Purification and characterization of an exo-type β-N-acetylglucosaminidase from Pseudomonas fluorescens JK-0412

Park, J.K.; Kim, W.J.; Park, Y.I.

doi:10.1111/j.1365-2672.2010.04879.x

Cited by 15 publications

(15 citation statements)

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“…[18]NahA38.8- Symbiobacterium thermophilum [47]ExoII30.71.1 V. furnissii [68]NagA3-0.3 Thermotoga maritima [84]CbsA3-0.3 Thermotoga neapolitana [84]--521.5- Enterobacter sp. [85]--67.2- Vibrio alginolyticus [65]--40.5- Talaromyces emersonii [86]--272.0 Streptomyces cerradoensis [40]NagA-5.3245 Pseudomonas fluorescens [3]-0.03- Trichinella spiralis [64] a These values (μmol min −1 mg −1 ) were determined using p NPGlcNAc as substrate …”

Section: Resultsmentioning

confidence: 99%

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Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance

et al. 2017

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BackgroundEnzymatic degradation of chitin has attracted substantial attention because chitin is an abundant renewable natural resource, second only to lignocellulose, and because of the promising applications of N-acetylglucosamine in the bioethanol, food and pharmaceutical industries. However, the low activity and poor tolerance to salts and N-acetylglucosamine of most reported β-N-acetylglucosaminidases limit their applications. Mining for novel enzymes from new microorganisms is one way to address this problem.ResultsA glycoside hydrolase family 20 (GH 20) β-N-acetylglucosaminidase (GlcNAcase) was identified from Microbacterium sp. HJ5 harboured in the saline soil of an abandoned salt mine and was expressed in Escherichia coli. The purified recombinant enzyme showed specific activities of 1773.1 ± 1.1 and 481.4 ± 2.3 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and N,N'-diacetyl chitobiose, respectively, a V max of 3097 ± 124 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and a K i of 14.59 mM for N-acetylglucosamine inhibition. Most metal ions and chemical reagents at final concentrations of 1.0 and 10.0 mM or 0.5 and 1.0% (v/v) had little or no effect (retaining 84.5 − 131.5% activity) on the enzyme activity. The enzyme can retain more than 53.6% activity and good stability in 3.0–20.0% (w/v) NaCl. Compared with most GlcNAcases, the activity of the enzyme is considerably higher and the tolerance to salts and N-acetylglucosamine is much better. Furthermore, the enzyme had higher proportions of aspartic acid, glutamic acid, alanine, glycine, random coils and negatively charged surfaces but lower proportions of cysteine, lysine, α-helices and positively charged surfaces than its homologs. These molecular characteristics were hypothesised as potential factors in the adaptation for salt tolerance and high activity of the GH 20 GlcNAcase.ConclusionsBiochemical characterization revealed that the GlcNAcase had novel salt–GlcNAc tolerance and high activity. These characteristics suggest that the enzyme has versatile potential in biotechnological applications, such as bioconversion of chitin waste and the processing of marine materials and saline foods. Molecular characterization provided an understanding of the molecular–function relationships for the salt tolerance and high activity of the GH 20 GlcNAcase.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0358-1) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

“…Chitooligosaccharides can be produced from the degradation of chitin by hydrochloric acid [3] or chitinases (EC 3.2.1.14) [2]. Chitin is second only to lignocellulose in natural abundance, but more than 80,000 tons of chitin from marine sources per year is unutilized chitinous waste [2].…”

Section: Introductionmentioning

confidence: 99%

Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance

et al. 2017

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“…Stm Hex reaction products were examined by HPLC using chitooligosaccharide (DP2–DP6) substrates. Nag2 from V. harveyii 650 required 25 h for complete hydrolysis of DP6 (Suginta et al ., ), while Stm Hex required 1 h. N ‐acetyl‐β‐glucosaminidase from Pseudomonas fluorescens JK‐0412 (Park et al ., ) and Aeromonas hydrophila SUWA‐9 (Lan et al ., ) attained complete DP5 hydrolysis by 24 and 7 h, respectively, and Stm Hex completed the same in 1 h. NagC from S. thermoviolaceus exhibited activity only on DP2–DP5 substrates and not on DP6 substrate (Kubota et al ., ). When colloidal chitin and α‐chitin were used as substrates, Stm Hex did not release any products even after prolonged reaction (data not shown), indicating that the enzyme was not active on polymeric chitin, similar to hexosaminidases reported from V. furnissii and Penaeus japonicus (Keyhani & Roseman, ; Koga et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Chitooligosaccharides are converted toN-acetylglucosamine byN-acetyl-β-hexosaminidase fromStenotrophomonas maltophilia

