Pseudomonas diminuta

Although Brevundimonas (Pseudomonas) diminuta (ATCC 19146) is most commonly used for steriliziug-grade filter vaUdation, iu certain appHcations other bacteria are used. For example, when it is necessary to demonstrate removal of mycoplasma in appHcations involving sera and tissue culture media, membranes having a smaller pore size rating, eg, 0.1 p.m, are frequentiy used. For these membranes,laidlawii may be employed for vaHdation purposes (9). 

The phosphotriesterase from Pseudomonas diminuta was shown to catalyze the enantioselective hydrolysis of several racemic phosphates (21), the Sp isomer reacting faster than the Rp compound [65,66]. Further improvements using directed evolution were achieved by first carrying out a restricted alanine-scan [67] (i.e. at predetermined amino acid positions alanine was introduced). Whenever an effect on activity/ enantioselectivity was observed, the position was defined as a hot spot. Subsequently, randomization at several hot spots was performed, which led to the identification of several highly (S)- or (R)-selective mutants [66]. A similar procedure was applied to the generation of mutant phosphotriesterases as catalysts in the kinetic resolution of racemic phosphonates [68]. 

The degradation of isoquinoline by Pseudomonas diminuta strain 7 is initiated by an oxidoreductase that contains [2Fe-2S] centers and the cofactor molybdopterin cytosine dinucleotide (Lehmann et al. 1994). 

Lehmann M, Tshisuaka B, Fetzner S, Roger P, Lingens F (1994) Purification and characterization of isoquinoline 1-oxidoreductase from Pseudomonas diminuta 7, a molybdenum-containing hydroxylase. JBiol Chem 269 11254-11260. 

The oxidoreductase from Pseudomonas diminuta strain 7 that carries out hydroxylation of isoquinoline at C2 is a molybdenum enzyme containing [Fe-S] centers, which is comparable to the aldehyde oxidoreductase from Desulfovibrio gigas (Lehmann et al. 1994). 

The degradation of isoquinoline by Alcaligenes faecalis strain Pa and Pseudomonas diminuta strain 7 (Roger et al. 1990, 1995) is mediated by an oxidoreductase that produces 1,2-dihydroiso-quinoline-l-one, followed by ring fission with the production of o-phthalate and oxidation to 3,4-dihydroxybenzoate (Figure 3.38). The oxidoreductase is purified and like most typical aza-rene oxidoreductases contains, per mole, 0.85 g atoms of Mo, 3.9 g atoms of Fe, and acid-labile S (Lehmann et al. 1994). 

Roger P, A Erben, E Lingens (1990) Microbial metabolism of quinoline and related compounds IV. Degradation of isoquinoline by Alcaligenes faecalis Pa and Pseudomonas diminuta 1. Biol Chem Hoppe-Seyler 370 1183-1189. 

Roger P, G Bar, E Lingens (1995) Two novel metabolites in the degradation pathway of isoquinoline by Pseudomonas diminuta 1. FEMS Microbiol Lett 129 281-286. 

Mulbry WW, JS Karns, PC Kearney, JO Nelson, CS McDaniel, JR Wild (1986) Identification of a plasmid-borne Parathion hydrolase gene from Flavobacterium sp. by Southern hybridization with opd from Pseudomonas diminuta. Appl Environ Microbiol 51 926-930. 

Serdar CM, DT Gibson, DM Munnecke, JH Lancaster (1982) Plasmid involvement in Parathion hydrolysis in Pseudomonas diminuta. Appl Environ Microbiol 44 246-249. 

Dumas DP, IR Wild, FM Raushel (1989) Diisopropylfluorophosphate hydrolysis by an organophosphate anhydrase from Pseudomonas diminuta. Biotechnol Appl Biochem 11 235-243. 

Roger, P. Bar, G. and Lingens, F., Novel Metabolites in the Degradation Pathway of Isoquinoline by Pseudomonas-Diminuta-7. FEMS Microbiol Lett, 1995. 129(2-3) pp. 281-285. 

Roger, P. Erben, A., and Lingens, F., Microbial Metabolism of Quinoline and Related Compounds. IV. degradation of isoquinoline by alcaligenes faecalis pa and pseudomonas diminuta 7. Biol Chem Hoppe Seyler, 1990. 371(6) pp. 511-513. 

Lehmann, M. Tshisuaka, B. Fetzner, S., et al., Purification and Characterization of Isoquinoline 1-Oxidoreductase From Pseudomonas-Diminuta-7, A Novel Molybdenum-Containing Hydroxylase. J Biol Chem, 1994. 269(15) pp. 11254-11260. 

D.P. Dumas, H.D. Durst, W.G. Landis, F.M. Raushel, and J.R. Wild, Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta. Arch. Biochem. Biophys. 277, 155-159 (1990). 

K.I. Dave, C.E. Miller, and J.R. Wild, Characterization of. organophosphorous hydrolases and the genetic manipulation of. the phosphotriesterase from pseudomonas diminuta. Chem. Biol. Intract. 87, 55-68 (1993). 

A phosphotriesterase isolated from the soil bacterium Pseudomonas diminuta is the best characterized enzyme of this type. There is evidence for the presence of two active site Zn2+ ions in vivo. A crystal structure of the dinuclear Cd2+ form is available in which the metal ions are bridged by a carbamylated Lys-amino group with a metal-metal distance of 3.8 A [ 18]. Substrate hydrolysis follows a SN2 type reaction and nucleophilic attack of M-OH is likely, but mechanistic details are not yet clear. 

Havens and Rase reported the immobilization of an enzyme to degrade a specific organophosphate. The organophosphate was an agricultural grade material (parathion). The enzyme was harvested from recombinant Pseudomonas diminuta and immobilized by emulsifying a solution with a prepolymer. The product of the reaction was reported to have excellent stability and the method was proposed for cleanup of small spills of parathion. 

The test should have been correlated with a bacterial challenge test (BDT). The FDA considers a sterilizing grade filter as one that produces a sterile filtrate when challenged with 10 Pseudomonas diminuta per cm2 of filter surface. 

Caldwell SR, Raushel FM. Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta. Biotechnol Bioeng 1991 37 103-109. 

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