Degradation of polyester polyurethane by newly isolated Pseudomonas aeruginosa strain MZA-85 and analysis of degradation products by GC–MS

In this report, a polyester polyurethane (PU) degrading bacterium, designated as strain MZA-85, was isolated from soil through enrichment. The bacterium was identified through 16S rRNA gene sequencing; it was completely matched with Pseudomonas aeruginosa type strain. The degradation of PU film pieces by P. aeruginosa strain MZA-85 was investigated by scanning electron microscopy (SEM), Fourier transformed infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM micrographs of PU film pieces, treated with strain MZA-85, revealed changes in the surface morphology. FTIR spectrum showed increase in organic acid functionality and corresponding decrease in ester functional group. GPC results revealed increase in polydispersity, which shows that long chains of polyurethane polymer are cleaved into shorter chains by microbial action. The bacterium was found to produce cell associated esterases based on p-Nitrophenyl acetate (pNPA) hydrolysis assay. 1,4-Butanediol and adipic acid monomers were detected by gas chromatography–mass spectrometry (GC–MS), which were produced as a result of hydrolysis of ester linkages in PU by cell bound esterases. Strain MZA-85 not only depolymerized PU but also mineralized it into CO₂ and H₂O, as indicated by increase in cells growth in the presence of degradation products as well as detection of CO₂ evolution through Sturm test. From the results presented above, it is finally concluded that P. aeruginosa strain MZA-85, as well as its enzymes, can be applied in the process of biochemical monomerization for the pure monomers recycling.     

Degradation of 4′-dimethylaminoazobenzene-2-carboxylic acid by Pseudomonas stutzeri

4-Dimethylaminoazobenzene-2-carboxylic acid (DMBC) was utilized as a necessary carbon and nitrogen source by Pseudomonas stutzeri IAM 12097. o-Aminobenzoic acid (o-ABA), N,N-dimethyl-p-phenylenediamine (DMPA) and cathecol were identified as intermediates of DMBC degradation. DMBC was degraded at a concentration below 70 mol dm^–3. The ability to utilize DMBC in P. stutzeri was lost spontaneously to some extent. When P. stutzeri was cured of plasmid DNA (approximately 8 MDal) by treatment with mitomycin C, acridine orange, and chloramphenicol, DMBC was not utilized by the resultant strain. These facts suggest that the degradative ability on DMBC in P. stutzeri is controlled by plasmid DNA. Correspondence to: C. Yatome     

Studies on the Biosynthesis of α-Putrescinylthymine in Bacteriophage φW-14-Infected Pseudomonas acidovorans

The alpha-putrescinylthymine (putThy) in bacteriophage phiW-14 DNA is synthesized at the mononucleotide level: it is labeled by uracil or deoxyuridine but not by thymidine, and it appears in the acid-soluble pool of infected cells before the onset of phage DNA synthesis. The methylene group at the C-5 position of the pyrimidine moiety of putThy is derived in vivo from a C(1) unit. Extracts of a phage infected thymidine auxotroph of the host, Pseudomonas acidovorans, apparently contain a phage-specific thymidylate synthetase and a phage-specific activity which forms 5-hydroxymethyl dUMP from N(5), N(10)-methylene-tetrahydrofolate and dUMP.     

Conversion of (−)-Carvotanacetone and (+)-Carvotanacetone by Pseudomonas ovalis,Strain 6–1

As a part of series on the biochemical reduction of terpenes, the conversion of (?)-carvotanacetone (I) and (+)-carvotanacetone (II) by Pseudomonas ovalis, strain 6–1, has been studied.

By the action of the microorganism, I was reduced to give (+)-carvomenthone (III), (+)-neocarvomenthol (IV), and (?)-carvomenthol (V), whereas II was also reduced to (?)-isocarvomenthone (VI), (?)-carvomenthone (VII), (?)-isocarvomenthol (VIII), and (?)-neoisocarvomenthol (IX); of which III, VI and IX are the major products.

The metabolic pathways of I and II and mechanism of stereospecific hydrogenation are also discussed.     

