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Takashi Kawaguchi - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Aspergillus aculeatus β-glucosidase 1 accelerating cellulose hydrolysis with Trichoderma cellulase system
AMB Express, 2015Co-Authors: Yutaro Baba, Junichi Sumitani, Shuji Tani, Takashi KawaguchiAbstract:Aspergillus aculeatus β-glucosidase 1 (AaBGL1), which promotes cellulose hydrolysis by Trichoderma cellulase system, was characterized and compared some properties to a commercially supplied orthologue in A. niger (AnBGL) to elucidate advantages of recombinant AaBGL1 (rAaBGL1) for synergistic effect on Trichoderma enzymes. Steady–state kinetic studies revealed that rAaBGL1 showed high catalytic efficiency towards β-linked glucooligosaccharides. Up to a degree of polymerization (DP) 3, rAaBGL1 prefered to hydrolyze β-1,3 linked glucooligosaccharides, but longer than DP 3, preferred β-1,4 glucooligosaccharides (up to DP 5). This result suggested that there were different formation for subsites in the catalytic cleft of AaBGL1 between β-1,3 and β-1,4 glucooligosaccharides, therefore rAaBGL1 preferred short chain of laminarioligosaccharides and long chain of cellooligosaccharides on hydrolysis. rAaBGL1 was more insensitive to glucose inhibition and more efficient to hydrolyze the one of major transglycosylation product, gentiobiose than AnBGL, resulting that rAaBGL1 completely hydrolyzed 5% cellobiose to glucose faster than AnBGL. These data indicate that AaBGL1 is valuable for the use of cellulosic biomass conversion.
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crystal structures of glycoside hydrolase family 3 β glucosidase 1 from Aspergillus aculeatus
Biochemical Journal, 2013Co-Authors: Kentaro Suzuki, Junichi Sumitani, Toru Nishimaki, Shuji Tani, Takashi Kawaguchi, Takayoshi Wakagi, Shinya FushinobuAbstract:GH3 (glycoside hydrolase family 3) BGLs (β-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 ( Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N -glycan chains) in the dimer. The largest N -glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.
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construction of a recombinant trichoderma reesei strain expressing Aspergillus aculeatus β glucosidase 1 for efficient biomass conversion
Biotechnology and Bioengineering, 2012Co-Authors: Hikaru Nakazawa, Junichi Sumitani, Shuji Tani, Takashi Kawaguchi, Tetsushi Kawai, Noriko Ida, Yosuke Shida, Yoshinori Kobayashi, Hirofumi Okada, Yasushi MorikawaAbstract:To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes. Biotechnol. Bioeng. 2012;109: 92–99. © 2011 Wiley Periodicals, Inc.
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construction of a recombinant trichoderma reesei strain expressing Aspergillus aculeatus β glucosidase 1 for efficient biomass conversion
Biotechnology and Bioengineering, 2012Co-Authors: Hikaru Nakazawa, Junichi Sumitani, Shuji Tani, Takashi Kawaguchi, Tetsushi Kawai, Noriko Ida, Yosuke Shida, Yoshinori Kobayashi, Hirofumi Okada, Yasushi MorikawaAbstract:To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.
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Agrobacterium tumefaciens -mediated transformation of Aspergillus aculeatus for insertional mutagenesis
AMB Express, 2011Co-Authors: Emi Kunitake, Junichi Sumitani, Shuji Tani, Takashi KawaguchiAbstract:Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus.
Sine Larsen - One of the best experts on this subject based on the ideXlab platform.
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Structural and Biochemical Studies Elucidate the Mechanism of Rhamnogalacturonan Lyase from Aspergillus aculeatus
Journal of molecular biology, 2010Co-Authors: M.h. Jensen, Sine Larsen, Harm Otten, Ulla Christensen, Torben Vedel Borchert, Lars Lehmann Hylling Christensen, Leila Lo LeggioAbstract:Abstract We present here the first experimental evidence for bound substrate in the active site of a rhamnogalacturonan lyase belonging to family 4 of polysaccharide lyases, Aspergillus aculeatus rhamnogalacturonan lyase (RGL4). RGL4 is involved in the degradation of rhamnogalacturonan-I, an important pectic plant cell wall polysaccharide. Based on the previously determined wild-type structure, enzyme variants RGL4_H210A and RGL4_K150A have been produced and characterized both kinetically and structurally, showing that His210 and Lys150 are key active-site residues. Crystals of the RGL4_K150A variant soaked with a rhamnogalacturonan digest gave a clear picture of substrate bound in the − 3/+ 3 subsites. The crystallographic and kinetic studies on RGL4, and structural and sequence comparison to other enzymes in the same and other PL families, enable us to propose a detailed reaction mechanism for the β-elimination on [-,2)-α- l -rhamno-(1,4)-α- d -galacturonic acid-(1,-]. The mechanism differs significantly from the one established for pectate lyases, in which most often calcium ions are engaged in catalysis.
