water content (Figure, b1).
On the other hand, for the water-immiscible solvents, it exhibits multi-layer adsorption isotherm curve, i.e. BET(Brunauer-Emmett-Teller) type (Figure, b2) having no plateau. Figure, b3 is the case of the protein in n-alkane. When the free water content is raised from absolutely zero, the bound water rises gradually up to the monolayer (point A), then the bound water increases up to the multi-layer (point B). When the water content is further raised, the free water increases so that the water around the protein becomes free water phase, and finally the biosystem reaches heterogenous state of W/O emulsion where water phase contains the protein molecules.
The effect of trace amount of water on the activity of suspended enzyme in various organic solvents is capable to be systematically evaluated to some extent by "water activity", a_w. a_w is a thermodynamic parameter. It is defined as the ratio of the partial vapor pressure in the gas phase, p_w , over the saturated vapor pressure in the liquid phase, p_w*. In a closed vessel when the vessel is kept at a fixed temperature:a_w ≡ p_w⁄p_w*. For the case of enzyme particles: a_w = γ_w x_w where γ_w is the activity coefficient and x_w is the mole fraction of water in the organic solvent. By putting enzyme powder, reaction mixture and saturated mineral salt solution separately in a closed chamber such as desiccator for a long time at a specified temperature, both the enzyme powder and reaction mixture having the same a_w can be obtained. Various a_w can be obtained through changing saturated aqueous solutions of different mineral salts. Various values of a_w of saturated mineral aqueous solution are available from Ref. 13. When the bioreaction is executed by mixing the enzyme powder and the reaction mixture after the equilibrium, the initial reaction rates are attained. Profiles concerning the initial rates vs. a_w are similar even if organic solvents are changed.

Kinds of enzymes, usage states, their purity and change in their properties

Kind of enzymes

A number of enzymes are applied to the microaqueous biosystem including esterases (especially derived from porcine lever), lipases (derived from various biological species including porcine lever and microorganisms), proteases (Thermolysin, α- chymotrypsin, etc.), peroxidases (derived from horseradish), phenol oxidases, and alcohol dehydrogenases (especially derived from yeast and horse).

state of enzymes' usage

A number of states of these enzymes have been developed which are active in organic solvents. These are summarized as follows:
(1) Free but solubilized at molecular level (Some enzymes are soluble in glycerol or dimethyl sulfoxide)
(2) Solubilized as polyethylene glycol derivative (PEG-enzyme complex), or complexes with suitable surfactants (called as lipid-coated enzyme, or surfactant-modified enzyme, or surfactant-enzyme complex
(3) Dispersed as fine particles
(4) Entrapped in reverse micelle or inverted micelle
(5) Enzyme is solubilized in water phase of micropores of porous particles which are dispersed in water-immiscible organic solvent
(6) Enzyme are entrapped in hydrophobic gels
(7) Microbial cells (wet or dry) which contain the enzyme(s) are dispersed in water-immiscible organic solvent. Alternatively, the microbial cells are entrapped in carrier particles which are dispersed in water-immiscible solvent. In general, microorganisms (Escherichia coli or Saccharomyces cerevisiae, or other typical microbial species) don not disperse in organic solvents. However, there are some groups of bacteria whose wet cells disperse uniformly in organic solvents due to their highly lipophilic cell walls (they have much amount of mycolic acid, a very lipophilic acid component of cell wall, which are characteristics of Corynebacterium sp. Mycobacterium sp. Rhodococcus sp. and Nocardia sp., etc.)
As listed above, many techniques have been developed so far, and hence it is nowadays possible to achieve states of active enzymes in any organic solvents. Techniques（2）and（4）give us enzyme preparations having high activities, but they are difficult with respect to recovery and/or continuous operation of reaction. Although Technique（3）requires pretreatment(s) or addition of activity-accelerating reagent(s), they are suitable as an industrial biocatalyst because of easy recovery/reuse or continuous operation. Since in Technique（7）separation/purification of the targeted enzyme from wet microbial cells are unnecessary, it is very promising if the targeted enzyme(s) can be accumulated in large amounts by genetic engineering.

Activation of enzyme

Although some enzymes exhibit low activities in organic solvents even if they are uniformly dispersed, their activities rise up dramatically when substance such as surfactant, fatty acid, hydrocarbon, sugar alcohols (arabitol, sorbitol, etc. Refer to the above figure) have been added in the aqueous enzyme solution in advance, followed by lyophilization. Technique (2) seems to belong to this approach.

Purity of enzyme preparation

Even though biotechnologists pay little attention to in their studies on microaqueous biosystem, purity or grade of enzyme preparation is a quite serious factor. As described in Section "States of water molecule in microaqueous biosystem", activity of enzyme depends on trace amount of water, and inert materials or impurities included in the crude enzyme preparation are not soluble in organic solvent, the targeted enzyme molecules are still surrounded by much amount of foreign inert material (Figure, a). Thus, enzyme activity is strongly affected by kinds and amount of the inert impurities. When crude enzyme powders are suspended, they exhibit sufficient activities (Figure, a), but when pure enzyme powders (at the highest purity, they are crystalline) are used, the enzyme shows enough and stable activity only when the following three factors (Figure, b), i.e. the best water content, suitable activity enhancer and appropriate carrier powder (such as cerite), are carefully optimized. In this case, the pure enzyme does not disperse in organic solvent, and its activity is still low if it is not deposited uniformly on the surface of carrier particles.