Screening and Characterization of Fructosyl-Valine–Utilizing Marine Microorganisms

We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from the marine environment and of fructosyl-amine oxidase from a marine yeast. Using fructosyl-valine (Fru-Val), a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically in a minimal medium supplemented with Fru-Val as the sole nitrogen source. Among these strains, a yeast strain identified as Pichia sp. N1-1, produced a Fru-Val–oxidizing enzyme. The enzyme was purified in its active form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H₂O₂ with the oxidation of Fru-Val. By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase. This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment and of fructosyl-amine oxidase from budding yeast. Received October 21, 1999; accepted September 12, 2000     

Studies on the “Salvage” Synthesis of Ribonucleosides and their 5′-Phosphates by Microorganisms

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (ΔG) at 20°C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

Studies on the “Salvage” Synthesis of Ribonucleosides and Their 5′-Phosphates by Microorganisms

Studies on the “salvage” synthesis of ribonucleosides and their 5′-phosphates from nucleic acid bases by microorganisms were undertaken. After screening test of less than one hundred strains of type culture, it was found that inosine was produced from hypoxanthine by Arthrobacter ureafaciens, A. simplex, Flavobacterium aquatile and F. suaveolens.

In certain conditions, inosine was further oxidized and hydrolyzed into xanthosine, uric acid and etc.

As for the conditions of cultivation and reaction, the components of the medium and pH of the culture medium were important factors.

Using the standard method, the yield of inosine from hypoxanthine by F. suaveolens reached more than 60%, and the conversion was stoichiometric and any other by-products were not detected.

Inosine, xanthosine, guanosine and uridine were produced from adenine, xanthine, guanine and uracil, respectively, by F. suaveolens.     

Degradation of caspase-activated DNase by the ubiquitin–proteasome system

DNA fragmentation is one of the most characteristic features of apoptotic cells and caspase-activated DNase (CAD) is considered to be a major nuclease responsible for DNA fragmentation. CAD forms a complex with its inhibitor (ICAD), which is also required for the functional folding of CAD, leading to CAD stabilization in cells. In this paper, we investigated the involvement of the ubiquitin–proteasome system in CAD stability. The expression and ubiquitination of CAD was remarkably increased by MG132 treatment in the absence of ICAD. These results suggest that CAD protein may be preferentially degraded by the ubiquitin–proteasome system in the absence of ICAD to maintain protein quality control.     

β-TrCP-Mediated Ubiquitination and Degradation of PHLPP1 Are Negatively Regulated by Akt

