Non-aqueous enzymology has emerged as a potentially novel area in industrial biocatalysis. Whole cells and their corresponding enzymes show great promise for the future development of cost-effective solvent bioconversion or remediation processes. These possibilities represent future avenues of research for both microbiologists and enzymologists.
by Dr S. Kumar, Dr R. Karan and Dr S.K. Khare

There are numerous advantages to using enzymes in low water / organic solvent media. These include increased solubility of non-polar substrates, ease of product recovery, a thermodynamic equilibrium in favour of synthesis, elimination of microbial contamination and absence of undesirable side reactions [1]. In general, enzymes give low rates of reaction in organic solvents and are even denatured in some cases. The stabilisation of enzymes in a non-aqueous medium can be achieved by chemical modification, immobilisation, medium engineering, cross-linked enzyme crystals, site directed mutagenesis and directed evolution [2]. However, in recent years a new class of microorganisms that are tolerant to organic solvents has emerged, which produce enzymes that are naturally stable in organic solvents.

Solvent-tolerant microorganisms
The toxic effect of organic solvents on microorganisms is well documented. Some microbes do tolerate such solvents at a low concentration. In this context, solvent-tolerant microbes are defined as, “organisms that can grow in the presence of large volume (10-50% volume of the medium) of low-polar organic solvents” [3]. The first solvent-tolerant strain of Pseudomonas putida was reported by Inoue and Horikoshi in 1989. This was able to grow in media containing more than 50% (v/v) toluene or high concentrations of cyclohexane, xylene, styrene and heptanol [4]. Since then, many solvent-tolerant microorganisms have been isolated and characterised. These micro-organisms mainly belong to the genera Pseudomonas, Bacillus, Flavobacterium, Rhodococcus and Enterobacter, an example is shown in Figure 1. This organism circumvents the toxic effects of solvents using various
adaptation strategies including:

• Modification in the cell membrane by (i) a shift in the ratio of saturated to unsaturated fatty acids (ii) isomerisation of the cis-isomers of unsaturated fatty acids to the trans-isomers (iii) change in fatty acid composition.

• Biotransformation and degradation of toxic organic solvents into non-toxic compounds.

• Prevalence of efflux pumps for various solvents [5].

Enzymes from solvent-tolerant microbes
Since the cells of solvent-tolerant microorganisms are adapted to function in solvent rich cellular environments, it is logical that their enzymes should also be adapted to function in the presence of solvents. Some of the industrially important enzymes from solvent-tolerant microbes, such as lipases, proteases, and amylases, have been reported to display remarkable stability and efficient catalysis in non-aqueous media [6].

The generic features of these enzymes include (i) better stability in hydrophobic solvents, especially alkanes, (ii) the monomeric nature of proteins with molecular weight ranging from 20 kDa to 80 kDa, (iii) hydrophobic surfaces, (iv) marked presence of disulphide bonds, (v) and often metalloprotein structures. The hydrophobic surface of aminopeptidase from Pseudomonas aeruginosa is shown as wireframe model in Figure 2.

Hydrophobic interaction chromatography has been used for the efficient purification of solvent tolerant enzymes. Interestingly their enzymatic characteristics vary from strain to strain. However, the common characteristic is their stability in alkanes, especially in long chain aliphatic hydrocarbons, benzene, toluene, and alcohols.

Proteases
Proteases that are stable in organic solvents are desirable for effective peptide synthesis. Protease-catalysed synthesis has several advantages over chemical processes, e.g. regio- and stereo-selectivity, absence of racemisation, non-requirement of side-chain protection and mild reaction conditions. Solvent-stable proteases have been reported from P. aeruginosa PST-01, P. aeruginosa PseA, Bacillus cereus BG1 and Bacillus pumilus 115b [7]. A protease from solvent-tolerant P. aeruginosa PST-01 has been effectively used for the synthesis of dipeptides, e.g. Cbz–Arg–Leu–NH2 and Cbz–Lys–Phe–NH2, with equilibrium yields of more than 60% in dimethyl formamide medium. This protease has two internal disulphide bonds and large number of hydrophobic amino acids at the surface, which are supposedly responsible for its stability in organic solvent [8].

Lipases
Lipases are the most sought-after enzymes from the view point of solvent stability, because of their esterification and transesterification reactions, which are favoured in non-aqueous media [9]. The products of these reactions are industrially important e.g. flavour esters, cocoa butter equivalents, human milk fat substitute ‘Betapol’, structured lipids and biodiesel. The natural substrates of lipase are insoluble in aqueous media. This means that the reaction must take place in an organic solvent medium. Lipases from solvent-tolerant P. aeruginosa PseA, P. aeruginosa LST-03, and Bacillus sphaericus 205Y have been found to be active and stable in range of hydrophobic solvents. An end application has been established by synthesising ethyl butyrate ester in n-hexane medium using  Burkholderia multivorans V2 lipase.