Suma

Ankati

Podile

2013

FEMS Microbiol Lett

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The Stenotrophomonas maltophilia k279a (Stm) Hex gene encodes a polypeptide of 785 amino acid residues, with an N-terminal signal peptide. StmHex was cloned without signal peptide and expressed as an 83.6 kDa soluble protein in Escherichia coli BL21 (DE3). Purified StmHex was optimally active at pH 5.0 and 40 °C. The Vmax, Km and kcat/Km for StmHex towards chitin hexamer were 10.55 nkat (mg protein)(-1), 271 μM and 0.246 s(-1) mM(-1), while the kinetic values with chitobiose were 30.65 nkat (mg protein)(-1), 2365 μM and 0.082 s(-1) mM(-1), respectively. Hydrolytic activity on chitooligosaccharides indicated that StmHex was an exo-acting enzyme and yielded N-acetyl-D-glucosamine (GlcNAc) as the final product. StmHex hydrolysed chitooligosaccharides (up to hexamer) into GlcNAc within 60 min, suggesting that this enzyme has potential for use in large-scale production of GlcNAc from chitooligosaccharides.

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“…AS-Esc9 displays high galactosidase activity and might help to produce low-lactose milk or dairy products for lactose-intolerant people (Husain 2010). Because of the biological properties of chitin and its derivatives, the putative N-acetylglucosaminidase of AS-Esc15 might be used in the pharmaceutical industry to produce N-acetylglucosamine (Park et al 2011). In addition to the putative glycoside hydrolases found, other putative enzymes encoded by insert fragments might be of interest to industry, such as the xylose isomerase, which might be used in the manufacture of high-fructose corn syrup to convert glucose to fructose (Antrim et al 1979).…”

Section: Discussionmentioning

confidence: 99%

New carbohydrate-active enzymes identified by screening two metagenomic libraries derived from the soil of a winter wheat field

2014

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Aims Soils are rich, diversified environments where β‐glucosidases abound because of their importance in organic matter degradation. The aim of this work was to discover new β‐glucosidases by constructing two metagenomic DNA libraries from soil samples collected in winter and spring from a field of winter wheat. Methods and Results Both libraries were screened on esculin‐supplemented medium so as to isolate candidates showing β‐glucosidase activity. Candidate analysis revealed seven putative β‐glycosidases and two putative glycosyltransferases, displaying 25 to 82% identity to known enzymes. The putative β‐glycosidases belong to families GH1, GH3 and GH20 and the two putative glycosyltransferases, probably, to new families. In characterization tests performed on bacteria in suspension or spread on agar plates, some candidates appeared to hydrolyse several natural and synthetic substrates. These tests also highlighted interesting industrial characteristics, such as the activity of four β‐glycosidases under alkaline conditions and the esculin‐hydrolysing activity of a β‐glucosidase candidate in the presence of glucose. Conclusions Seven putative β‐glycosidases and two putative glycosyltransferases were found by functional screening of two metagenomic DNA libraries derived from agricultural soil. Significance and Impact of the Study This study has identified β‐glycosidases and putative glycosyltransferases that have or may have interesting industrial characteristics.

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Purification and characterization of an exo-type β-N-acetylglucosaminidase from Pseudomonas fluorescens JK-0412

Cited by 15 publications

References 37 publications

Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance

Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance

Chitooligosaccharides are converted toN-acetylglucosamine byN-acetyl-β-hexosaminidase fromStenotrophomonas maltophilia

New carbohydrate-active enzymes identified by screening two metagenomic libraries derived from the soil of a winter wheat field

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