红灯食烷菌(Alcanivorax hongdengensis)黄素结合单加氧酶(AlmA)的基因克隆及其烷烃诱导表达

【目的】研究海洋烷烃降解菌新种模式菌株Alcanivorax hongdengensis A-11-3降解长链烷烃的分子机制。【方法】PCR克隆编码黄素结合单加氧酶的基因序列,利用生物信息学软件对序列进行分析,运用RT-PCR和实时荧光定量PCR技术分析基因在不同烷烃诱导下的表达水平。【结果】从菌株A-11-3中克隆获得了两个黄素结合单加氧酶基因片段(almA1和almA2)。它们编码的氨基酸序列与菌株Acinetobacter sp.DSM17874的AlmA同源性分别为58.6%和53.2%。实时荧光定量PCR分析表明,almA1基因只在长链烷烃(C28-C32)的诱导下上调表达,而almA2基因中能在更宽范围的长链烷烃(C24-C34)和支链烷烃诱导下上调表达。两者均在C9-C22的烷烃诱导下没有上调表达。【结论】黄素结合单加氧酶可能是A-11-3降解长链烷烃和支链烷烃的关键酶。     

Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23

An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 °C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladAα_B23, ladAβ_B23, and ladB _B23, on its chromosome, whose protein products share significant amino acid sequence identities, 49.8, 34.4, and 22.7 %, respectively, with that of ladA alkane monooxygenase gene found on a plasmid of Geobacillus thermodetrificans NG 80-2. Each of the three genes, ladAα_B23, ladAβ_B23, and ladB _B23, was heterologously expressed individually in an alkB1 deletion mutant strain, Pseudomonas fluorescens KOB2Δ1. It was found that all three genes were functional in P. fluorescens KOB2Δ1, and partially restored alkane degradation activity. In this study, we suggest that G. thermoleovorans B23 utilizes multiple LadA-type alkane monooxygenases for the degradation of a broad range of alkanes.     

Degradation of oil tank sludge using long-chain alkane-degrading bacteria

Bacteria degrading a very long-chain alkane, n-tetracosane, were isolated from enrichment culture of soil in Okinawa. Phylogenetic analysis of their16S rRNA sequences revealed that they belong to classes Gammaproteobacteria and Actinomycetes. Three isolates belonging to the genera Acinetobacter sp., Pseudomonas sp., and Gordonia sp. showed a stable growth on n-tetracosane and had a wide range of assimilation of aliphatic hydrocarbons from C₁₂ to C₃₀, while not on alkanes shorter than C₈. Of the isolates, Gordonia sp. degraded oil tank sludge hydrocarbons efficiently by solving the sludge in a hydrophobic solvent, while Acinetobacter sp. showed little degradation, possibly due to the difference in the mechanism of hydrophobic substrate incorporation between proteobacteria and actinobacteria. The data suggested that non-heme di-iron monooxygenases of the AlkB-type, not bacterial CYP153 type cytochrome P450 alkane hydroxylase, was involved in the alkane degradation.     

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)⁹ addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF6^8,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced^10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.     

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with ¹³C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent β-oxidation and the reverse Wood–Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills.     

The Genome of the Moderate Halophile Amycolicicoccus subflavus DQS3-9A1T Reveals Four Alkane Hydroxylation Systems and Provides Some Clues on the Genetic Basis for Its Adaptation to a Petroleum Environment

The moderate halophile Amycolicicoccus subflavus DQS3-9A1^T is the type strain of a novel species in the recently described novel genus Amycolicicoccus, which was isolated from oil mud precipitated from oil produced water. The complete genome of A. subflavus DQS3-9A1^T has been sequenced and is characteristic of harboring the genes for adaption to the harsh petroleum environment with salinity, high osmotic pressure, and poor nutrient levels. Firstly, it characteristically contains four types of alkane hydroxylases, including the integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153, a long-chain alkane monooxygenase (LadA) and propane monooxygenase. It also accommodates complete pathways for the response to osmotic pressure. Physiological tests proved that the strain could grow on n-alkanes ranging from C10 to C36 and propane as the sole carbon sources, with the differential induction of four kinds of alkane hydroxylase coding genes. In addition, the strain could grow in 1–12% NaCl with the putative genes responsible for osmotic stresses induced as expected. These results reveal the effective adaptation of the strain DQS3-9A1^T to harsh oil environment and provide a genome platform to investigate the global regulation of different alkane metabolisms in bacteria that are crucially important for petroleum degradation. To our knowledge, this is the first report to describe the co-existence of such four types of alkane hydroxylases in a bacterial strain.     

