恶臭假单胞菌NA-1菌株烟酸羟基化酶活性的诱导和转化条件的研究

恶臭假单胞菌NA-1菌株的培养和产酶特性与已报道的产酶菌株粘质沙雷氏菌(Serratiamarcescens)IFO12648和荧光假单胞菌(Psudomonasfluorescens)TN5有所不同,主要反映在最适碳源及浓度、最适诱导剂浓度和最适培养温度等方面。最适的转化条件是温度为30℃,pH为7.0,烟酸的浓度为3%。采用初步优化后的条件和流加底物的方式进行4L上罐生产,恶臭假单胞菌NA-1菌株的6-羟基烟酸产率可达到108.39gL。     

The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1

Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r(2)=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19+/-1.5x10(-4) mg indigo (mg protein)(-1) min(-1) was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0+/-0.2x10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions.     

Benzoate-dependent induction from the OP2 operator-promoter region of the TOL plasmid pWWO in the absence of known plasmid regulatory genes.

Expression of the lower catabolic pathway of the TOL plasmid pWWO requires an aromatic acid inducer and the product of the xylS regulatory gene. Pseudomonas putida cells transformed with a plasmid containing the operator-promoter region of the lower pathway (OP2 [or Pm]), upstream from the catechol 2,3-dioxygenase structural gene, showed enzyme induction in the absence of known TOL plasmid regulatory genes. Induction was not seen in transformed Escherichia coli cells or in a P. putida mutant lacking chromosomally encoded benzoate catabolic functions.     

Acetopyruvate hydrolase production by Pseudomonas putida O1--optimization of batch and fed-batch fermentations

The main objective of this work was the optimization of the production of the beta-ketolase, acetopyruvate hydrolase, from Pseudomonas putida O1. Orcinol was used as an inducer for enzyme production. The growth medium was optimized in two steps. In the first step, screening for optimal glucose concentration was performed. In the second step, a central composite design was used to optimize carbon and nitrogen sources in the medium. After this optimization procedure, a medium was obtained which produced seven times more biomass than the initial medium. Acetopyruvate hydrolase enzyme production was optimized by determining the optimal time of feed and amount of orcinol, using statistical methods. In a subsequent step, the maximal orcinol-degradation rate was determined. The results obtained were used to find an optimal feeding profile for enzyme production. By using the optimized fed-batch process, acetopyruvate hydrolase activity was enhanced from 10 units l(-1)to 400 units l(-1), in comparison with previously reported fermentation experiments. Productivity could even be increased by a factor of 75, to a value of 20 units l(-1 )h(-1).     

Inducible degradation of hydroxyproline in Pseudomonas putida: pathway regulation and hydroxyproline uptake

Studies in Pseudomonas putida of the inducible degradation of hydroxyproline to alpha-ketoglutarate have indicated that either of the two epimers, hydroxy-l-proline or allohydroxy-d-proline, acts as an inducer of all the pathway enzymes. In a mutant lacking the first enzyme of the sequence, hydroxyproline-2-epimerase, which interconverts these two hydroxyproline epimers, either epimer is still equally active as an inducer of the remaining three enzymes, suggesting that each epimer has intrinsic inducer activity. The second and third enzymes of the sequence were induced coordinately. The induction process appeared to be insensitive to catabolite repression under a number of experimental conditions. The induced enzymes were stable even under conditions of nitrogen starvation and other conditions designed to increase protein turnover. In addition to inducing the degradative enzymes, the two hydroxyproline epimers were also found to induce an uptake system that concentrates hydroxyproline intracellularly. Either amino acid induced the uptake system for its epimer as well as for itself.     

Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose

Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.     

Production of Aspergillus terreus alpha-L-rhamnosidase by solid state fermentation

AIMS: The study of production of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase in solid state fermentation using wheat bran, washed sugar cane bagasse and polyurethane foam as substrates or supports for the enzyme production. METHODS AND RESULTS: Cultures were carried out in Petri dishes under controlled temperature and humidity. Naringin or rhamnose were the enzyme inducers and carbon sources. The enzyme activity to inducer ratio was appreciably greater when using sugar cane bagasse or polyurethane foam than wheat bran. The influence of inoculum size, inducer, airflow, humidity and temperature were determined. Under optimum conditions, about four units of enzyme per ml nutrient solution were obtained after 4-6 d. CONCLUSIONS: The activity to inducer ratio was higher, and the cultivation time was shorter in solid state fermentation than those observed in submerged cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Solid cultures, using naringin as inducer, can be appropriate alpha-L-rhamnosidase production.     