Other industrially useful enzymes
Solvent-tolerant microorganisms are just as useful in producing cholesterol esterase and cholesterol oxidases [10]. The cholesterol oxidases, which are active and stable in the presence of organic solvents or detergents, are in demand for applications in the measurement of cholesterol concentrations in food, as well as serum and other clinical samples. They also have important applications in the bioconversion of 3β-hydroxysteroids in the presence of organic solvents. Cholesterol oxidases from Burkholderia cepacia strain ST-200 showed a 3-3.5 times higher cholesterol oxidation rate in the presence of organic solvents, with log P values in the range of 2.1–4.2. The structural gene encoding this cholesterol oxidase has been successfully cloned and expressed in E. coli [11]. Solvent-stable cyclodextrin glucanotransferase (CGTase), esterases and aminopeptidases, which seem to be useful for organic synthesis and industrial biocatalysis, are also reported. It is worthwhile to mention that halophiles also produce an array of enzymes, some of which are endowed with significant solvent stability.

Applications in bioremediation and biotransformation
The persistence of many solvents in contaminated sites is indicative of the lack of natural systems that can efficiently degrade these compounds. Organic solvent-tolerant bacteria can be invaluable  for such processes. The efficacy of solvent-tolerant bacteria for biotransformations in biphasic systems is highlighted in a recent review by Heipieper et al. [12]. The bioremediation of crude-oil-polluted sea water by an immobilised hydrocarbon-degrading bacterial strain is an example that proves the point. To summarise, solvent-tolerant microorganisms and their enzymes can be vitally useful in bioremediation and biotransformation.

Future perspectives
The full potential of this class of microbes and their enzymes is yet to be realised. Some of the following areas will be of great interest in coming years:
(i) the elucidation of a detailed structure-function relationship of solvent stable enzymes (ii) the development of generic designs for engineering native proteins for solvent-stability on the basis of the above (iii) an in-depth investigation of the kinetics and mechanism of reactions (iv) exploring further usage in organic synthesis, solvent bioremediation and biotransformation (v) cloning genes of solvent-tolerant enzymes and their overexpression (vi) metagenomic approaches to mine novel solvent-tolerant enzymes from unculturable microbes.

References
1. Klibanov MA. Improving enzymes by using them in organic solvents, Nature 2001; 409: 241-246.
2. Gupta MN and Roy I. Enzymes in organic media: forms, functions and applications. Eur J Biochem 2004; 271: 2575–2583.
3. Aono R and Inoue A. Organic solvent tolerance in microorganisms. In: Extremophiles: Microbial life in extreme environments (Ed. Horikoshi, K. and Grant, D. W.), Wiley-Liss, inc. 1998; 287-310.
4. Inoue A and Horikoshi K. A Pseudomonas thrives in high concentrations of toluene. Nature 1989; 338: 264-266.
5. Isken S and de Bont JAM. Bacteria tolerant to organic solvents. Extremophiles 1998; 2: 229–238.
6. Gupta A and Khare SK. Enzymes from solvent-tolerant microbes: Useful biocatalysts for non-aqueous enzymology, Crit Rev Biotechnol 2009; 29(1): 44-54.
7. Rahman RNZRA, Mahamad S, Salleh AB and Basri M. A new organic solvent tolerant protease from Bacillus pumilus 115b. J Ind Microbiol Biotechnol 2007; 34: 509–517.
8. Ogino H, Uchiho T, Doukyu N, Yasuda M, Ishimi K and Ishikawa H. Effect of exchange of amino acid residues of the surface region of the PST-01 protease on its organic solvent-stability. Biochem Bioph Res Comm 2007; 358: 1028–1033.
9. Khare SK, Snape J and Nakajima M. Application of enzyme and membrane technology in the processing of fats and oils. In: Methods in Non-aqueous Enzymology. (Ed. Gupta, M. N.), Basel: Birkhauser-Verlag. 2000; 52–69.
10. Sardessai YN and Bhosle S. Industrial potential of organic solvent tolerant bacteria Biotechnol Prog 2004; 20: 655-660.
11. Doukyu N and Aono R. Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200. Appl Microbiol Biotechnol 2001; 57: 146–152.
12. Heipieper HJ, Neumann G, Cornelissen  and Meinhardt F. Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems. Appl Microbiol Biotechnol 2007; 74: 961–973.

The authors
Sumit Kumar, Ram Karan and S.K. Khare*
Enzyme and Microbial Biochemistry laboratory
Department of Chemistry
Indian Institute of Technology
Delhi
India

*Author for correspondence
Dr. S.K. Khare
Enzyme and Microbial Biochemistry laboratory
Department of Chemistry
Indian Institute of Technology, Delhi
Hauz Khas, New Delhi-110016, India
Tel: +91 11 2659 6533
email: skkhare@chemistry.iitd.ac.in