The amino acid analogue L-serine hydroxamate, which is bacteriostatic for Escherichia coli, has been shown to inhibit protein synthesis. The antimetabolite is a competitive inhibitor of seryl-transfer ribonucleic acid (tRNA) synthetase with a K; value of 30 uM. Mutants resistant to L-serine hydroxamate have been selected, and three were shown to have seryl-tRNA synthetases with increased K, values. One mutant contains a 3-phosphoglycerate dehydrogenase which is insensitive to inhibition by L-serine.

The structural analogue of l -serine, l -serine hydroxamate, inhibited the growth of Escherichia coli K-12. Of the other amino acid hydroxamates tested, only l -lysine hydroxamate reduced the rate of growth. Inhibition of growth by l -serine hydroxamate was rapidly reversed by the addition of l -serine to the bacterial culture or by removal of the analogue by filtration. The reversal of inhibition was specific for l -serine. l -Alanine, glycine, or adenine had no effect on an inhibited culture. No evidence for active transport of the analogue was obtained.

Conditions for the gelation k-carrageenan, which is a new polymer for immobilization of enzymes and microbial cells, were investigated in detail. k-Carrageenan was easily induced to gel by contact with metal ions, amines, amino acid derivatives, and water-miscible organic solvents. By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated. Several kinds of enzymes and microbial cells were easily immobilized with high enzyme activities. Immobilized preparations were easily tailor-made to various shape such as cube, bead, and membrane. The obtained immobilized preparations were stable, and columns packed with them were used for continuous enzyme reaction for a long period. Their operational stabilities were enhanced by hardening with glutaraldehyde and hexamethylenediamine.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonasfluorescens B52 and P.fluorescens SIK Wi, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.Triacylglycerol acylhydrolase (EC 3.1.1.3) catalyzes the hydrolysis of triacylglycerol to glycerol and fatty acid and is generally called lipase. This enzyme is important in the food industry, and there is also current interest in the application of lipases to the production of chiral compounds. Extracellular lipases are produced by a variety of microorganisms: fungi, yeasts, and bacteria, including actinomycetes (60, 64). The lipase genes have been cloned from fungi (4, 21, 57, 58, 66), a yeast (32), Pseudomonas species (1, 6, 27, 29, 31, 34, 61a, 65), and staphylococci (19, 38). Serratia marcescens, an enteric bacterium, produces several extracellular enzymes which participate in the degradation of high-molecular-weight compounds, e.g., serine protease (67), chitinase (30), phospholipase (17), and nuclease (2, 7). Their genes have been cloned from S. marcescens, and the deduced amino acid sequences indicated that these proteins are secreted through a mechanism containing the secAY gene products with the aid of typical N-terminal signal peptides as described previously (51). Interestingly, the S. marcescens metalloprotease has been recently reported to be excreted through a mechanism similar to that of Escherichia coli ot-hemolysin having no N-terminal signal peptide (40).S. marcescens has been long known to produce extracellular lipase (64), but there is little knowledge of its enzyme. Very recently, we showed it to be stable in some organic solvents and to be applicable to the synthesis of (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester by asymmetric hydrolysis of racemic trans-3-(4-methoxyphenyl)glycidic acid methyl ester, a key intermediate in the synthesis of diltiazem hydrochloride that is useful as a coronary vasodilator (44). Our final goal is the construction of a lipase-hyperproducing strain of S. marcescens. This paper deals with the molecular cloning of the S. MATERIALS AND METHODSStrains and plasmids. E. coli K-12 DH5 (53) was used as a host for the construction of plasmids. The wild-typ...

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