Purification and Characterization of a Thermostable Xylanase from Fomitopsis pinicola

Shin, Keum; Jeya, Marimuthu; Lee, Jung-Kul; Kim, Yeong-Suk

doi:10.4014/jmb.1003.03031

Cited by 29 publications

(15 citation statements)

References 33 publications

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“…The LqCel7B K m value for pNP-G5 is within the range reported for other GH7 cellobiohydrolases (22)(23)(24)(25)(26). The inhibitory effect of cellobiose, which is commonly observed in GH7 enzymes from fungi (15,16,(26)(27)(28), on the hydrolytic activity of LqCel7B was investigated using pNP-G5 (Fig. S2F), and a K i value of 1.40 mM was measured.…”

Section: Resultsmentioning

confidence: 99%

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Kern

McGeehan

Streeter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

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Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.gribble | carbohydrate degrading enzymes

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Section: Resultsmentioning

confidence: 99%

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Kern

McGeehan

Streeter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

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“…β‐Xylosidase enzyme is also interesting in industry because of its involvement in the degradation of hemicellulose by hydrolyzing its main heteroglycan (xylan) (Ohta and others ; Shin and others ). Xylan presents a d ‐xylopyranose skeleton linked by β‐ d ‐(1‐4) bonds, which may contain residues of l ‐arabinofuranose, 4‐O‐methyl‐ d ‐glucuronic, acetate groups and other substituents depending on the nature of the plant material (Zhou and others ).…”

Section: Introductionmentioning

confidence: 99%

Screening of β‐Glucosidase and β‐Xylosidase Activities in Four Non‐Saccharomyces Yeast Isolates

López

Mateo

Maicas

2015

Journal of Food Science

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The finding of new isolates of non-Saccharomyces yeasts, showing beneficial enzymes (such as β-glucosidase and β-xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non-Saccharomyces yeasts. Four isolates were selected because of their both high β-glucosidase and β-xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB-medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β-glucosidase and β-xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β-glucosidase and β-xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3-folds) when wines were treated with non-Saccharomyces isolates. In detail, terpineol, 4-vinyl-phenol and 2-methoxy-4-vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2-phenyl ethanol than those inoculated with other yeasts.

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“…Aryl saccharides have been used as chromogenic substrates in cellulase research for over 50 years [9][10][11]. Chromogenic and fluorogenic cellobiosides and lactosides have been very helpful tools in research on H. jecorina Cel7A and the homologous endoglucanase Cel7B, as well as on GH7 enzymes from other organisms, ranging from simple applications such as expression assays and monitoring of protein purification, to high-throughput screening, pH-and temperaturedependence profiling, enzyme kinetics and inhibition studies [12][13][14][15][16][17][18][19][20]. However, similar applications have not been readily available for H. jecorina Cel6A and other GH6 enzymes, because of poor hydrolysis of the heteroglycosidic bond and/or preferential cleavage at homoglycosidic bonds in the substrate.…”

Section: Introductionmentioning

confidence: 99%

Rational design, synthesis, evaluation and enzyme–substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases

Nerinckx

Piens

et al. 2012

The FEBS Journal

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Methylumbelliferyl‐β–cellobioside (MUF–G2) is a convenient fluorogenic substrate for certain β–glycoside hydrolases (GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes (GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF–G2 in the +1 subsite of Hypocrea jecorina Cel6A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl‐β–cellobiosides [6–chloro‐4–methyl‐ (ClMUF); 6–chloro‐4‐trifluoromethyl‐ (ClF3MUF); 4–phenyl‐ (PhUF); 6–chloro‐4–phenyl‐ (ClPhUF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6A was 10–150 times higher than with MUF–G2, although it was still three orders of magnitude lower than with H. jecorina Cel7B. The 4–phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while ClMUF–G2 could be used for determination of kcat and KM for H. jecorina Cel6A and Thermobifida fusca Cel6A. Crystal structures of H. jecorina Cel6A D221A mutant soaked with the MUF‐, ClMUF‐ and ClPhUF‐β–cellobioside substrates show that the modifications turned the umbelliferyl group ‘upside down’, with the glycosidic bond better positioned for protonation than with MUF–G2. Database Structural data have been submitted to the Protein Data Bank under accession numbers pdb http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4AU0, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4AX7, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4AX6 Structured digital abstract • http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296 • http://www.uniprot.org/uniprot/P07987 and http://www.uniprot.org/uniprot/P07987 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (View Interaction: http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8401623, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8401640)

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Purification and Characterization of a Thermostable Xylanase from Fomitopsis pinicola

Cited by 29 publications

References 33 publications

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Screening of β‐Glucosidase and β‐Xylosidase Activities in Four Non‐Saccharomyces Yeast Isolates

Rational design, synthesis, evaluation and enzyme–substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases

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