Alan William Bunch

Microbial Cyanide Metabolism

Publisher Summary This chapter focuses on cyanide metabolism in micro-organisms. It is noted that cyanide is a relatively common product of microbial as well as plant metabolism. Cyanide production by micro-organisms has many characteristics typical of secondary metabolism. In addition, it is probably the simplest secondary metabolic system and a continued investigation of cyanide formation should greatly aid a better understanding of microbial secondary metabolism. Cyanide degradation by Chromobacterium violaceum, or C. violaceum, is known to synthesize at least three enzymes capable of metabolizing cyanide. These include rhodanese, γ-cyano-α-aminobutyric acid synthase, and β-cyanoalanine synthase. The concentrations of all three enzymes increase in the post-cyanogenic period. The buildup of β-cyanoalanine is particularly noteworthy in bacteria grown under conditions of high cyanogenesis. The suspensions of harvested C. violaceum cells are also able to form β-cyanoalanine when incubated with cyanide and serine.

Biotransformation of nitriles by rhodococci.

Rhodococci have been shown to be capable of a very wide range of biotransformations. Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities. Initial investigations used relatively simple aliphatic nitriles. These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes. Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles. This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g. acrylamide production from acrylonitrile).The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released). It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules. The genes for many nitrile-hydrolysing enzymes have been identified and sequenced. The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.

Biocatalysts for clean industrial products and processes.

Biocatalysis inherently offers the prospect of clean industrial processing and has become an accepted technology throughout most sectors. The convergence of biology and chemistry has enabled a plethora of industrial opportunities to be targeted, while discoveries in biodiversity and the impact of molecular biology and computational science are extending the range of natural and engineered biocatalysts that can be customised for clean industrial requirements.

The nitrilases of Rhodococcus rhodochrous NCIMB 11216

Rhodococcus rhodochrous NCIMB 11216 grows on propionitrile or benzonitrile as the sole source of carbon and nitrogen. The possibility that different nitrile-hydrolyzing enzymes were produced under these two growth conditions was investigated. Nitrilase activity in whole cell suspensions from either bacteria grown on propionitrile or benzonitrile were capable of biotransforming a wide range of nitriles. The propionitrile-induced nitrile degrading activity hydrolyzed 3-cyanobenzoate and both the nitrile groups in 1,3-dicyanobenzoate. In contrast, the benzonitrile-induced activity hydrolyzed only one of the nitrile groups in 1,3-dicyanobenzoate, but did not affect 3-cyanobenzoate. Both nitrilases biotransformed α-cyano-o-tolunitrile to produce 2-cyanophenylacetic acid. The nitrilases were purified by fast protein liquid chromatography and the n-terminus of each enzyme sequenced. SDS-PAGE analysis identified a subunit molecular weight of 45.8 kDa for each nitrilase. The n-terminal sequences showed significant similarity with other sequenced nitrilases and with the exception of a single amino acid were identical with each other. Both nitrilases had temperature and pH optima of 30°C and 8.0, respectively. The propionitrile-induced nitrilase had a Km for benzonitrile of 20.7 mm and a Vmax of 12.4 μmol min−1 mg−1 protein whereas the benzonitrile-induced nitrilase had a Km for benzonitrile of 8.83 mm and a Vmax of 0.57 μmol min−1 mg−1 protein.

Vancomycin production in batch and continuous culture

Production of the glycopeptide antibiotic vancomycin by two Amycolatopsis orientalis strains was examined in batch shake flask culture in a semidefined medium with peptone as the nitrogen source. Different growth and production profiles were observed with the two strains; specific production (Yp/x) was threefold higher with strain ATCC 19795 than with strain NCIMB 12945. A defined medium with amino acids as the nitrogen source was developed by use of the Plackett–Burman statistical screening method. This technique identified certain amino acids (glycine, phenylalanine, tyrosine, and arginine) that gave significant increased specific production, whereas phosphate was identified as inhibitory for high specific vancomycin production. Experiments made with the improved medium and strain ATCC 19795 showed that vancomycin production kinetics were either growth dissociated or growth associated, depending on the amino acid concentration. In chemostat culture at a constant dilution rate (0.087 h−1), specific vancomycin production rate (qvancomycin) decreased linearly as the medium phosphate concentration was increased from 2 to 8 mM. In both phosphate and glucose limited chemostats, qvancomycin was a function of specific growth rate; the maximum value was observed at D = 0.087 h−1 (52% of the maximum specific growth rate). Under phosphate limited growth conditions, qvancomycin was threefold higher (0.37 mg/g dry weight/h) than under glucose limitation (0.12 mg/g dry weight/h).

Survivability of bacteria ejected from icy surfaces after hypervelocity impact.

