Purification, crystallization and characterization of <i>N</i>-acetylneuraminate lyase from <i>Escherichia coli</i>

Aisaka, Kazuo; Igarashi, Ayuko; Yamaguchi, Kazuo; Uwajima, Takayuki

doi:10.1042/bj2760541

Cited by 56 publications

(78 citation statements)

References 26 publications

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“…In contrast, GiCLO particles were the most sensitive to heat treatment but showed resistance to extremes of pH. This is consistent with previous research on the soluble form of these enzymes (10,(25)(26)(27)(28)(29)(30). Native soluble BLA has been shown to be stable up to 75°C and between pH 6.0 and 8.5 (29).…”

Section: Discussionsupporting

confidence: 80%

“…No structural or stability data are currently available for OpdA from A. radiobacter; however, other similar phosphotriesterases have been shown to be stable up to 50°C and between pH 6.0 and 10.0 (31,32). The isoelectric points of BLA and NanA are 6.9 and 4.5, respectively (27,29), while the isoelectric point of a well-characterized homologous phosphotriesterase from Brevundimonas diminuta is 8.3 (33). Therefore, OpdA is proposed to be more stable at alkaline pH.…”

Section: Discussionmentioning

confidence: 97%

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In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

Venning-Slater

Hooks

Rehm

2014

Appl Environ Microbiol

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Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the in vivo self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable ␣-amylase from Bacillus licheniformis, N-acetyl-D-neuraminic acid aldolase (NanA) from Escherichia coli, and organophosphohydrolase (OpdA) from Agrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The ␣-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert N-acetylmannosamine and pyruvate to Nacetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 97%

In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

Venning-Slater

Hooks

Rehm

2014

Appl Environ Microbiol

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“…However, the original study using the E. coli deletion strains indicated that the nature of the C-5 amino substituent in Sia does not affect transport or degradation (60). The aldolases from C. perfringens and E. coli are capable of cleaving a range of neuraminic acid derivatives with different substituents at C-5, including formyl, succinyl, and glycolyl neuraminic acids (1,48). Regarding the NanT permease, it has been established that many bacteria, both gram negative and gram positive, exhibit an active proton symporter-type mechanism (59).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Bacterial YhcH Protein Indicates a Role in Sialic Acid Catabolism

et al. 2005

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The yhcH gene is part of the nan operon in bacteria that encodes proteins involved in sialic acid catabolism. Determination of the crystal structure of YhcH from Haemophilus influenzae was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. The structure was determined at 2.2-Å resolution by multiple-wavelength anomalous diffraction. The protein fold is a variation of the double-stranded ␤-helix. Two antiparallel ␤-sheets form a funnel opened at one side, where a putative active site contains a copper ion coordinated to the side chains of two histidine and two carboxylic acid residues. A comparison to other proteins with a similar fold and analysis of the genomic context suggested that YhcH may be a sugar isomerase involved in processing of exogenous sialic acid.

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“…Aldolase cleavage was determined both spectrophotometrically and chromatographically (HPLC). The first method measured the decrease in absorbance at 340 nm corresponding to the oxidation of NADH produced by lactate dehydrogenase (LDH) when pyruvate appeared as a consequence of the hydrolysis of Neu5Ac into such compound and of ManNAc produced by LpNAL (1,43). The standard reaction medium (1 ml) for the above-described assay, which was carried out in a Shimadzu UV-2401 PC spectrophotometer, contained 150 M NADH, 0.5 U LDH, 10 mM Neu5Ac, and 1.5 g of purified LpNAL in 20 mM phosphate buffer (pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization of a Novel N -Acetylneuraminate Lyase from Lactobacillus plantarum WCFS1

Sánchez-Carrón

García-García

López-Rodríguez

et al. 2011

Appl Environ Microbiol

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N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of Nacetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D-mannosamine (ManNAc). In nature, Nacetylneuraminate lyase occurs mainly in pathogens. However, this paper describes how an N-acetylneuraminate lyase was cloned from the human gut commensal Lactobacillus plantarum WCFS1 (LpNAL), overexpressed, purified, and characterized for the first time. This novel enzyme, which reaches a high expression level (215 mg liter ؊1 culture), shows similar catalytic efficiency to the best NALs previously described. This homotetrameric enzyme (132 kDa) also shows high stability and activity at alkaline pH (pH > 9) and good temperature stability (60 to 70°C), this last feature being further improved by the presence of stabilizing additives. These characteristics make LpNAL a promising biocatalyst. When its sequence was compared with that of other, related (real and putative) NALs described in the databases, it was seen that NAL enzymes could be divided into four structural groups and three subgroups. The relation of these subgroups with human and other mammalian NALs is also discussed.

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Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli

Cited by 56 publications

References 26 publications

In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

Crystal Structure of the Bacterial YhcH Protein Indicates a Role in Sialic Acid Catabolism

Molecular Characterization of a Novel N -Acetylneuraminate Lyase from Lactobacillus plantarum WCFS1

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