Laboratory evolution of a soluble,self-sufficient,highly active alkane hydroxylase

We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.     

Characterization and Two-Dimensional Crystallization of Membrane Component AlkB of the Medium-Chain Alkane Hydroxylase System from Pseudomonas putida GPo1

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C₁₂E₈]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-d-maltopyranoside (DM), n-dodecyl-β-d-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism.     

Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis

LadA, a monooxygenase catalyzing the oxidation of n-alkanes to 1-alkanols, is the key enzyme for the degradation of long-chain alkanes (C₁₅–C₃₆) in Geobacillus thermodenitrificans NG80-2. In this study, random- and site-directed mutagenesis were performed to enhance the activity of the enzyme. By screening 7,500 clones from random-mutant libraries for enhanced hexadecane hydroxylation activity, three mutants were obtained: A102D, L320V, and F146C/N376I. By performing saturation site-directed mutagenesis at the 102, 320, 146, and 376 sites, six more mutants (A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, and F146N/N376I) were generated. Kinetic studies showed that the hydroxylation activity of purified LadA mutants on hexadecane was 2–3.4-fold higher than that of the wild-type enzyme, with the activity of F146N/N376I being the highest. Effects of the mutations on optimum temperature, pH, and heat stability of LadA were also investigated. A complementary study showed that Pseudomonas fluorescens KOB2Δ1 strains expressing the LadA mutants grew more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutants in vivo. Structural changes resulting from the mutations were analyzed and the correlation between structural changes and enzyme activity was discussed. The mutants generated in this study are potentially useful for the treatment of environmental oil pollution and in other bioconversion processes.     

Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase

 A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO) is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol conversion by the bare FeO⁺ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH₄) complex with an OFe—CH₄ bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital interactions between a C _3v - or D _2d -distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered structure. The generated intermediate involves an HO—Fe—CH₃ moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments on regioselectivity of enzyme-catalyzed alkane hydroxylations. Received: 15 September 1997 / Accepted: 20 December 1997     

Functional characterization of two alkane hydroxylases in a versatile <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain NY3

Pseudomonas aeruginosa strain NY3 has an extraordinary capacity to utilize a wide range of substrates, including n–alkanes of lengths C₅ to C₃₄, aromatic compounds, phenols, diesel and crude oil, and it can produce a variety of small bioactive molecules, including rhamnolipids, which can enhance its metabolic capacity for hydrophobic organic pollutants. This capacity makes NY3 a good candidate for use in environmental pollution remediation. Alkane hydroxylases catalyze both the initial and rate-limiting step of the terminal oxidation of n–alkanes. To better understand the genetic mechanisms by which P. aeruginosa NY3 degrades such a wide range of n–alkanes, two putative coding genes of alkane hydroxylases were functionally characterized using a gene-knockout approach with three different degradation systems. The single n–alkane test indicated that the hydroxylase AlkB2 acted in the early growth phase and played a major role in the utilization of C₁₂–C₁₈. However, a double mutant showed a trend towards recovery when C₂₀–C₂₄ were used as sole carbon source. This suggests that there are other enzymes capable of utilizing n–alkanes longer than C₂₀. Tests of both artificial n–alkanes mixture and crude oil-containing waste water showed similar results, suggesting that both AlkB1 and AlkB2 are involved in n–alkane degradation, and, moreover, that AlkB2 plays a major role. Finally, given the wider functional range of both AlkBs in the mixture of n–alkanes compared to that of single n–alkanes, these results hint at co-metabolism.     

