Rapid differentiation between short-chain-length and medium-chain-length polyhydroxyalkanoate-accumulating bacteria with spectrofluorometry

Wu, Hui‐Lin; Sheu, Der-Shyan; Lee, Chia-Yin

doi:10.1016/s0167-7012(02)00232-4

Cited by 45 publications

(27 citation statements)

References 18 publications

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“…The presence of PHAs was evidenced by the observation of cells under a microscope, and by staining the biopolymer inside the cells with Nile red as reported by Wu et al [21]. A FT-IR analysis was also done by scanning the sample with a Perkin Elmer 1420 FT-IR spectrometer from 650 to 4,000 cm ¡1 as reported by Hong et al [6].…”

Section: Microorganism and Culture Mediummentioning

confidence: 98%

Biosynthesis and characterization of polyhydroxyalkanoates in the polysaccharide-degrading marine bacterium Saccharophagus degradans ATCC 43961

González‐García

Nungaray

Córdova

et al. 2008

J Ind Microbiol Biotechnol

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The marine bacterium Saccharophagus degradans was investigated for the synthesis of polyhydroxyalkanoates (PHAs), using glucose as the sole source of carbon in a two-step batch culture. In the first step the microorganism grew under nutrient balanced conditions; in the second step the cells were cultivated under limitation of nitrogen source. The biopolymer accumulated in S. degradans cells was detected by Nile red staining and FT-IR analysis. From GC-MS analysis, it was found that this strain produced a homopolymer of 3-hydroxybutyric acid. The cellular polymer concentration, its molecular mass, glass transition temperature, melting point and heat of fusion were 17.2+/-2.7% of dry cell weight, 54.2+/-0.6 kDa, 37.4+/-6.0 degrees C, 165.6+/-5.5 degrees C and 59.6+/-2.2 J g(-1), respectively. This work is the first report determining the capacity of S. degradans to synthesize PHAs.

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Section: Microorganism and Culture Mediummentioning

confidence: 98%

Biosynthesis and characterization of polyhydroxyalkanoates in the polysaccharide-degrading marine bacterium Saccharophagus degradans ATCC 43961

González‐García

Nungaray

Córdova

et al. 2008

J Ind Microbiol Biotechnol

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“…Colony/cell staining and gene detection are often used as front-line methods for high throughput screening and identification of novel microbes with PHA production potential owing to the relative ease of sample preparation and short analysis time. In colony/cell staining method, Nile red or Nile blue A dye is directly added into the solid growth medium [122], liquid cell cultures [123,124] or onto heat-fixed smeared cells [125]. Under UV illumination, Nile red and Nile blue A dye stains PHA to give a pink/red/yellow/orange appearance enabling the PHA-producing microbes to be identified and isolated.…”

Section: Detection Of Pha In Biomass and Pha Production Capacitymentioning

confidence: 99%

Start a Research on Biopolymer Polyhydroxyalkanoate (PHA): A Review

et al. 2014

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“…Liquid cultures were centrifuged at 5,000 ϫ g, washed with distilled water, and lyophilized overnight. About 10 mg of lyophilized cells was subjected to methanolysis in the presence of 15% sulfuric acid as previously described (40). The resulting methyl esters of constituent 3-hydroxyalkanoic acids were assayed by GC according to the method of Brandl et al (5).…”

Section: Methodsmentioning

confidence: 99%

Altering the Substrate Specificity of Polyhydroxyalkanoate Synthase 1 Derived from Pseudomonas putida GPo1 by Localized Semirandom Mutagenesis

Sheu

Lee

2004

J Bacteriol

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The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 Pp , class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA ؊ as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB ؊ 4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB ؊ 4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB ؊ 4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13 C nuclear magnetic resonance spectrum.

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Rapid differentiation between short-chain-length and medium-chain-length polyhydroxyalkanoate-accumulating bacteria with spectrofluorometry

Cited by 45 publications

References 18 publications

Biosynthesis and characterization of polyhydroxyalkanoates in the polysaccharide-degrading marine bacterium Saccharophagus degradans ATCC 43961

Biosynthesis and characterization of polyhydroxyalkanoates in the polysaccharide-degrading marine bacterium Saccharophagus degradans ATCC 43961

Start a Research on Biopolymer Polyhydroxyalkanoate (PHA): A Review

Altering the Substrate Specificity of Polyhydroxyalkanoate Synthase 1 Derived from Pseudomonas putida GPo1 by Localized Semirandom Mutagenesis

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