Unsaturated fatty acids having structural features which are different from those of the monoenoic acids normally synthesized by Escherichia coli can serve as growth factors for an auxotroph requiring unsaturated fatty acids. These analogues were incorporated into the phospholipids, as shown by gas-liquid and thin-layer chromatographic analysis of the phospholipid fatty acid composition. Some of these fatty acids were cisA5-and cis-A'-tetradecenoic, cis-,A"-eicosenoic, cis, ciA-jA '14-eicosadienoic, cis,cis,cis-A"14,17-eicosatrienoic, trans-,A'-and trans-A"1-octadecenoic acids. Although partial degradation of some of these analogues to shorter even-chain homologues occurred, chain elongation of the exogenous fatty acids was not detected. Trans-olefinic acids were utilized without stereochemical or positional isomerization. These studies provide a basis for exploring the properties of the fatty acids and phospholipids required for the formation, structure, and function of membranes. A previous report described the isolation and characterization of a mutant of Escherichia coli K-12 which requires exogenous unsaturated fatty acids for growth (18). The biochemical defect of this mutant was shown to be the loss of the fihydroxydecanoyl thioester dehydrase which is responsible for the formation of cis-A3-decenoylacyl carrier protein (ACP). The latter compound is the first unsaturated fatty acid intermediate in the sequence of conversions leading to cis-A9hexadecenoic and cis-All-octadecenoic acids, the long chain monoenoic acids found in the cell (15). The present investigation concerns the specificity of the unsaturated fatty acid requirement of the mutant. This requirement has been examined by using a variety of analogues as growth supplements. These replacements were chosen so that their structures differed, in one or more features, from the unsaturated fatty acids synthesized in wild-type E. coli K-12. It is hoped that a controlled alteration in bacterial fatty acid composition will make possible a clearer understanding of the role of the apolar residues of phospholipids in biological structure and function. MATERIALS AND METHODS Chemnicals. cis-A9-Tetradecenoic, cis-A9-hexadecenoic, cis-A9-octadecenoic, trans-A9-hexadecenoic, trans-Y9-octadecenoic, cis-A5-eicosenoic, 1 2-hydroxy cis-,A9-octadecenoic, and branched-chain and straightchain saturated fatty acids, all >990/,, were obtained from Applied Science Laboratories, State College, Pa. The following fatty acids were products, all >99%c, of the Hormel Institute, Austin, Minn.: cis-A5-tetradecenoic, cis-Al"-octadecenoic, trans-All-octadecenoic, cis-All-eicosenoic, cis-A6-octadecenoic, cis-A'3-docosenoic, cis-A15-tetracosenoic, cis, cis-All 14 eicosadienoic, and cis, cis,cis-A"l '417-eicosatrienoic acids. We purchased 9 and 10 (mixture) monohydroxyoctadecanoic acid (>98%) from K and K Laboratories, Plainview, N.Y.; cis,cis-A912-octadecadienoic acid (>99%;) was purchased from Mann Research Laboratories, New York, N.Y.; and decenoic acid (79%c trans-A2-, 11%Ccis-A3-,...

Enzymes can catalyze various reactions with high selectivity and are involved in many important biological processes. However, the general instability of enzymes against high temperature often limits their application. To address this, we synthesized a trehalose-based hydrogel in two steps from commercial starting materials with minimal purification procedures. Mono- and multi-functional trehalose monomers were cross-linked by redox-initiated radical polymerization to form a hydrogel. Phytase, an important enzyme utilized in animal feedstock, was employed to study the effectiveness of the trehalose hydrogel to stabilize proteins against heat. Addition of the phytase solution to the hydrogel resulted in enzyme internalization as confirmed by confocal microscopy. The phytase in the hydrogel retained 100% activity upon heating at 90 °C compared to 39% when the hydrogel was absent. The enzyme could also be recovered from the hydrogel. The trehalose hydrogel synthesis reported herein should be readily scalable for thermal stabilization of a wide variety of enzymes.

The utilization of glycerol as a carbon source for growth by Klebsiella aerogenes, strain 2103, involves separate aerobic (sn-glycerol-3-phosphate or G3P) and anaerobic (dihydroxyacetone or DHA) pathways of catabolism. Enzyme and transport activities of the aerobic pathway are elevated in cells grown under oxygenated conditions on glycerol or G3P. Anaerobic growth on G3P as carbon source requires the presence of an exogenous hydrogen acceptor such as fumarate; cells thus grown also are highly induced in the G3P pathway. Anaerobic growth on glycerol requires no exogenous hydrogen acceptors; cells thus grown are highly induced in the DHA pathway but almost uninduced in the G3P pathway and the addition of fumarate electron acceptors has no effect on the relative levels of the two pathways. When both glycerol and G3P are provided anaerobically with fumarate, the DHA pathway is still preferentially induced, which probably accounts for the exclusive utilization of glycerol until its exhaustion. These observations suggest the presence of a regulatory control of G3P pathway imposed by the operation of the DHA pathway. Klebsiella aerogenes, strain 1033, dissimilates glycerol by two separate pathways. In one pathway, the nutrient is phosphorylated by an adenosine 5'-triphosphate (ATP)-dependent kinase to sn-glycerol-3-phosphate (G3P). G3P is then converted to dihydroxyacetone phosphate by dehydrogenases characteristic of flavoenzymes (Fig. 1). In the other pathway, glycerol is converted to dihydroxyacetone (DHA) by an NAD-linked dehydrogenase. DHA is then phosphorylated to DHA phosphate (DHAP) by an ATP-dependent kinase. Evidence from in vivo isotopic tracer studies, as well as in vitro measurements of enzyme activities, indicates that the G3P pathway is responsible for aerobic, and the DHA pathway is responsible for anaerobic, degradation of glycerol (7, 9, 13). The presence of the latter pathway in K. aerogenes apparently permits the cell to grow anaerobically on glycerol in the absence of exogenous hydrogen acceptors, since Escherichia coli, lacking the enzymes of this pathway, is unable to do so. In the work to be described below, it is shown that K. aerogenes, like E. coli, can grow directly on G3P without prior hydrolysis, and that anaerobic growth on G3P requires exogenous hydrogen acceptors. All the enzymes of the glp regulon (a collection of operons, the expression of which is inducible by G3P) described for E.

Glycerol:NAD+2-OXIDOREDUCTASE (EC 1.1.1.6) was purified to homogeneity from a mutant of Escherichia coli K12 that uses this enzyme, instead of ATP:glycerol 3-phosphotransferase (EC 2.7.1.30), as the first enzyme for the dissimilation of glycerol. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows a subunit of 39,000 daltons. During electrophoresis under nondenaturing conditions, the protein migrates as two bands. These two forms, both of which are enzymatically active, appear to be dimers and octomers of the same subunit. The optimal pH for the oxidation of glycerol is about 10, and that for the reduction of dihydroxyacetone is about 6. Glycerol dehydrogenation is highly activated by NH4+, K+, or Rb+, but strongly inhibited by N-ethylmalemide, 8-hydroxyquinoline, 1,10-phenanthroline, Cu2+, and Ca2+. The enzyme exhibits a broad substrate specificity. In addition to glycerol, it act on 1,2-propanediol and several of its analogs.