Jaap Kingma

An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli.

The inner and outer membranes of gram-negative bacteria are usually obtained from spheroplasts. The lysozyme treatments normally used for converting cells to spheroplasts were originally developed with exponential phase cells and have proven to be ineffective with cells grown under other conditions. A procedure has therefore been developed which renders variously grown cells completely susceptible to lysozyme. This procedure has been tested on various strains of Escherichia coli at all stages of growth in minimal medium, from the early exponential to the late stationary phase. It has been tested on stationary phase cultures which ceased to grow because of limiting aeration, limiting carbon source, limiting amino acids, and limiting nicotinic acid. Very efficient conversion of cells to spheroplasts was observed in all cases. The resulting spheroplasts are an excellent source for subsequent membrane separations.

Bioconversions of aliphatic compounds by Pseudomonas oleovorans in multiphase bioreactors : background and economic potential

Pseudomonas oleovorans can grow on linear alkanes and alkenes in the hexane to dodecane range by virtue of enzymes encoded by the alk genes. By introducing selected alk genes into Pseudomonas strains and by supplying alkanes in the growth medium as a bulk liquid phase, specific alkane oxidation products can be accumulated in the alkane phase. We review the genetics and enzymology of the alk system and the potential of bioconversions in two-liquid-phase bioreactors, and suggest that such systems might eventually allow the biotechnological production of intermediate value compounds.

The effect of toluene on the structure and permeability of the outer and cytoplasmic membranes of escherichia coli

The effect of toluene on Escherichia coli has been examined. In the presence of Mg2+, toluene removes very little protein, phospholipid, or lipopolysacharide from E. coli. In the absence of Mg2+, or in the presence of EDTA, toluene removes considerably more cell material, including several specific cytoplasmic proteins such as malate dehydrogenase (EC 1.1.1.37). In contrast, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutamate dehydrogenase (EC 1.4.1.4) are not released at all under the same conditions. Cells treated with toluene in the presence of Mg2+ remain relatively impermeable to pyridne nucleotides, while cells treated with toluene in the presence of EDTA become permeable to these compounds. Freeze-fracture electron microscopy shows that toluene causes considerable damage to the cytoplasmic membrane, while the outer membrane remains relatively intact. These results indicate that the permeability characteristics of toluene-treated cells depend at least partly on the state of the outer membrane after the toluene treatment.

Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis.

ABSTRACT Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative α/β-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.

Pseudomonas oleovorans as a tool in bioconversions of hydrocarbons: growth, morphology and conversion characteristics in different two-phase systems

Abstract The bioconversion of hydrocarbons by Pseudomonas oleovorans has been studied in two-phase systems. In these systems, the hydrocarbon substrate is present in sufficient amounts to form the bulk apolar phase. High cell densities (up to 20 mg dry mass per ml water phase) are reached when the apolar phase consists of n-octane, 1-octene or 1-decene. There is considerable cell damage after incubation for 50–70 h. Loss of cell viability and membrane damage as observed by freeze-fracture electron microscopy correlate with a loss of hydrocarbon oxidation, measured as the conversion of 1-octene to 1,2-epoxyoctane. The final yield of oxidized hydrocarbon in the apolar substrate phase can be increased substantially by replacing the damaged cells with freshly grown cells. Yields up to 150 mg 1,2-epoxyoctane per ml 1-octene and up to 20–25 mg 1,2-epoxyoctane per ml culture were obtained with four cycles of the cell renewal procedure. Several other substrates in addition to octene were tested in the optimized two-phase system. Of these, 1-decene was converted into (R)-1,2-epoxydecane with an optical purity of 60%, while allylbenzene was converted into chiral 1,2-epoxy-3-phenylpropane. Some of the future applications of the conversion products are discussed.

Formation of polyester blends by a recombinant strain ofPseudomonas oleovorans: Different poly(3-hydroxyalkanoates) are stored in separate granules

WhenPseudomonas oleovorans (GPo1) is grown on sodium octanoate under ammonium limiting conditions, it is able to accumulate a copolyester consisting of medium chain length 3-hydroxyalkanoic acids (PHAm). 3-Hydroxybutyrate is only incorporated in trace amounts. WhenP. oleovorans is equipped with the PHB biosynthetic genes ofAlcaligenes eutrophus (GPo1[pVK101::PP1]), it forms a polyester containing major amounts of 3-hydroxybutyrate. The resulting polymer however is a blend of PHAm and PHB, rather than a copolymer of 3-hydroxybutyrate and medium chain length 3-hydroxyalkanoic acids [11]. To establish whether PHAm and PHB molecules are stored in the same or separate granules by this recombinantP. oleovorans strain, we studied polymer forming cells by freeze-fracture electron microscopy. This approach is possible because previous freeze-fracture electron microscopy studies on PHAm and PHB accumulating strains have shown that PHAm and PHB granules can be distinguished from each other: PHAm granules from mushroom-like structures, whereas PHB granules from needle structures during freeze-fracturing. In this paper we show that stationary phase cells of GPo1[pVK101::PP1] contained both mushroom and needle-like structures, indicating that PHAm and PHB chains were stored in separate granules. To be able to determine whether the separation of PHAm and PHB is complete, the respective granules were separated on sucrose gradients. A total cell extract of GPo1[pVK101::PP1] which was subjected to sucrose gradient centrifugation revealed two white bands of different densities: the upper band with a density of 1.05 g/mL consisted exclusively of PHAm granules, while the lower band with a density of 1.19 g/mL consisted of PHB granules only. Thus, when bacteria synthesize both PHAm and PHB, the resulting polymer chains are segregated completely and stored in separate granules.

