The membrane reactions of Pseudomonas putida S12 to environmental stress were investigated. Cells reacted to the addition of six different heavy metals with an increase in the ratio of trans to cis unsaturated fatty acids. A correlation among the increase in the trans/cis ratio, the toxic effects of the heavy metals, and nonspecific permeabilization of the cytoplasmic membrane, as indicated by an efflux of potassium ions, was measured. Cells previously adapted to toxic concentrations of toluene exhibited increased tolerance to all applied concentrations of zinc compared with nonadapted cells. Cells exposed to different temperatures grew optimally at 30؇C. The degree of saturation of the membrane fatty acids of these cells decreased with decreasing temperature. An increase in the trans/cis ratio of unsaturated fatty acids took place only at higher temperatures. Osmotic stress, expressed as reduced water activity, was obtained by using different types of solutes. Only in the presence of toxic concentrations of sodium chloride or sucrose did the trans/cis ratio increase. At no applied water activity a significant effect of glycerol on the trans/cis ratio was measured. When cells were exposed to different pHs, a distinct optimum cis/trans isomerase activity was measured at pHs between 4.0 and 5.0, whereas at higher or lower pHs no reaction occurred. This optimum coincided with a loss of viability between pH 4 and 5.

The acceptor specificity of Streptococcus mutans GS‐5 glucosyltransferase‐D (GTF‐D) was studied, particular the specificity toward non‐saccharide compounds. Dihydroxy aromatic compounds like catechol, 4‐methylcatechol, and 3‐methoxycatechol were glycosylated by GTF‐D with a high efficiency. Transglycosylation yields were 65%, 50%, and 75%, respectively, using 40 mM acceptor and 200 mM sucrose as glucosyl donor. 3‐Methoxylcatchol was also glycosylated, though at a significantly lower rate. A number of other aromatic compounds such as phenol, 2‐hydroxybenzaldehyde, 1,3‐dihydroxybenzene, and 1,2‐phenylethanediol were not glycosylated by GTF‐D. Consequently GTF‐D aromatic acceptors appear to require two adjacent aromatic hydroxyl groups. In order to facilitate the transglycosylation of less water‐soluble acceptors the use of various water miscible organic solvents (cosolvents) was studied. The flavonoid catechin was used as a model acceptor. Bis‐2‐methoxyethyl ether (MEE) was selected as a useful cosolvent. In the presence of 15% (v/v) MEE the specific catechin transglucosylation activity was increased 4‐fold due to a 12‐fold increase in catechin solubility. MEE (10–30% v/v) could also be used to allow the transglycosylation of catechol, 4‐methylcatechol, and 3‐methoxycatechol at concentrations (200 mM) otherwise inhibiting GTF‐D transglycosylation activity. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 363–369, 2000.

The (+)-catechin transglucosylating activities of several glucosyltransferases (GTFs) from the genus Streptococcuswere compared. For this purpose, a mixture of four GTFs fromStreptococcus sobrinus SL-1 and recombinant GTF-B and GTF-D from Streptococcus mutans GS-5 expressed inEscherichia coli were studied. It was shown that after removal of α-glucosidase activity, GTF-D transglucosylated catechin with the highest efficiency. A maximal yield (expressed as the ratio of moles of glucoside formed to moles of catechin initially added) of 90% was observed with 10 mM catechin and 100 mM sucrose (Km , 13 mM) in 125 mM potassium phosphate, pH 6.0, at 37°C. 1H and 13C nuclear magnetic resonance spectroscopy revealed the structures of two catechin glucosides, (+)-catechin-4′-O-α-d-glucopyranoside and (+)-catechin-4′,7-O-α-di-d-glucopyranoside. Fructose accumulation during glucosyl transfer from sucrose to the acceptor competitively inhibited catechin transglucosylation (Ki , 9.3 mM), whereas glucose did not inhibit catechin transglucosylation. The addition of yeasts was studied in order to minimize fructose inhibition by means of fructose removal. For this purpose, the yeasts Pichia pastoris and the mutantSaccharomyces cerevisiae T2-3D were selected because of their inabilities to utilize sucrose. Addition of P. pastoris or S. cerevisiae T2-3D to the standard reaction mixture resulted in a twofold increase in the duration of the maximum GTF-D transglucosylation rate. The addition of the yeasts also stimulated sucrose utilization by GTF-D.

Screening for novel thioglucoside hydrolase activity resulted in the isolation of Sphingobacterium sp. strain OTG1 from enrichment cultures containing octylthioglucoside (OTG). OTG was hydrolysed into octanethiol and glucose by cell free extracts. Besides thioglucoside hydrolysis, several other glucoside hydrolase activities were detected in the Sphingobacterium sp. strain OTG1 cell free extract. By adding beta-glucosidase inhibitors it was possible to discriminate between these different activities. Ascorbic acid and D-gluconic acid lactone inhibited the hydrolysis of p-nitrophenyl beta-glucoside, but did not affect octyl- and octylthioglucoside hydrolase activity. Besides OTG, various other thioglucosides were hydrolysed by the novel thioglucosidase, with almost the same activities regardless of the nature of the aglycone, including the myrosinase model substrate sinigrin (a glucosinolate). Sinigrin could also be used as a growth substrate by Sphingobacterium sp. strain OTG1, although at concentrations exceeding 0.15 mM degradation was not complete.

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