Three dehalogenases and physiological restraints in the biodegradation of haloalkanes by Arthrobacter sp. strain HA1.

Arthrobacter sp. strain HA1 utilizes 18 C2-to-C8 1-haloalkanes for growth and synthesizes an inducible 1-bromoalkane debrominase of unknown physiological function (R. Scholtz, T. Leisinger, F. Suter, and A.M. Cook, J. Bacteriol. 169:5016-5021, 1987) in addition to an inducible 1-chlorohexane halidohydrolase which dehalogenates some 50 substrates, including alpha, omega-dihaloalkanes. alpha, omega-Dihaloalkanes were utilized by cultures of strain HA1 under certain conditions only. C9 and C8 homologs prevented growth. At suitable concentrations, C7-to-C5 homologs could serve as sole sources of carbon and energy for growth. C4 and C3 homologs could be utilized only in the presence of a second substrate (e.g., butanol), and the C2 homolog was not degraded. Kinetics of growth and substrate utilization indicated that cells of strain HA1 growing in butanol-salts medium could be used to test whether compounds induced the 1-chlorohexane halidohydrolase. No gratuitous induction of synthesis of the enzyme was observed. Many enzyme substrates (e.g., bromobenzene) did not induce synthesis of the enzyme, though the enzyme sequence to degrade the product (phenol) was present. Some inducers (e.g., bromomethane) were enzyme substrates but not growth substrates. In an attempt to find a physiological role for the 1-bromoalkane debrominase, we observed that several long-chain haloaliphatic compounds (greater than C9; e.g., 1-bromohexadecane and 1-chlorohexadecane) were utilized for growth and that induced cells could dehalogenate several 1-haloalkanes (at least C4 to C16). The dehalogenation of the long-chain compounds could not be assayed in the cell extract, so we presume that a third haloalkane dehalogenase was present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of 1-chlorohexane halidohydrolase, a dehalogenase of wide substrate range from an Arthrobacter sp.

1-Chlorohexane halidohydrolase from Arthrobacter sp. strain HA1 was purified to homogeneity by fractional precipitation, ion-exchange chromatography, gel filtration, and high-performance liquid chromatography gel filtration. The enzyme was a monomer with a molecular weight of about 37,000; its amino acid composition and N-terminal sequence were determined. The enzyme had a broad optimum around pH 9.5, a temperature optimum near 50 degrees C, an activation energy of 40 kJ/mol, and a molecular activity of 0.9 kat/mol. The substrate range of the enzyme included at least 50 halogenated compounds. 1-Chloroalkanes (C3 to C10), 1-bromoalkanes (C1 to C9), and 1-iodoalkanes (C1 to C7), but no 1-fluoroalkane, were substrates. Subterminally substituted, branched-chain, and nonsaturated haloalkanes were dehalogenated. Some halogenated aromatic substrates, e.g., bromobenzene and benzyl bromide, were hydrolyzed. Several alpha,omega-dihaloalkanes were subject to double dehalogenation. Thus, 1,2-dibromoethane was hydrolyzed first to 2-bromoethanol and then to 1,2-dihydroxyethane. Crude extracts of strain HA1 were found to contain a debrominase that cleaved bromoalkanes with long alkyl chains.     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

Dehalogenation of haloalkanes byRhodococcus erythropolis Y2

Phodococcus erythropolis Y2 produced two types of dehalogenase: a hydrolytic enzyme, that is an halidohydrolase, which was induced by C₃ to C₆ 1-haloalkane substrates, and at least one oxygenase-type dehalogenase induced by C₇ to C₁₆ 1-haloalkanes andn-alkanes. The oxygenase-type activity dehalogenated C₄ to C₁₈ 1-chloroalkanes with an optimum activity towards 1-chlorotetradecane. The halidohydrolase catalysed the dehalogenation of a wide range of 1- and ,-disubstituted haloalkanes and ,-substituted haloalcohols. In resting cell suspensions of hexadecane-grownR. erythropolis Y2 the oxygenase-type dehalogenase had a specific activity of 12.9 mU (mg protein)^–1 towards 1-chlorotetradecane (3.67 mU mg^–1 towards 1-chlorobutane) whereas the halidohydrolase in 1-chlorobutane-grown batch cultures had a specific activity of 44 mU (mg protein)^–1 towards 1-chlorobutane.The significance of the two dehalogenase systems in a single bacterial strain is discussed in terms of their contribution to the overall catabolic potential of the organism.     