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Aspergillus aculeatus beta-1,4-Galactanase: Substrate Recognition and Relations to Other Glycoside Hydrolases in Clan GH-A
Biochemistry, 2002Co-Authors: Carsten Ryttersgaard, Leila Lo Leggio, Pedro M. Coutinho, Bernard Henrissat, Sine LarsenAbstract:The three-dimensional structure of Aspergillus aculeatus β-1,4-galactanase (AAGAL), an enzyme involved in pectin degradation, has been determined by multiple isomorphous replacement to 2.3 and 1.8 A resolution at 293 and 100 K, respectively. It represents the first known structure for a polysaccharidase with this specificity and for a member of glycoside hydrolase family 53 (GH-53). The enzyme folds into a (β/α)8 barrel with the active site cleft located at the C-terminal side of the barrel consistent with the classification of GH-53 in clan GH-A, a superfamily of enzymes with common fold and catalytic machinery but diverse specificities. Putative substrate−enzyme interactions were elucidated by modeling of β-1,4-linked galactobioses into the possible substrate binding subsites. The structure and modeling studies identified five potential subsites for the binding of galactans, of which one is a pocket suited for accommodating the arabinan side chain in arabinogalactan, one of the natural substrates. A com...
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A stepwise optimization of crystals of rhamnogalacturonan lyase from Aspergillus aculeatus
Acta Crystallographica Section D Biological Crystallography, 2002Co-Authors: Renuka Kadirvelraj, Sakari Kauppinen, Jens-christian N. Poulsen, Pernille Harris, Sine LarsenAbstract:Recombinant rhamnogalacturonan lyase from Aspergillus aculeatus has been crystallized by a stepwise procedure and X-ray diffraction data have been collected. The crystals were grown using hanging-drop vapour-diffusion and microseeding techniques. Crystals were obtained showing a flat plate morphology. The crystallization conditions were 20% PEG 4000, 9% PEG 400, 0.1 M (NH4)2SO4 and 0.1 M sodium acetate pH 4.4. These crystals diffracted to a resolution of 1.5 A. The unit-cell parameters are a = b = 77.0, c = 170.8 A with the possible space group P43212 or P41212. There is most likely to be one molecule in the asymmetric unit, leading to a calculated solvent content of approximately 47% for the crystals.
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Crystallization and preliminary X-ray studies of β-1,4-galactanase from Aspergillus aculeatus
Acta Crystallographica Section D Biological Crystallography, 1999Co-Authors: Carsten Ryttersgaard, Henrik Dalbøge, Jens-christian N. Poulsen, Stephan Christgau, Thomas Sandal, Sine LarsenAbstract:Recombinant β-1,4-galactanase from Aspergillus aculeatus has been crystallized and characterized by X-ray diffraction. Crystals were obtained in hanging drops by vapour-diffusion under the conditions 30% PEG 400, 0.2 M CaCl2 and 0.1 M Na HEPES buffered to pH 7.5. The crystals diffract to 2.3 A resolution and belong to one of the orthorhombic space groups I222 or I212121. The unit-cell dimensions are a = 60.42, b = 88.94 and c = 129.08 A. With one molecule in the asymmetric unit, the corresponding solvent content is ∼48%.
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Crystallization and preliminary X-ray studies of beta-1, 4-galactanase from Aspergillus aculeatus.