PHLPP1 belongs to a novel family of Ser/Thr protein phosphatases that serve as tumor suppressors by negatively regulating Akt signaling. Our recent studies have demonstrated that loss of PHLPP expression occurs at high frequency in colorectal cancer. In this study, we identified PHLPP1 as a proteolytic target of a β-TrCP-containing Skp-Cullin 1-F-box protein (SCF) complex (SCF^β-TrCP) E3 ubiquitin ligase in a phosphorylation-dependent manner. Overexpression of wild-type but not ΔF-box mutant β-TrCP leads to decreased expression and increased ubiquitination of PHLPP1, whereas knockdown of endogenous β-TrCP has the opposite effect. In addition, we show that the β-TrCP-mediated degradation requires phosphorylation of PHLPP1 by casein kinase I and glycogen synthase kinase 3β (GSK-3β), and activation of the phosphatidylinositol 3-kinase/Akt pathway suppresses the degradation of PHLPP1 by inhibiting the GSK-3β activity. Furthermore, expression of a degradation-deficient PHLPP1 mutant in colon cancer cells results in a more effective dephosphorylation of Akt and inhibition of cell growth. Taken together, our findings demonstrate a key role for β-TrCP in controlling the level of PHLPP1, and activation of Akt negatively regulates this degradation process.Hyperactivation of phosphatidylinositol 3-kinase/Akt signaling is commonly associated with human cancers (1, 5, 27). Inability to terminate the growth and survival signals mediated by Akt is one of the major mechanisms contributing to the development of cancer (1, 22, 32). The activation of Akt involves two phosphorylation steps: it is first phosphorylated at the activation loop (Thr308) within the kinase core by PDK-1 and subsequently at the hydrophobic motif (Ser473) in the C terminus by the TORC2 complex (22). Since the activity of Akt is tightly controlled by phosphorylation, dephosphorylation of Akt leads to effective signaling termination by inactivating the kinase. Recently, a novel family of Ser/Thr protein phosphatases, PHLPP, has been identified to fulfill the role of a negative regulator for Akt via direct dephosphorylation (3, 14). Two isoforms of PHLPP, namely PHLPP1 and PHLPP2, are found in this phosphatase family. Although the two isoforms of PHLPP share their ability to dephosphorylate Akt, each PHLPP preferentially regulates a subset of Akt isoforms in human lung cancer cells (3). Several lines of evidence suggest that PHLPP functions as a tumor suppressor. For example, overexpression of PHLPP in glioblastoma and colon cancer cells inhibits tumorigenesis in xenografted nude mice (14, 20), while decreased PHLPP expression correlates with increased metastastic potential in breast cancer cells (26). Furthermore, our recent studies have shown that downregulation of both PHLPP isoforms occurs at high frequency in colorectal cancer clinical samples (20). Loss of tumor suppressor expression can be caused by alterations at the gene level such as loss of heterozygosity or gene methylation. However, dysregulation of protein degradation pathways has also been implicated as a reason for downregulation of tumor suppressors (2, 6, 16).The ubiquitin (Ub) proteasome pathway controls degradation of the majority of eukaryotic proteins (12). β-TrCP belongs to a large family of F-box-containing proteins, and it serves as the substrate recognition subunit in the SCF (Skp1-Cullin 1-F-box protein) Ub-E3 ligase protein complex (4). By regulating the proteolytic process of its substrates, β-TrCP plays an important role in controlling cell cycle and cancer biogenesis (10). It is believed that β-TrCP-mediated ubiquitination requires phosphorylation of its substrates (35). A consensus binding motif with the sequence of DSG(X)_2-nS (so-called “phospho-degron”) has been proposed, in which the two serine residues are phosphorylated prior to binding to β-TrCP (4). However, variations of this motif, including replacement of the serine residues with phosphomimetic residues (e.g., Glu or Asp) in the substrate sequence, have been shown to be equally effective in mediating association with β-TrCP (31, 34).In this study, we report the identification of PHLPP1 as a proteolytic target of β-TrCP. We show that the degradation process of PHLPP1 depends on casein kinase I (CK1)- and glycogen synthase kinase 3 (GSK-3)-mediated phosphorylation, and activation of Akt negatively regulates PHLPP1 turnover. In addition, a PHLPP1 phosphorylation/degradation mutant antagonizes Akt more effectively in colon cancer cells.     

Growth of Pseudomonas chlororaphis on apolyester–polyurethane and the purification andcharacterization of a polyurethanase–esterase enzyme

A Pseudomonas chlororaphis was found to degrade and utilize apolyester polyurethane as a sole carbon and energy source. Polyurethane utilization by P.chlororaphis followed simple Michaelis–Menten kinetics. The K_s and μ_max values were 0.802 mg·ml⁻¹ and 1.316 doublings·h⁻¹, respectively. The enzymes from P. chlororaphis responsible for polyurethanedegradation were found to be extracellular. Analysis of the polyurethane degrading proteins, usingnon-denaturing polyacrylamide gel electrophoresis, revealed three active protein bands with R_f values of 0.25, 0.417 and 0.917. A polyurethane degrading enzyme was purifiedand displayed esterase activity. This enzyme was inhibited by phenylmethylsulfonyl fluoride andhad a molecular weight of 27,000 daltons.     

Enzymatic Degradation of Colistin Isolation and Identification of α-N-Acyl α,γ-Diaminobutyric Acid and Colistin Nonapeptide

Colistin, a fatty acyl peptide antibiotic, was attacked by proteolytic enzymes such as papain, ficin and bromelain, and as degradation product, a peptide portion retaining the ring structure of colistin was liberated. In contrast, an analogous antibiotic polymyxin B showed a characteristic resistance to the catalytic activity of papain.

Colistin nonapeptide and α-N-fatty acyl α,γ-diaminobutyric acid were obtained as products from the above enzymatic hydrolyzates of colistin and their chemical and physicochemical properties were investigated.