A novel synthesis of branched high-molecular-weight (C40+) long-chain alkanes

Many biological and geochemical questions remain concerning the structures, functions, and properties of naturally occurring high-molecular-weight (C40+) alkanes with various mid-chain alkylation patterns. Above C40, these alkanes are exceedingly difficult to separate and purify, and syntheses can be blocked by the low solubility of intermediates. To overcome these problems, a facile three-step synthesis employing the alkylation of 1,3-dithiane with a suitable alpha,omega-dibromoalkane was developed. Bisalkylation of the bis(dithianyl)alkane intermediate with the appropriate 1-bromoalkane and subsequent desulfurization with Raney nickel furnished the desired long-chain alkane. Long-chain alkanes modified at mid-chain and/or symmetrically near the chain termini (or unmodified, i.e., long-chain n-paraffins) are accessible by the selection of appropriate bromoalkanes. Nine mid-chain methylated (C38H78 to C53H108), one symmetrical terminal-chain dimethylated (C40H82), and four linear (C44H90 to C58H118) long-chain alkanes were synthesized by using this approach. High-temperature gas chromatography (HTGC) was found to have important advantages for evaluating the purity of the synthetic high-molecular-weight alkanes.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Aerobic vinyl chloride metabolism in Mycobacterium aurum L1.

Mycobacterium aurum L1, capable of growth on vinyl chloride as a sole carbon and energy source, was previously isolated from soil contaminated with vinyl chloride (S. Hartmans et al., Biotechnol. Lett. 7:383-388, 1985). The initial step in vinyl chloride metabolism in strain L1 is catalyzed by alkene monooxygenase, transforming vinyl chloride into the reactive epoxide chlorooxirane. The enzyme responsible for chlorooxirane degradation appeared to be very unstable and thus hampered the characterization of the second step in vinyl chloride metabolism. Dichloroethenes are also oxidized by vinyl chloride-grown cells of strain L1, but they are not utilized as growth substrates. Three additional bacterial strains which utilize vinyl chloride as a sole carbon and energy source were isolated from environments with no known vinyl chloride contamination. The three new isolates were similar to strain L1 and were also identified as Mycobacterium aurum.     

Physiological function of alcohol dehydrogenases and long-chain (C(30)) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus

A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C(30) fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).     

Properties of rat liver N-acylethanolamine amidohydrolase

Rat liver microsomes and mitochondria contain an amidohydrolase which catalyzes the hydrolysis of N-acylethanolamine to ethanolamine and fatty acid. The enzyme is active over a wide range of pH, does not require divalent cations, and is inhibited by sulfhydryl-reactive agents. The detergents Triton X-100, sodium cholate, and sodium dodecyl sulfate are also inhibitory, but sodium taurodeoxycholate has little effect and was therefore used to solubilize the enzyme. The solubilized enzyme exhibits high substrate specificity for long-chain amides of ethanolamine. Amides of propanolamine or higher homologs are hydrolyzed at a drastically slower rate, and isomers prepared from long-chain amine and short-chain hydroxy acid are neither substrates nor inhibitors of the enzyme. Neither ceramide (N-acylsphingosine) nor N,O-diacylethanolamine is hydrolyzed. Both particulate and soluble enzyme preparations also catalyze the synthesis of N-acylethanolamine from ethanolamine and fatty acid, probably by the amidohydrolase acting in reverse.     

Aerobic vinyl chloride metabolism in Mycobacterium aurum L1.

Mycobacterium aurum L1, capable of growth on vinyl chloride as a sole carbon and energy source, was previously isolated from soil contaminated with vinyl chloride (S. Hartmans et al., Biotechnol. Lett. 7:383-388, 1985). The initial step in vinyl chloride metabolism in strain L1 is catalyzed by alkene monooxygenase, transforming vinyl chloride into the reactive epoxide chlorooxirane. The enzyme responsible for chlorooxirane degradation appeared to be very unstable and thus hampered the characterization of the second step in vinyl chloride metabolism. Dichloroethenes are also oxidized by vinyl chloride-grown cells of strain L1, but they are not utilized as growth substrates. Three additional bacterial strains which utilize vinyl chloride as a sole carbon and energy source were isolated from environments with no known vinyl chloride contamination. The three new isolates were similar to strain L1 and were also identified as Mycobacterium aurum.     