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. II. Isolation and properties of blocked mutants

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. II. Isolation and properties of blocked mutants. J. Bacteriol. 91:1155-1160. 1966.-Mutants of Pseudomonas putida blocked in early reactions of the pathway for oxidation of d-mandelate were isolated and partially characterized. The specific genetic lesions in these mutants made normal inducer-metabolites of the pathway nonmetabolizable. Under the conditions of gratuitous enzyme synthesis so obtained, it could be shown that the d and l isomers of mandelate are equipotent inducers, and that the synthesis of the first five enzymes of the mandelate pathway is coordinate. Further experiments with the blocked mutants showed that benzoylformate, the third intermediate of the pathway, acts as an inducer without prior conversion to mandelate, and that there is no inducible, concentrating permease for mandelate.     

Kinetic analysis of bacterial bioluminescence

Bioluminescence from the lux-based bacterial reporter Pseudomonas fluorescens HK44 was experimentally investigated under growth substrate-rich and limiting conditions in batch, continuous stirred tank (CSTR), and turbidostat reactors. A mechanistically based, mathematical model was developed to describe bioluminescence based on 1) production and decay of catalytic enzymes, and 2) reactant cofactor availability. In the model, bioluminescence was a function of inducer, growth substrate, and biomass concentration. A saturational dependence on growth substrate concentration accommodated dependence on cofactor availability and inducer concentration to accommodate enzyme production was incorporated in the model. Under growth substrate and inducer limiting conditions in the batch reactor and CSTR, bioluminescence was found to decrease in response to cellular energy limitations. The effective lux system enzyme decay rate was determined in independent measurements to be 0.35 hr(-1) and the model captured most of the bioluminescent behavior, except at long growth times and high cell density.     

Diketone cleaving enzyme Dke1 production by Acinetobacter johnsonii--optimization of fermentation conditions

The main objective of this work was the optimization of the production of the novel dioxygenase diketone cleaving enzyme (Dke1) from Acinetobacter johnsonii. Acetylacetone was used as an inducer for enzyme production. In the first step, the growth medium was optimized by using screening designs for finding the optimal carbon and nitrogen source. In the second step, a genetic algorithm was used to optimize the concentrations of all medium components. After six generations the stopping criterion was reached and a growth medium was obtained which produced sixteen times more enzyme than the starting medium. In the next step, an addition profile for the inducer acetylacetone was developed to further increase enzyme production by using a genetic algorithm. In this case, after four generations the stopping criterion was fulfilled. By using the obtained optimal addition profile Dke1 activity was enhanced from 826 to 2584Ul(-1). In comparison to the starting conditions activity could even be increased by a factor of 50.     

Oxidation of methyl tert-butyl ether by alkane hydroxylase in dicyclopropylketone-induced and n-octane-grown Pseudomonas putida GPo1

The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of P. putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells. Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high K(s) value (20 to 40 mM) for MTBE.     

Kinetic analysis of a tod-lux bacterial reporter for toluene degradation and trichloroethylene cometabolism

Kinetics of toluene and trichloroethylene (TCE) degradation and bioluminescence from the bioreporter Pseudomonas putida B2 and TVA8 were investigated utilizing batch and continuous culture, respectively. Degradation was modeled using a Michaelis-Menten expression for the competition of two substrates for a single enzyme system, and bioluminescence was modeled assuming a luciferase enzyme saturational dependence on toluene as the inducer and growth substrate. During the batch experiments, bioluminescence increased at approximately 90 namp/min for initial toluene concentrations of 10 to 50 mg/L, but more slowly at higher toluene concentrations, suggesting maximum promoter induction at below 10 mg/L and toxic effects above 50 mg/L toluene. TCE degradation did not occur until toluene depletion, presumably due to competition between toluene and TCE for the toluene dioxygenase enzyme. During continuous culture, bioluminescence transiently increased, then gradually decreased in response to increasing step changes in toluene feed concentration. Bioluminescence in the CSTR appeared to be limited by growth substrate and/or inducer.     