Application of long-chain alcohols as faecal markers to estimate diet composition of horses and cattle fed with herbaceous and woody species

Utilization of long-chain alcohols (LCOH) as diet composition markers in horses and cattle was assessed in a study conducted with 12 mature crossbreed mares (385±47 kg BW) and six adult non-lactating cows (499±36 kg BW) of Asturiana de los Valles breed. The LCOH data were combined with alkane and long-chain fatty acid (LCFA) data to test the applicability of combining these markers to estimate diet composition. Animals were randomly divided into groups of three animals and received a daily total amount of 1.0 kg dry matter/100 kg BW of diets composed of different proportions of ryegrass (Lolium perenne) and woody species (Ulex gallii and heather). Diet composition was estimated from even-chain LCOH (C₂₀-OH to C₃₀-OH) combined or not with alkane (C₂₅-C₃₁ and C₃₃) and/or LCFA (C₂₂-FA to C₂₈-FA, C₃₀-FA, C₃₂-FA and C₃₄-FA) concentrations in diet components and faeces by least-squares procedures, using marker faecal concentrations uncorrected for incomplete faecal recovery (FR0) or corrected using mean recoveries across diets within animal species (FR1). Results showed large differences between plant species in their LCOH profiles, and that these markers offered additional discriminatory information to that provided by alkanes and LCFA. The LCOH markers were incompletely recovered in the faeces of both animal species. In cattle, LCOH FR tended to increase with carbon-chain length in a linear manner in both diets (P<0.001), whereas in horses overall data showed a curvilinear relationship between these variables. Combination of LCOH, LCFA and alkanes resulted in more accurate diet estimates. Correction of faecal LCOH concentrations to incomplete FR led to more accurate diet composition estimates in both animal species. Results obtained in this study suggest the usefulness of LCOH markers combined with alkanes and LCFA to estimate diet composition of horses and cattle grazing mixed grassy–woody plant communities.     

Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5

Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.     

Alkane hydroxylase genes in psychrophile genomes and the potential for cold active catalysis

Background

Psychrophiles are presumed to play a large role in the catabolism of alkanes and other components of crude oil in natural low temperature environments. In this study we analyzed the functional diversity of genes for alkane hydroxylases, the enzymes responsible for converting alkanes to more labile alcohols, as found in the genomes of nineteen psychrophiles for which alkane degradation has not been reported. To identify possible mechanisms of low temperature optimization we compared putative alkane hydroxylases from these psychrophiles with homologues from nineteen taxonomically related mesophilic strains.

Results

Seven of the analyzed psychrophile genomes contained a total of 27 candidate alkane hydroxylase genes, only two of which are currently annotated as alkane hydroxylase. These candidates were mostly related to the AlkB and cytochrome p450 alkane hydroxylases, but several homologues of the LadA and AlmA enzymes, significant for their ability to degrade long-chain alkanes, were also detected. These putative alkane hydroxylases showed significant differences in primary structure from their mesophile homologues, with preferences for specific amino acids and increased flexibility on loops, bends, and α-helices.

Conclusion

A focused analysis on psychrophile genomes led to discovery of numerous candidate alkane hydroxylase genes not currently annotated as alkane hydroxylase. Gene products show signs of optimization to low temperature, including regions of increased flexibility and amino acid preferences typical of psychrophilic proteins. These findings are consistent with observations of microbial degradation of crude oil in cold environments and identify proteins that can be targeted in rate studies and in the design of molecular tools for low temperature bioremediation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1120) contains supplementary material, which is available to authorized users.     

Expanding the alkane oxygenase toolbox: new enzymes and applications

As highly reduced hydrocarbons are abundant in the environment, enzymes that catalyze the terminal or subterminal oxygenation of alkanes are relatively easy to find. A number of these enzymes have been biochemically characterized in detail, because the potential of alkane hydroxylases to catalyze high added-value reactions is widely recognized. Nevertheless, the industrial application of these enzymes is restricted owing to the complex biochemistry, challenging process requirements, and the limited number of cloned and expressed enzymes. Rational and evolutionary engineering approaches have started to yield more robust and versatile enzyme systems, broadening the alkane oxygenase portfolio. In addition, metagenomic approaches provide access to many novel alkane oxygenase sequences.     