Contrary to colistin, this colistin nonapeptide was inactive to Escherichia coli. NIHJ and to many other strains even at a concentration of 800 mcg/ml by the agar dilution method. As α-N-fatty acyl α,γ-diaminobutyric acid which is rest part of colistin was added to colistin nonapeptide, antimicrobial activity of colistin nonapeptide did not increase.     

Degradation of polypropylene–poly-L-lactide blend by bacteria isolated from compost

Abstract

Polypropylene (PP) degrading bacteria (P1 to P16) were isolated from compost using enrichment technique. Five isolates (P3, P6, P8, P10, and P13) were selected based on their degradation abilities. These isolates were identified as Bacillus spp. through biochemical characteristics and 16S rDNA sequence analysis. The isolates were tested for their ability to degrade blends of PP and poly-L-lactide (PLLA) (PP80 and PP80C6) in minimal media as well as in soil. In minimal media, the growth of bacteria increased with time, showing utilization of blend as carbon source. The protein content was estimated at the end of 15?days and maximum amount was secreted by isolate P8 indicating maximum potential to degrade polymers compared to other isolates. Scanning electron microscopy (SEM) results revealed the formation of biofilm on the polymer surface. Fourier-transform infrared spectroscopy (FTIR) analysis showed the formation of new bond at 2123?cm^?1 and breakage of old C=O ester bond at 1757?cm^?1 in case of polymer PP80C6. Thermogravimetric analysis (TGA) showed decrease in thermal stability of polymers after degradation. The carbon dioxide evolved from sample was measured and biodegradation degree was also calculated. The degree of biodegradation shown by the isolate P8 was 12% and the P6 was 10%. The results demonstrated that Bacillus species isolated from composted samples in this study provided promising evidence for the biodegradation of polypropylene and poly-L-lactide (PP-PLLA) blends in the environment.     

Effect of visible light on catalytic hydrolysis of p-nitrophenyl palmitate by the Pseudomonas cepacia lipase immobilized on sol–gel support

This paper demonstrates Pseudomonas cepacia lipase catalyzed hydrolysis of p-nitrophenyl palmitate under irradiation of light with wavelengths of 250–750 nm. The reaction follows Michaelis–Menten Kinetics and the light irradiation increases the overall rate of hydrolysis. Using Lineweaver–Burk plot K _M and V _max values for the reaction in presence of light are found to be 39.07 and 66.67 mM/min/g, respectively; while for the same reaction under dark condition, the values are 7.08 and 10.21 mM/min/g. The linear form of enzyme dependent rate of reaction confirms that no mass-transfer limitations are present and the reaction is a kinetically controlled enzymatic reaction.     

Degradation of Soluble Amyloid β-Peptides 1–40, 1–42, and the Dutch Variant 1–40Q by Insulin Degrading Enzyme from Alzheimer Disease and Control Brains

Insulin degrading enzyme (IDE) is a metalloprotease that has been involved in amyloid peptide (A) degradation in the brain. We analyzed the ability of human brain soluble fraction to degrade A analogs 1–40, 1–42 and the Dutch variant 1–40Q at physiological concentrations (1 nM). The rate of synthetic ¹²⁵I-A degradation was similar among the A analogs, as demonstrated by trichloroacetic acid precipitation and SDS-PAGE. A 110 kDa protein, corresponding to the molecular mass of IDE, was affinity labeled with either ¹²⁵I-insulin, ¹²⁵I-A 1–40 or ¹²⁵I-A 1–42 and both A degradation and cross-linking were specifically inhibited by an excess of each peptide. Sensitivity to inhibitors was consistent with the reported inhibitor profile of IDE. Taken together, these results suggested that the degradation of A analogs was due to IDE or a closely related protease. The apparent Km, as determined using partially purified IDE from rat liver, were 2.2 ± 0.4, 2.0 ± 0.1 and 2.3 ± 0.3 M for A 1–40, A 1–42 and A 1–40Q, respectively. Comparison of IDE activity from seven AD brain cytosolic fractions and six age-matched controls revealed a significant decrease in A degrading activity in the first group, supporting the hypothesis that a reduced IDE activity may contribute to A accumulation in the brain.     