Acta crystallographica. Section D Biological crystallography, 1999Co-Authors: Carsten Ryttersgaard, Stephan Christgau, Thomas Sandal, J Poulsen, H Dalbøge, Sine LarsenAbstract:Recombinant beta-1,4-galactanase from Aspergillus aculeatus has been crystallized and characterized by X-ray diffraction. Crystals were obtained in hanging drops by vapour-diffusion under the conditions 30% PEG 400, 0.2 M CaCl2 and 0.1 M Na HEPES buffered to pH 7.5. The crystals diffract to 2.3 A resolution and belong to one of the orthorhombic space groups I222 or I212121. The unit-cell dimensions are a = 60.42, b = 88.94 and c = 129.08 A. With one molecule in the asymmetric unit, the corresponding solvent content is approximately 48%.
Junichi Sumitani - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Aspergillus aculeatus β-glucosidase 1 accelerating cellulose hydrolysis with Trichoderma cellulase system
AMB Express, 2015Co-Authors: Yutaro Baba, Junichi Sumitani, Shuji Tani, Takashi KawaguchiAbstract:Aspergillus aculeatus β-glucosidase 1 (AaBGL1), which promotes cellulose hydrolysis by Trichoderma cellulase system, was characterized and compared some properties to a commercially supplied orthologue in A. niger (AnBGL) to elucidate advantages of recombinant AaBGL1 (rAaBGL1) for synergistic effect on Trichoderma enzymes. Steady–state kinetic studies revealed that rAaBGL1 showed high catalytic efficiency towards β-linked glucooligosaccharides. Up to a degree of polymerization (DP) 3, rAaBGL1 prefered to hydrolyze β-1,3 linked glucooligosaccharides, but longer than DP 3, preferred β-1,4 glucooligosaccharides (up to DP 5). This result suggested that there were different formation for subsites in the catalytic cleft of AaBGL1 between β-1,3 and β-1,4 glucooligosaccharides, therefore rAaBGL1 preferred short chain of laminarioligosaccharides and long chain of cellooligosaccharides on hydrolysis. rAaBGL1 was more insensitive to glucose inhibition and more efficient to hydrolyze the one of major transglycosylation product, gentiobiose than AnBGL, resulting that rAaBGL1 completely hydrolyzed 5% cellobiose to glucose faster than AnBGL. These data indicate that AaBGL1 is valuable for the use of cellulosic biomass conversion.
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crystal structures of glycoside hydrolase family 3 β glucosidase 1 from Aspergillus aculeatus
Biochemical Journal, 2013Co-Authors: Kentaro Suzuki, Junichi Sumitani, Toru Nishimaki, Shuji Tani, Takashi Kawaguchi, Takayoshi Wakagi, Shinya FushinobuAbstract:GH3 (glycoside hydrolase family 3) BGLs (β-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 ( Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N -glycan chains) in the dimer. The largest N -glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.
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construction of a recombinant trichoderma reesei strain expressing Aspergillus aculeatus β glucosidase 1 for efficient biomass conversion
Biotechnology and Bioengineering, 2012Co-Authors: Hikaru Nakazawa, Junichi Sumitani, Shuji Tani, Takashi Kawaguchi, Tetsushi Kawai, Noriko Ida, Yosuke Shida, Yoshinori Kobayashi, Hirofumi Okada, Yasushi MorikawaAbstract:To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes. Biotechnol. Bioeng. 2012;109: 92–99. © 2011 Wiley Periodicals, Inc.
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construction of a recombinant trichoderma reesei strain expressing Aspergillus aculeatus β glucosidase 1 for efficient biomass conversion
Biotechnology and Bioengineering, 2012Co-Authors: Hikaru Nakazawa, Junichi Sumitani, Shuji Tani, Takashi Kawaguchi, Tetsushi Kawai, Noriko Ida, Yosuke Shida, Yoshinori Kobayashi, Hirofumi Okada, Yasushi MorikawaAbstract:To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.
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Agrobacterium tumefaciens -mediated transformation of Aspergillus aculeatus for insertional mutagenesis
AMB Express, 2011Co-Authors: Emi Kunitake, Junichi Sumitani, Shuji Tani, Takashi KawaguchiAbstract:Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus.
Yasushi Morikawa - One of the best experts on this subject based on the ideXlab platform.
