Methyl-β-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming mycobacteria

Abstract

Modified b-cyclodextrins have been shown previously to enhance sterol conversion to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by growing Mycobacterium spp. The enhancement effect was mainly attributed to steroid solubilization by the formation of inclusion complexes with modified cyclodextrins. In this work, the influence of randomly methylated b-cyclodextrin (MCD) on the growth, AD-and ADD-producing activity, cell wall (CW) composition and ultrastructure of sterol-transforming Mycobacterium sp. VKM Ac-1816D was studied. The specific growth rate of the strain on glycerol increased in the presence of MCD (20-100 mM). Washed cells grown in the presence of MCD (20-40 mM) expressed 1.6-fold higher ADD-producing activity than did the cells grown without MCD, and their adhesiveness differed. Electron microscopy showed MCD-mediated CW exfoliation and accumulation of membrane-like structures outside the cells, while preserving cells intact. The analysis of CW composition revealed both a decrease in the proportion of extractable lipids and a considerable shift in fatty acid profile resulting from MCD action. The MCD-mediated enhancement of mycolic and fatty acids content was observed outside the cells. The total secreted protein level rose 2.4-fold, and the extracellular 3-hydroxysteroid oxidase activity 3.2-fold. The composition of the CW polysaccharide was not altered, while the overall proportion of the carbohydrates in the CW of the MCD-exposed mycobacteria increased. The results showed that the multiple mechanisms of MCD-mediated intensification of sterol to AD(D) conversion by mycobacteria include not only solubilization of steroids, but also the increase of CW permeability for both steroids and soluble nutrients, disorganization of the lipid bilayer and the release of steroid-transforming enzymes weakly associated with the CW.

Effect of Tween 80 on 9α-steroid hydroxylating activity and ultrastructural characteristics of Rhodococcus sp. cells

“…In addition, the observed by SEM and TEM exfoliation of the electron dense capsule-like material and its partial disappearance from the Tween 80-grown possibly further contributes to lowering the cell surface hydrophobicity. Similar loss of hydrophobicity with parallel loss of surface lipids and mycolic acids were reported for the closely related mycobacteria and corynebacteria (Donova et al 2007;Puech et al 2001).…”

“…Aqueous-organic solvent two-phase systems where the water-immiscible organic solvent contained the steroid substrate are also applied (Angelova et al 2005). Only a few reports, however, associate the effect of the compounds applied to enhance the steroid substrates solubilization (such as lecitin, cyclodextrin and bis(2-ethylhexyl)phthalate) with their impact on the cell viability and morphology, microbial cell wall and cellular membranes (de Carvalho et al 2004a, b;Donova et al 2007;Rumijowska et al 1997).…”

Functional reconstruction of bovine P450scc steroidogenic system in <i>Escherichia coli</i>

“…Besides, CDs may facilitate the transport of poorly soluble hydrophobic substances to and from microbial cells, thus functioning as effective substrate/product delivery systems. As reported, CDs themselves do not penetrate through bacterial cell membranes, but can disrupt the outermost cell wall layers of the gram-positive bacteria [36]. The detail study of MCD effect on the cells of E. coli is out of the purposes of the current work, and can be investigated especially.…”

Improvement of Steroid Biotransformation with Hydroxypropyl-β-Cyclodextrin Induced Complexation

“…It indicated that there was a slight improvement of the A. simplex TCCC 11037 cell's catalytic activity after being incubated in HP-β-CD solution. This could be ascribed to the interaction between CDs and cell membrane or cell wall by virtue of either their complexing ability and/or surface activity as mentioned by Donova and Nikolayeva [21]. Thus, HP-β-CD would be an excellent additive to retain the enzyme stability than DMF or ethanol in phosphate buffer.…”