Some aspects of the regulation of the production of extracellular proteolytic enzymes by a marine bacterium

A highly proteolytic Gram-negative, rod-shaped bacterium was isolated from the gills of fresh plaice and the effect of culture conditions on the production of proteolytic enzymes was investigated. When the organism, strain SA 1, was grown in the presence of complex mixtures of proteins and amino acids, both endopeptidase and aminopeptidase activity was demonstrated in the cell-free culture medium. However, synthesis of these enzymes was not observed when the organism was grown in a mineral medium with lactate or succinate as the only carbon and energy source. Synthesis of both endopeptidase and aminopeptidase was induced by the presence of amino acids in the medium. Of the amino acids tested, l-phenylalanine was found to be the best single inducer for the production of endopeptidase. When in addition one or more different amino acids were added, endopeptidase production was found to increase with increasing complexity of the mixture, up to a maximum which was obtained with five different amino acids. Production of the aminopeptidase was optimal when l-glutamic acid was used as a single inducer. For this enzyme the amount of enzyme activity released in the medium decreased with increasing complexity of the amino acid mixture. Endopeptidase as well as aminopeptidase activity was found to accumulate in the medium at the end of the logarithmic growth phase, when the culture was no longer growing exponentially. When the stationary phase was reached, enzyme production stopped. Production of both enzymes was immediately halted upon addition of chloramphenicol and was found to be repressed by glucose and lactate. These results suggest that synthesis of proteolytic extracellular enzymes by the organism studied is controlled by an efficient regulatory mechanism, in which growth rate is an important parameter.     

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 g l⁻¹) in the fermentation medium further increased naringinase production (8.9 IU ml⁻¹) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.     

Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid

A new enzyme, phenylacetyl-CoA ligase (AMP-forming) (PA-CoA ligase, EC 6.2.1-) involved in the catabolism of phenylacetic acid (PAA) in Pseudomonas putida is described and characterized. PA-CoA ligase was specifically induced by PAA when P. putida was grown in a chemically defined medium in which phenylacetic acid was the sole carbon source. Hydroxyl, methyl-phenylacetyl derivatives, and other PAA close structural molecules did not induce the synthesis of this enzyme and neither did acetic, butyric, succinic, nor fatty acids (greater than C5 atoms carbon length). PA-CoA ligase requires ATP, CoA, PAA, and MgCl2 for its activity. The maximal rate of catalysis was achieved in 50 mM HCl/Tris buffer, pH 8.2, at 30 degrees C and under these conditions, the Km calculated for ATP, CoA, and PAA were 9.7, 1.0, and 16.5 mM, respectively. The enzyme is inhibited by some divalent cations (Cu2+, Zn2+, and Hg2+) and by the sulfhydryl reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-chloromercuribenzoate. PA-CoA ligase was purified to homogeneity (513-fold). It runs as a single polypeptide in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a molecular mass of 48 +/- 1 kDa. PA-CoA ligase does not use as substrate either 3-hydroxyphenylacetic, 4-hydroxyphenylacetic, or 3,4-dihydroxyphenylacetic acids and shows a substrate specificity different from other acyl-CoA-activating enzymes. The enzyme is detected in P. putida from the early logarithmic phase of growth and is repressed by glucose, suggesting that PA-CoA ligase is a specific enzyme involved in the utilization of PAA as energy source.     

Indigo formation by microorganisms expressing styrene monooxygenase activity.

The transformation of indole to indigo by microorganisms expressing styrene monooxygenase (SMO) has been studied. Styrene and indole are structurally very similar, and thus we looked at a variety of styrene-degrading strains for indole transformation to indigo. Two strains, Pseudomonas putida S12 and CA-3, gave a blue color on solid media when grown in the presence of indole. Indole induces its own transformation on solid media but is a poor inducer in liquid media. Styrene is the best inducer of indole transformation in both strains. Arginine represses styrene consumption and indigo formation rates in P. putida S12 compared to phenylacetic acid-grown cells, while the opposite effect is seen for P. putida CA-3. Characterization of an SMO- and styrene oxide isomerase (SOI)-negative transposon mutant of P. putida CA-3 and an SOI-negative N-methyl-N'-nitro-N-nitrosoguanidine mutant of P. putida S12 reveals the involvement of both SMO and SOI in indole transformation to indigo. Both strains stoichiometrically produce high-purity indigo from indole.     