Isolation and Characterization of two New Microbial Strains Capable of Degradation of the Naturally Occurring Organophosphonate––Ciliatine

Air-born mixed fungal and bacterial culture capable of complete degradation of ciliatine was isolated. The utilization of the natural organophosphonate proceeded in the phosphate independent manner. Enzymatic activity involved in ciliatine degradation studied in the fungal cell-free extract proved to be distinct from bacterial pathway described before.     

Degradation Mechanism of Poly(vinyl alcohol) by Successive Reactions of Secondary Alcohol Oxidase and β-Diketone Hydrolase from Pseudomonas sp.

The secondary alcohol oxidase from Pseudomonas sp. catalyzed the oxidation of various vinyl alcohol oligomers with the molecular weight of 220 to 1500 and of β-ketols such as 5-hydroxy-3-heptanone, 4-hydroxy-2-nonanone, 3-hydroxy-5-nonanone, 6-hydroxy-4-nonanone, 7-hydroxy-5-dodecanone, and 8-hydroxy-6-tridecanone. β-Diketone hydrolase from the same strain catalyzed the hydrolysis of various aliphatic β-diketones and some aromatic β-diketones such as 1-phenyl-1,3-butanedione and 1-phenyl-2,4-pentanedione. 4,6-Nonanediol, used as a low molecular weight model of poly(vinyl alcohol) (PVA), was oxidized to 4,6-nonanedione by way of 6-hydroxy-4-nonanone by secondary alcohol oxidase. 4,6-Nonanedione was hydrolyzed to 2-pentanone and n-butyric acid by β-diketone hydrolase. These reactions were stoichiometric.

The presence of the β-diketone structure in PVA oxidized by secondary alcohol oxidase was confirmed by spectral experiments. The absorption due to β-diketone structure in the oxidized PVA decreased as it was hydrolyzed by β-diketone hydrolase. The ratio of the amount of carboxyl groups in the degraded PVA to that of carbonyl groups in the oxidized PVA became more than 0.5. A pathway for the enzymatic degradation of PVA was proposed.     

Degradation of insulin in vitro by liver and epididymal adipose tissue from obese–hyperglycaemic mice

The insulin-degrading activity of liver supernatants and epididymal adipose-tissue homogenates from genetically obese–hyperglycaemic mice (ob ob) and their lean litter mates was studied by measurement of radioactive trichloroacetic acid-soluble degradation products of the insulin molecule. Optimum assay conditions for the decomposition of the hormone were devised. The properties of the degrading activity suggested the presence of enzymic insulin destruction in both the liver and epididymal adipose tissue. There was no difference in insulin degradation in liver samples from obese and lean mice when the results were related to the protein content of the supernatants. The epididymal adipose-tissue homogenates from obese mice displayed about eightfold higher degrading activity per unit of protein than did homogenates from lean animals. The physiological significance of this finding is discussed in the light of the increased fat depots, hyperphagia, raised serum insulin concentrations and increased insulin tolerance previously recorded in this strain of mice.     

Biodegradation of p-nitrophenol by Pseudomonas aeruginosa HS-D38 and analysis of metabolites with HPLC–ESI/MS

Pseudomonas aeruginosa strain HS-D38 was capable of mineralizing p-nitrophenol (PNP) as the sole source of carbon, nitrogen and energy. Degradation of 200 mg L^?1 PNP was examined in different media including: (i) MSM (mineral salts medium, no carbon and nitrogen source); (ii) addition of 1% ammonium chloride as additional nitrogen source (ANM); and (iii) addition of 1% glucose as a carbon source (ACM). Complete degradation of 200 mg L^?1 PNP was achieved in 12 h in MSM. Additional ammonium chloride accelerated the PNP degradation, but additional glucose inhibited this process. This strain metabolized as high concentration as 300 and 500 mg L^?1 of PNP in 14 h and 24 h, respectively, in MSM. The degradation was accompanied by release of stoichiometric amount of nitrate from PNP. During the bacterial growth on PNP, hydroquinone and 1,2,4-benzenetriol were observed as the key degradation intermediates by using a combination of techniques, including HPLC–DAD and LC–ESI/MS compared with the authentic standards. These results indicated that PNP was degraded via a hydroquinone pathway.