Change in properties of enzyme

Dependency of the reaction rate on substrate concentration obeys Michaelis -Menten equation even in the microaqueous biosystem just like in aqueous solution. However, properties of the enzymatic reaction in the microaqueous biosystem changes as follows:
(1) Thermal stability (half-life) Heat inactivation occurs by change in 3D structure of the enzyme protein (active folding →inactive folding), and the change is associated with water. The change does not occur in anhydrous inorganic solvent so that the enzyme is quite heat stable at this condition . This means that the value of the half-life is very much longer in anhydrous solvent than in the aqueous solution. In microaqueous biosystem, when water content is increased, it approaches to the one in aqueous solution. It is also affected by the kinds of organic solvents.
(2) Substrate specificity
The substrate specificity of an enzyme is evaluated with the parameter, k_cat⁄K_m. Its value changes in microaqueous biosystem.
(3) Enantioselectivity or stereospecificity The enantioselectivity or stereoselectivity is estimated by the ratio of the substrate specificity of R-form to that of S-form of the substrate in question and is evaluated with E value defined by E ≡ (k_cat⁄K_m )_R⁄(k_cat⁄K_m)_S . The greater the E value is, the higher the optical purity（enantiomeric excess, ee value） of the product is. It is said that higher E value than 100 is preferable industrially. The E values change for various organic solvent.

Organic solvents

Various organic solvents are used for biocatalytic reactions. Those are classified into three kinds from viewpoint of mutual solubility of water and the solvents (Refer to below table).

Table 2. Organic solvents used in microaqueous biosystem

	Name of organic solvent
(1) water-miscible organic solvents	methanol, ethanol, ethylene glycol, glycerol, N,N'-dimethylformamide, dimethyl-sulfoxide, acetone, formaldehyde, acetonitrile, dioxane, etc.
(2) water-immiscible organic solvents (water solubility [g/L] at temperature)	* alcohols: (n-, iso-) propyl alcohol, (n-, s-, t-) butyl alcohol, (n-, s-, t-)-amyl alcohol, n-octanol, etc. *esters: methyl acetate, ethyl acetate (29.4, 25℃; 37.8, 40℃), n-butyl acetate, hexyl acetate, etc. * alkyl halides and aromatic halides: methylene chloride (2, 30℃), chloroform, carbon tetrachloride, 1,2-dichloroethane, trichloroethane (0.4, 40℃), chlorobenzene, (o-, m-, p-) dichlorobenzene, etc. * ketones: methyl ethyl ketone, etc.
(3) water-insoluble organic solvents (hydrocarbons) (water solubility [ppm] at temperature)	* acyclic hydrocarbons (alkanes): n-hexane (320, 40℃), n-heptane (310, 30℃), n-octane, isooctane, (180, 30℃), etc. * alicyclic hydrocarbons: cyclohexane (160, 30℃), etc. * aromatic hydrocarbons: benzene (600, 25℃; 1200, 40℃), toluene (300, 25℃; 880, 30℃), etc.

Among them (3) n-alkanes (aliphatic hydrocarbons) will be the best in view of the least toxicity against the enzyme. Activities and stabilities of enzymes in various organic solvents have been studied as 'medium engineering'. The properties of organic solvent affecting on an enzymatic reaction are hydrophobic parameter (or polar parameter), dielectric constant, etc.
A hydrophobic parameter is a value that quantifies the tendency of a molecule or substance to prefer a non-aqueous environment over an aqueous one. The hydrophobic parameter is expressed by log P , where P is a kind of partition coefficient defined by:

P ≡ (The solubility of the organic solvent in question in 1-octanol)/(The solubility of the organic solvent in question in water)

If the value of log P is less than 2, the enzyme activity is small, if it is between 2and 4, the activity is medium, and if it is greater than 4, the activity is high. The three divisions roughly correspond to the three classifications in Table 2.

Microaqueous Bioreactor

When one has developed an excellent biocatalyst which has high activity and very stable in microaqueous biosystem for the synthesis of useful product from lipophilic substrate, one can construct an industrial bioreactor process to manufacture it on large scale.
The major difference from an ordinary bioreactor system involving hydrophilic substrate in aqueous solution is to control water content at the optimal condition. For example, as shown in Tabel 1, lipase catalyzes ester synthesis and transesterification reactions. Because water is produced in the ester synthesis from a fatty acid and an alcohol as the reaction proceeds. High yield is not realized if the formed water is not effectively removed, but excessive dehydration up to remove enzyme-bound water results in loss of catalytic activity. One conceived strategy of the optimal moisture control for batch operation of the reaction may be to remove efficiently at early and middle phases keeping moisture value at suitable level so as to achieve the highest reaction rate because the water concentration is relatively low at those phases, followed by decreasing it extensively at final stage to achieve the high yield of the product even if the rate decrease is scarified.
In order to remove the water content in bio-esterification reaction catalyzed by lipase, several techniques are known such as reduction of pressure at ambient temperature, distillation at reduce pressure, azeotropic distillation, vacuum azeotropic distillation, purging of dry gas, pervaporation, etc. The pervaporation is a sort of membrane separation. If the pressure of the back side (i.e. the opposite side of the reaction mixture) of hydrophilic membrane is reduced, water molecules are selectively removed even if the reaction medium is low-boiling solvent. In the transesterification reaction by lipase such as lipase-catalyzed glycerolysis of fats, the reaction rate decreases down to nearly zero if the water concentration is very low, but hydrolysis reaction that is a side rection, takes place if the water concentration is high so that there is an optimal moisture level at the trade-off between the reaction rate and the yield.

See also

• Enzyme
• Microorganism
• Catalysis
• Biocatalysis
• Water
• Bioreactor

References

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