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extracellular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

delta1-piperideine-2-carboxylate reductase of Pseudomonas putida.

Pseudomonas putida metabolizes D-lysine to delta 1-piperideine-2-carboxylate and L-pipecolate. The second step of this catabolic pathway is catalyzed by delta 1-piperideine-2-carboxylate reductase. This enzyme was isolated and purified from cells grown on DL-lysine as substrate. The enzyme was very unstable, resulting in low recovery of activity and low purity after a six-step purification procedure. The enzyme had a pH optimum of 8.0 to 8.3. The Km values for delta 1-piperideine-2-carboxylate and NADPH were 0.23 and 0.13 mM, respectively. NADPH at concentrations above 0.15 mM was inhibitory to the enzyme. Delta 1-pyrroline-5-carboxylate, pyroglutamate, and NADH were poor substrates or coenzyme for delta 1-piperideine-2-carboxylate reductase. The enzyme reaction from delta 1-piperideine-2-carboxylate to L-pipecolate was irreversible. EDTA, sodium pyrophosphate, and dithiothreitol at concentrations of 1 mM protected the enzyme during storage. The enzyme was inhibited almost totally by Zn2+, Mn2+, Hg2+ Co2+, and p-chloromercuribenzoate at concentrations of 0.1 mM. The enzyme had a molecular weight of about 200,000. Both D-lysine and L-lysine were good inducers for the enzyme. Neither delta1-piperideine-2-carboxylate nor L-pipecolate was an effective inducer for the enzyme. P. putida cells grew on D-lysine only after a 5- to 8-h lag, which could be abolished by adding a supplement of 0.01% alpha-ketoglutarate or other readily metabolizable compounds. Such a supplement also converted the noncoordinate induction of this enzyme and pipecolate oxidase, both of the D-lysine pathway, to coordinacy. However, this effect was not observed if the enzyme pair was from different pathways of lysine metabolism in this organism (i.e., the D- and L-lysine pathways).     

Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

Overexpression of salicylate hydroxylase and the crucial role of lys(163) as its NADH binding site

Expression systems for the sal gene encoding salicylate hydroxylase from Pseudomonas putida S-1 were examined and some constructs were expressed in these systems. By cultivation of Escherichia coli BL21 (DE3)/pSAH8 in LB medium at 37 degrees C with isopropyl-b-D-thiogalactopyranoside as the inducer, salicylate hydroxylase was overexpressed mainly in the form of inclusion bodies. Lower temperature cultivation at 20 degrees C after induction resulted in a large amount of the enzyme in the soluble form. The E. coli clone harboring the recombinant plasmid produced a 45 kDa protein that appeared to be electrophoretically and immunochemically identical to the P. putida enzyme and contained the same N-terminal amino acid sequence. This recombinant DNA product also exhibited properties characteristic of a flavoprotein and was fully functional as salicylate hydroxylase. Based on chemical modification of the salicylate hydroxylase from P. putida, Lys163 was previously proposed to be the NADH binding site. In this study, to obtain a better understanding of the predicted role of Lys163, this residue in the active center of salicylate hydroxylase was replaced with Arg, Gly, or Glu by conventional site-directed mutagenesis. Kinetic studies using these mutant enzymes and the recombinant enzyme revealed increases in apparent K(m) values for NADH in the order of wild-type enzyme > K163R > K163G > K163E, with some decreases in V(max). Examination of the recombinant enzyme and K163G indicated that the pH dependency of K(m) on NADH with pK(a) 10.5 is lost by mutation despite the lack of changes in V(max) values, suggesting a requirement for the lysine residue as the NADH binding site. Based on these results, Lys163 is proposed to play a role in the binding of NADH at the active site through an ionic bond rather than playing a role in catalysis.