Phosphorus scavenging in the unicellular marine diazotroph Crocosphaera watsonii

Through the fixation of atmospheric nitrogen and photosynthesis, marine diazotrophs play a critical role in the global cycling of nitrogen and carbon. Crocosphaera watsonii is a recently described unicellular diazotroph that may significantly contribute to marine nitrogen fixation in tropical environments. One of the many factors that can constrain the growth and nitrogen fixation rates of marine diazotrophs is phosphorus bioavailability. Using genomic and physiological approaches, we examined phosphorus scavenging mechanisms in strains of C. watsonii from both the Atlantic and the Pacific. Observations from the C. watsonii WH8501 genome suggest that this organism has the capacity for high-affinity phosphate transport (e.g., homologs of pstSCAB) in low-phosphate, oligotrophic systems. The pstS gene (high-affinity phosphate binding) is present in strains isolated from both the Atlantic and the Pacific, and its expression was regulated by the exogenous phosphate supply in strain WH8501. Genomic observation also indicated a broad capacity for phosphomonoester hydrolysis (e.g., a putative alkaline phosphatase). In contrast, no clear homologs of genes for phosphonate transport and hydrolysis could be identified. Consistent with these genomic observations, C. watsonii WH8501 is able to grow on phosphomonoesters as a sole source of added phosphorus but not on the phosphonates tested to date. Taken together these data suggest that C. watsonii has a robust capacity for scavenging phosphorus in oligotrophic systems, although this capacity differs from that of other marine cyanobacterial genera, such as Synechococcus, Prochlorococcus, and Trichodesmium.     

Isolation and characterization of a thermostable intracellular enzyme with peroxidase activity from Bacillus sphaericus

During a screening for bacteria producing enzymes with peroxidase activity, a Bacillus sphaericus strain was isolated. This strain was found to contain an intracellular enzyme with peroxidase activity. The native enzyme had a molecular mass of above 300 kDa and precipitated at a salt concentration higher than 0.1 M. Proteolytic digestion with trypsin reduced the molecular mass of the active enzyme to 13 kDa (dimer) or 26 kDa (tetramer) and increased its solubility, allowing purification to homogeneity. Spectroscopic investigations showed the enzyme to be a hemoenzyme containing heme c as the covalently bound prosthetic group. The enzyme was stable up to 90 degrees C and at alkaline conditions up to pH 11, with a pH optimum at pH 8.5. It could be visualized by activity staining after SDS-PAGE and showed activity with a number of typical substrates for peroxidases, e.g., 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) diammonium salt, guaiacol and 2,4-dichlorophenol; however the enzyme had no catalase and cytochrome c peroxidase activity.     

Utilization of different fatty acyl-CoA thioesters by serine palmitoyltransferase from rat brain

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the first unique reaction of sphingolipid biosynthesis. Activities were determined with different fatty acyl-CoA substrates to describe the range of long-chain bases that could be made by rat brain microsomes. The activities were greatest with palmitoyl-CoA and palmitelaidoyl-CoA, followed by fully saturated homologs differing from these by only one carbon atom, and diminished considerably as the alkyl-chain length increased or decreased, or with the presence of a cis-double bond. These characteristics explain the predominance of long-chain bases with 18 carbon atoms in brain sphingolipids, and account for the minor variants such as the C17- and C20-long chain bases.     

A novel amidase (half-amidase) for half-amide hydrolysis involved in the bacterial metabolism of cyclic imides

A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K(m) = 6.2 mM; k(cat) = 5.76 s(-1)) and glutaramic acid (K(m) = 2.8 mM; k(cat) = 2.23 s(-1)). However, the substrates of known amidases such as short-chain (C(2) to C(4)) aliphatic amides, long-chain (above C(16)) aliphatic amides, amino acid amides, aliphatic diamides, alpha-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M(r) of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.     

Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2

Rhodococcus erythropolis strain Y2, isolated from soil by enrichment culture using 1-chlorobutane, was able to utilize a range of halogenated aliphatic compounds as sole sources of carbon and energy. The ability to utilize 1-chlorobutane was conferred by a single halidohydrolase-type haloalkane dehalogenase. The presence of the single enzyme in cell-free extracts was demonstrated by activity strain polyacrylamide gel electrophoresis. The purified enzyme was a monomeric protein with a relative molecular mass of 34 kDa and demonstrated activity against a broad range of haloalkanes, haloalcohols and haloethers. The highest activity was found towards alpha, omega disubstituted chloro- and bromo- C2-C6 alkanes and 4-chlorobutanol. The Km value of the enzyme for 1-chlorobutane was 0.26 mM. A comparison of the R. erythropolis Y2 haloalkane halidohydrolase with other haloalkane dehalogenases is discussed on the basis of biochemical properties and N-terminal amino acid sequence data.     

Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Design of new immobilized-stabilized carboxypeptidase a derivative for production of aromatic free hydrolysates of proteins

This paper presents stable carboxypeptidase A (CPA)-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced after immobilizing-stabilizing CPA on cross-linked 6% agarose beads, activated with low and high concentrations of aldehyde groups, and different immobilization times. The CPA-glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde as activation reactant. The most stabilized CPA-glyoxyl derivative was produced using 48 h of immobilization time and high activation grade of the support. This derivative was approximately 260-fold more stable than the soluble enzyme and presented approximately 42% of the activity of the soluble enzyme for the hydrolysis of long-chain peptides (e.g., cheese whey proteins previously hydrolyzed with immobilized trypsin and chymotrypsin) and of the small substrate N-benzoylglycyl-l-phenylalanine (hippuryl-l-Phe). These results were much better than those achieved using the conventional support, glutaraldehyde-agarose. Amino acid analysis of the products of the acid hydrolysis of CPA (both soluble and immobilized) showed that approximately four lysine residues were linked on the glyoxyl agarose beads, suggesting the existence of an intense multipoint covalent attachment between the enzyme and the support. The maximum temperature of hydrolysis was increased from 50 degrees C (soluble enzyme) to 70 degrees C (most stable CPA-glyoxyl derivative). The most stable CPA-glyoxyl derivative could be efficiently used in the hydrolysis of long-chain peptides at high temperature (e.g., 60 degrees C), being able to release 2-fold more aromatic amino acids (Tyr, Phe, and Trp) than the soluble enzyme, under the same operational conditions. This new CPA derivative greatly increased the feasibility of using this protease in the production of protein hydrolysates that must be free of aromatic amino acids.     

Metabolism of aromatic aldehydes as cosubstrates by the acetogen Clostridium formicoaceticum

When the acetogen Clostridium formicoaceticum was cultivated on mixtures of aromatic compounds (e.g., 4-hydroxybenzaldehyde plus vanillate), the oxidation of aromatic aldehyde groups occurred more rapidly than did O-demethylation. Likewise, when fructose and 4-hydroxybenzaldehyde were simultaneously provided as growth substrates, fructose was utilized only after the aromatic aldehyde group was oxidized to the carboxyl level. Aromatic aldehyde oxidoreductase activity was constitutive (activities approximated 0.8 U mg^–1), and when pulses of 4-hydroxybenzaldehyde were added during fructose-dependent growth, the rate at which fructose was utilized decreased until 4-hydroxybenzaldehyde was consumed. Although 4-hydroxybenzaldehyde inhibited the capacity of cells to metabolize fructose, lactate or gluconate were consumed simultaneously with 4-hydroxybenzaldehyde, and lactate or aromatic compounds lacking an aldehyde group were utilized concomitantly with fructose. These results demonstrate that (1) aromatic aldehydes can be utilized as cosubstrates and have negative effects on the homoacetogenic utilization of fructose by C. formicoaceticum, and (2) the consumption of certain substrates by this acetogen is not subject to catabolite repression by fructose. Received: 14 May 1998 / Accepted: 7 August 1998