Both the Saturnian and Jovian systems contain satellites with icy surfaces. If life exists on any of these icy bodies (in putative subsurface oceans for example) then the possibility exists for transfer of life from icy body to icy body. This is an application of the idea of Panspermia, wherein life migrates naturally through space. A possible mechanism would be that life,here taken as bacteria, could become frozen in the icy surface ofone body. If a high-speed impact occurred on that surface, ejectacontaining the bacteria could be thrown into space. It could thenmigrate around the local region of space until it arrived at a second icy body in another high-speed impact. In this paper we consider some of the necessary steps for such a process to occur,concentrating on the ejection of ice bearing bacteria in the initial impact, and on what happens when bacteria laden projectiles hit an icy surface. Laboratory experiments using high-speed impacts with a light gas gun show that obtaining icy ejecta with viable bacterial loads is straightforward. In addition to demonstrating the viability of the bacteria carried on the ejecta, we have also measured the angular and size distribution of the ejecta produced in hypervelocity impacts on ice. We have however been unsuccessful at transferring viablebacteria to icy surfaces from bacteria laden projectilesimpacting at hypervelocities.

Bacterial ethylene synthesis from 2-oxo-4-thiobutyric acid and from methionine

The ability of selected bacterial cultures to synthesize ethylene during growth in nutrient broth supplemented with methionine or 2-oxo-4-methylthiobutyric acid (KMBA) was examined. Although most cultures transformed KMBA into ethylene, only those of Escherichia coli SPAO and Chromobacterium violaceum were able to convert exogenously added methionine to ethylene. In chemically defined media, E. coli SPAO produced the highest amounts of ethylene from methionine and KMBA. This capability was affected by the nature of the carbon source and the type and amount of nitrogen source used for growth. When glutamate was used as sole source of carbon and nitrogen for growth, the activity of the ethylenogenic enzymes was reduced to 25% of that observed with cultures grown with glucose and NH4Cl. Neither methionine nor KMBA significantly affected the ethylenogenic capacity of E. coli SPAO. Menadione and paraquat, compounds that generate superoxide radicals, stimulated ethylene synthesis by harvested cells, but not by cell-free extracts of E. coli SPAO. In addition, cells of Pseudomonas aeruginosa, which produced no ethylene in culture in the presence of exogenously added KMBA, yet possessed the necessary enzymes in an active form, were able to synthesize ethylene from KMBA when incubated with menadione or paraquat.

A microcalorimetric comparison of the anti‐Streptococcus mutans efficacy of plant extracts and antimicrobial agents in oral hygiene formulations

T. D. MORGAN, A. E. BEEZER, J. C. MITCHELL AND A. W. BUNCH. 2001.

The formation and substrate specificity of bacterial lactonases capable of enantioselective resolution of racemic lactones

Abstract The substrate specificities of ϵ-caprolactone hydrolase from Acinetobacter NCIMB 9871 and δ-valerolactone hydrolase from Pseudomonas NCIMB 9872 were investigated. Both lactonases showed activity toward six- and seven-membered ring lactones. δ-Valerolactone hydrolase exhibited enantioselectivity in its activity. It showed a preference for the R enantiomer of δ-decanolactone and δ-nonalactone whereas ϵ-caprolactone hydrolase showed little enantioselectivity toward the lactone substrates tested. The δ-valerolactone hydrolase from Pseudomonas NCIMB 9872 may be useful for the resolution of racemic lactones, and hence may serve as an alternative route to chiral lactone synthesis.

Vancomycin production is enhanced in chemostat culture with biomass‐recycle

Production of the glycopeptide antibiotic vancomycin by Amycolatopsis orientalis ATCC 19795 was examined in phosphate-limited chemostat cultures with biomass-recycle, employing an oscillating membrane separator, at a constant dilution rate (D= 0. 14 h-1). Experiments made under low agitation conditions (600 rpm) showed that the biomass concentration could be increased 3.9-fold with vancomycin production kinetics very similar to that of chemostat culture without biomass-recycle. The specific production rate (qvancomycin) was maximal when the biomass-recycle ratio (R) was 0.13 (D= 0.087 h-1). When the dissolved oxygen tension dropped below 20% (air saturation), the biomass and vancomycin concentrations decreased and an unidentified red metabolite was released into the culture medium. Using increased agitation (850 rpm), used to maintain the dissolved oxygen tension above 20% air saturation, maximum increases in biomass concentration (7.9-fold) and vancomcyin production 1.6-fold (0.6 mg/g dry weight/h) were obtained when R was 0.44 (D= 0.056 h -1) compared to chemostat culture without biomass-recycle. Moreover, at this latter recycle ratio the volumetric vancomycin production rate was 14.7 mg/L/h (a 7-fold increase compared to chemostat culture without biomass-recycle). These observations encourage further research on biomass-recycling as a means of optimising the production of antibiotics.