Physiology and polyester formation of Pseudomonas oleovorans in continuous two-liquid-phase cultures.

Pseudomonas oleovorans is able to grow on linear aliphatic hydrocarbons of medium chain length as sole energy and carbon source. When nitrogen, sulfur, or magnesium is limiting, P. oleovorans produces an intracellular polyester poly(beta-hydroxyalkanoate) (PHA) from the excess alkanoic acid formed from the alkanes supplied in the medium. To study the effect of growth rate and exposure to bulk amounts of n-octane on the physiology and morphology of P. oleovorans, we have established continuous cultures of this organism in two-liquid phase media containing about 15% (v/v) n-octane. P. oleovorans was grown in an ammonium-limited single-stage chemostat at growth rates varying from D = 0.05 to D = 0.46 h-1. In contrast to batch cultures of P. oleovorans grown on n-octane, both rapidly and slowly growing cells remained fully viable during the entire continuous culture experiments, which typically lasted 200-300 h. The cellular morphology of these cells was studied as a function of time by freeze-fracture electron microscopy, which provided information on changes in membrane ultrastructure and revealed large and small PHA granules in slowly and rapidly growing cells, respectively. The cell density, cellular protein content, and PHA content were determined as a function of growth rate. The cell density decreased from 2.25 to 1.32 mg ml-1, while the PHA content of the cells decreased from 46.7% to 8.3% of the total cell dry weight when the dilution rate (= growth rate) increased from 0.09 to 0.46 h-1. The rest biomass concentration, defined as the difference between total biomass and PHA, was almost independent of the cellular growth rate. The cellular protein content relative to the rest biomass increased from 24% to 46% when the growth rate increased from 0.09 to 0.46 h-1, indicating that rapidly growing cells contain more protein than slowly growing cells, which correlates well with the qualitative data of the electron micrographs.

THE ALKANE OXIDATION SYSTEM OF PSEUDOMONAS-OLEOVORANS - INDUCTION OF THE ALK GENES IN ESCHERICHIA-COLI W3110(PGEC47) AFFECTS MEMBRANE BIOGENESIS AND RESULTS IN OVEREXPRESSION OF ALKANE HYDROXYLASE IN A DISTINCT CYTOPLASMIC MEMBRANE SUBFRACTION

The alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxyiase AlkB, is an integral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5–2% AlkB relative to the total cell protein, both in P. oleovorans and in recombinant Escherichia coli DH1. We present a study on the Induction and localization of the alkane hydroxylase in E. coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10–15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild‐type W3110. Freeze‐fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low‐density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both by SDS‐PAGE and enzyme activity measurements. A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low‐density membrane fraction, alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane. The major changes occurred in the unsaturated fatty acids: C16:1 and C18:1 increased at the expense of C17:0cyc and C19:0cyc*

REPLACEMENT OF TRYPTOPHAN RESIDUES IN HALOALKANE DEHALOGENASE REDUCES HALIDE BINDING AND CATALYTIC ACTIVITY

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues was studied by replacing Trp125 with phenylalanine, glutamine or arginine and Trp175 by glutamine using site-directed mutagenesis. All mutants except Trp125-->Phe showed a more than 10-fold reduced kcat and much higher Km values with 1,2-dichloroethane and 1,2-dibromoethane than the wild-type enzyme. Fluorescence quenching experiments showed a decrease in the affinity of the mutant enzymes for halide ions. The 2H kinetic isotope effect observed with the wild-type enzyme in deuterium oxide was lost in the active mutants, except the Trp125-->Phe enzyme. The results indicate that both tryptophans are involved in stabilizing the transition state during the nucleophilic substitution reaction that causes carbon-halogen bond cleavage.

Crystallization of Haloalkane Dehalogenase from Xanthobacter autotrophicus GJ10

Haloalkane dehalogenases are enzymes that release chloride or bromide from n-halogenated alkanes. X-ray quality crystals of haloalkane dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 64% saturated ammonium sulfate solutions (pH 6.2 to 6.4). The crystals diffract in the X-ray beam to at least 2.4 A resolution (1 A = 0.1 nm). Their space group is P2(1)2(1)2, with cell dimensions a = 94.1 A, b = 72.8 A, c = 41.4 A and alpha = beta = gamma = 90 degrees. There is one monomer (molecular weight 36,000) per asymmetric unit.