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construction of a recombinant trichoderma reesei strain expressing Aspergillus aculeatus β glucosidase 1 for efficient biomass conversion
Biotechnology and Bioengineering, 2012Co-Authors: Hikaru Nakazawa, Junichi Sumitani, Shuji Tani, Takashi Kawaguchi, Tetsushi Kawai, Noriko Ida, Yosuke Shida, Yoshinori Kobayashi, Hirofumi Okada, Yasushi MorikawaAbstract:To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes. Biotechnol. Bioeng. 2012;109: 92–99. © 2011 Wiley Periodicals, Inc.
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construction of a recombinant trichoderma reesei strain expressing Aspergillus aculeatus β glucosidase 1 for efficient biomass conversion
Biotechnology and Bioengineering, 2012Co-Authors: Hikaru Nakazawa, Junichi Sumitani, Shuji Tani, Takashi Kawaguchi, Tetsushi Kawai, Noriko Ida, Yosuke Shida, Yoshinori Kobayashi, Hirofumi Okada, Yasushi MorikawaAbstract:To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.
Shuji Tani - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Aspergillus aculeatus β-glucosidase 1 accelerating cellulose hydrolysis with Trichoderma cellulase system
AMB Express, 2015Co-Authors: Yutaro Baba, Junichi Sumitani, Shuji Tani, Takashi KawaguchiAbstract:Aspergillus aculeatus β-glucosidase 1 (AaBGL1), which promotes cellulose hydrolysis by Trichoderma cellulase system, was characterized and compared some properties to a commercially supplied orthologue in A. niger (AnBGL) to elucidate advantages of recombinant AaBGL1 (rAaBGL1) for synergistic effect on Trichoderma enzymes. Steady–state kinetic studies revealed that rAaBGL1 showed high catalytic efficiency towards β-linked glucooligosaccharides. Up to a degree of polymerization (DP) 3, rAaBGL1 prefered to hydrolyze β-1,3 linked glucooligosaccharides, but longer than DP 3, preferred β-1,4 glucooligosaccharides (up to DP 5). This result suggested that there were different formation for subsites in the catalytic cleft of AaBGL1 between β-1,3 and β-1,4 glucooligosaccharides, therefore rAaBGL1 preferred short chain of laminarioligosaccharides and long chain of cellooligosaccharides on hydrolysis. rAaBGL1 was more insensitive to glucose inhibition and more efficient to hydrolyze the one of major transglycosylation product, gentiobiose than AnBGL, resulting that rAaBGL1 completely hydrolyzed 5% cellobiose to glucose faster than AnBGL. These data indicate that AaBGL1 is valuable for the use of cellulosic biomass conversion.
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crystal structures of glycoside hydrolase family 3 β glucosidase 1 from Aspergillus aculeatus
Biochemical Journal, 2013Co-Authors: Kentaro Suzuki, Junichi Sumitani, Toru Nishimaki, Shuji Tani, Takashi Kawaguchi, Takayoshi Wakagi, Shinya FushinobuAbstract:GH3 (glycoside hydrolase family 3) BGLs (β-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 ( Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N -glycan chains) in the dimer. The largest N -glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.
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construction of a recombinant trichoderma reesei strain expressing Aspergillus aculeatus β glucosidase 1 for efficient biomass conversion
Biotechnology and Bioengineering, 2012Co-Authors: Hikaru Nakazawa, Junichi Sumitani, Shuji Tani, Takashi Kawaguchi, Tetsushi Kawai, Noriko Ida, Yosuke Shida, Yoshinori Kobayashi, Hirofumi Okada, Yasushi MorikawaAbstract:To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes. Biotechnol. Bioeng. 2012;109: 92–99. © 2011 Wiley Periodicals, Inc.
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construction of a recombinant trichoderma reesei strain expressing Aspergillus aculeatus β glucosidase 1 for efficient biomass conversion
Biotechnology and Bioengineering, 2012Co-Authors: Hikaru Nakazawa, Junichi Sumitani, Shuji Tani, Takashi Kawaguchi, Tetsushi Kawai, Noriko Ida, Yosuke Shida, Yoshinori Kobayashi, Hirofumi Okada, Yasushi MorikawaAbstract:To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.
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Agrobacterium tumefaciens -mediated transformation of Aspergillus aculeatus for insertional mutagenesis
AMB Express, 2011Co-Authors: Emi Kunitake, Junichi Sumitani, Shuji Tani, Takashi KawaguchiAbstract:Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus.