Comparative alkane chemistry of Parthenium argentatum (guayule) and some F1 hybrids

The leaf alkanes of Parthenium argentatum (guayule), P. tomentosum var. stramonium, P. fruticosum var. trilobatum, and the first filial (F₁) generations obtained from crosses with guayule were investigated by GC and mass spectrometry and shown to be useful in chemotaxonomic studies. The identified n-alkanes ranged from C₁₉ to C₄₀ with either n-C₂₉ or n-C₃₁ as the main component. The alkane chemistry of guayule with n-C₃₁ being the main component predominated in most of the F₁ hybrids. The presence of iso-branched alkanes (C₂₇, C₂₉, C₃₁) in P. tomentosum and its hybrids could be detected by GC/MS. These preliminary investigations indicate that epicuticular wax alkanes can be useful in inheritance studies of guayule and its hybrids.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Location of lysyl residues at the allosteric site of fructose 1,6-bisphosphatase

Various flavin analogs were used as alternate substrates or competitive inhibitors to characterize the FMN binding sites of the NADH- and NADPH-specific FMN oxidoreductases from Beneckea harveyi. Several polyhydroxyl compounds were found to be poor competitive inhibitors for the FMN sites of these enzymes. The FMN binding sites of the two enzymes were found to be quite similar. The NADH:FMN oxidoreductase binds FMN exclusively through the isoalloxazine ring. The methyl groups at positions 7 and 8 contribute significantly to this binding. Utilizing lumichrome as a competitive inhibitor of the FMN binding site and AMP as a competitive inhibitor of the NADH binding site, we were able to determine that the NADH:FMN oxidoreductase forms an active ternary complex with NADH binding first in an ordered mechanism. The NADPH oxidoreductase also binds FMN primarily through the isoalloxazine ring. Unlike their participation in reaction with the NADH-specfic enzyme, the methyl groups at positions 7 and 8 are not involved in binding. There was no significant binding of the ribityl phosphate moiety with either enzyme. Both enzymes have lower K_m values for lumiflavin than FMN.     

Isomers of long-chain alkane derivatives and nervous impulse blockage

Summary The potency to block nervous impulse of members of normal aliphatic homologous series of primary and secondary isomers of functional derivatives of alkanes was tested in bundles of a few axons from sciatic nerves of the toadBufo marinus. For the primary substituted functional derivates of pentane, the relative potency series was 1>HBr>Cl>COOCH₃>F>CH₂OH>COCH₃>OHNH₂>COOH. For the homologous series of primary alkanols, and from saturated Ringer's solutions, the time required to reversibly reduce the amplitude of the action potential to one-half its initial value were determined. The cut-off effect was detected at the level of dodecan-1-ol, while for the primary bromoalkanes it was bromooctane. However, solutions of the secondary isomer of the inactive primary homologues, such as tridecan-5-ol and tridecan-7-ol or 2-bromononane, were able to block nervous impulse conduction reversibly. From the concentration required for an equipotent effect it was calculated that the standard free energy for adsorption of primary alkanols was –705 cal mol^– CH₂. Similarly, for primary bromoalkanes a value of –733 cal mol^–1 CH₂ was obtained. The concentration required for an equipotent effect for primary isomer (either of alkanols or bromoalkanes) is lower than those obtained for the secondary isomers. Therefore, the latter are less potent than the former. Among secondary isomers the potency decreases as the functional group is moved away from the terminal carbon. The differential effect of structural isomers of long-chain alkane derivates around the point of cut-off cannot be explained in terms of differences in chemical properties, concentration in aqueous and membrane phases, or mean molecular volume. It is concluded that a volume related to that of the hydrophobic region of the agent and not its mean molecular volume should be responsible for an expansion of the target region.