著者

Keisuke Kojima Naoki Sunagawa Yoshihisa Yoshimi Theodora Tryfona Masahiro Samejima Paul Dupree Kiyohiko Igarashi

出版者

The Japanese Society of Applied Glycoscience

雑誌

Journal of Applied Glycoscience (ISSN:13447882)

巻号頁・発行日

pp.jag.JAG-2021_0017, (Released:2022-03-05)

被引用文献数

7

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Endo-type xylanases are key enzymes in microbial xylanolytic systems, and xylanases belonging to glycoside hydrolase (GH) families 10 or 11 are the major enzymes degrading xylan in nature. These enzymes have typically been characterized using xylan prepared by alkaline extraction, which removes acetyl sidechains from the substrate, and thus the effect of acetyl groups on xylan degradation remains unclear. Here, we compare the ability of GH10 and 11 xylanases, PcXyn10A and PcXyn11B, from the white-rot basidiomycete Phanerochaete chrysosporium to degrade acetylated and deacetylated xylan from various plants. Product quantification revealed that PcXyn10A effectively degraded both acetylated xylan extracted from Arabidopsis thaliana and the deacetylated xylan obtained by alkaline treatment, generating xylooligosaccharides. In contrast, PcXyn11B showed limited activity towards acetyl xylan, but showed significantly increased activity after deacetylation of the xylan. Polysaccharide analysis using carbohydrate gel electrophoresis showed that PcXyn11B generated a broad range of products from native acetylated xylans extracted from birch wood and rice straw, including large residual xylooligosaccharides, while non-acetylated xylan from Japanese cedar was readily degraded into xylooligosaccharides. These results suggest that the degradability of native xylan by GH11 xylanases is highly dependent on the extent of acetyl group substitution. Analysis of 31 fungal genomes in the Carbohydrate- Active enZymes database indicated that the presence of GH11 xylanases is correlated to that of carbohydrate esterase family 1 acetyl xylan esterases (AXEs), while this is not the case for GH10 xylanases. These findings may imply co-evolution of GH11 xylanases and CE1 AXEs.

著者

Kaori Matsuyama Naoki Sunagawa Kiyohiko Igarashi

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.37, no.4, pp.397-403, 2020-12-25 (Released:2020-12-25)

参考文献数

43

被引用文献数

1 4

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The study of Carbohydrate-Active enZymes (CAZymes) associated with plant cell wall metabolism is important for elucidating the developmental mechanisms of plants and also for the utilization of plants as a biomass resource. The use of recombinant proteins is common in this context, but heterologous expression of plant proteins is particularly difficult, in part because the presence of many cysteine residues promotes denaturation, aggregation and/or protein misfolding. In this study, we evaluated two phenotypes of methylotrophic yeast Pichia pastoris as expression hosts for expansin from peach (Prunus persica (L.) Batsch, PpEXP1), which is one of the most challenging targets for heterologous expression. cDNAs encoding wild-type expansin (PpEXP1_WT) and a mutant in which all cysteine residues were replaced with serine (PpEXP1_CS) were each inserted into expression vectors, and the protein expression levels were compared. The total amount of secreted protein in PpEXP1_WT culture was approximately twice that of PpEXP1_CS. However, the amounts of recombinant expansin were 0.58 and 4.3 mg l−1, corresponding to 0.18% and 2.37% of total expressed protein, respectively. This 13-fold increase in production of the mutant in P. pastoris indicates that the replacement of cysteine residues stabilizes recombinant PpEXP1.

著者

Sora Yamaguchi Naoki Sunagawa Mikako Tachioka Kiyohiko Igarashi Masahirog Samejima

出版者

The Japanese Society of Applied Glycoscience

雑誌

Journal of Applied Glycoscience (ISSN:13447882)

巻号頁・発行日

vol.67, no.3, pp.79-86, 2020-09-03 (Released:2020-09-03)

参考文献数

22

被引用文献数

4

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Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH family 7 (Cel7) and other carbohydrate-active enzymes during the degradation of cellulosic biomass. However, while the catalytic rate of enzymes generally becomes faster at higher temperatures, Cel6 CBHs are inactivated at lower temperatures than Cel7 CBHs, and this represents a limiting factor for industrial utilization. In this study, we produced a series of mutants of the glycoside hydrolase family 6 cellobiohydrolase PcCel6A from the fungus Phanerochaete chrysosporium, and compared their thermal stability. Eight mutants from a random mutagenesis library and one rationally designed mutant were selected as candidate thermostable mutants and produced by heterologous expression in the yeast Pichia pastoris. Comparison of the hydrolytic activities at 50 and 60 °C indicated that the thermal stability of PcCel6A is influenced by the number and position of cysteine residues that are not involved in disulfide bonds.

著者

Kiyohiko Igarashi Satoshi Kaneko Motomitsu Kitaoka Masahiro Samejima

出版者

The Japanese Society of Applied Glycoscience

雑誌

Journal of Applied Glycoscience (ISSN:13447882)

巻号頁・発行日

vol.67, no.2, pp.51-57, 2020-05-20 (Released:2020-05-20)

参考文献数

40

被引用文献数

2

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Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar Km and kcat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher Km value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.

著者

Sosyu Tsutsui Kiyoshi Sakuragi Kiyohiko Igarashi Masahiro Samejima Satoshi Kaneko

出版者

The Japanese Society of Applied Glycoscience

雑誌

Journal of Applied Glycoscience (ISSN:13447882)

巻号頁・発行日

vol.67, no.1, pp.17-22, 2020-02-20 (Released:2020-02-20)

参考文献数

29

被引用文献数

4

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Sugarcane bagasse is a useful biomass resource. In the present study, we examined the efficacy of ammonia pretreatment for selective release of hemicellulose from bagasse. Pretreatment of bagasse with aqueous ammonia resulted in significant loss of xylan. In contrast, pretreatment of bagasse with anhydrous ammonia resulted in almost no xylan loss. Aqueous ammonia or anhydrous ammonia-pretreated bagasse was then subjected to enzymatic digestion with a xylanase from the glycoside hydrolase (GH) family 10 or a xylanase from the GH family 11. The hydrolysis rate of xylan in bagasse pretreated with aqueous ammonia was approximately 50 %. In contrast, in the anhydrous ammonia-treated bagasse, xylan hydrolysis was > 80 %. These results suggested that anhydrous ammonia pretreatment would be an effective method for preparation of sugarcane bagasse for enzymatic hydrolysis to recover xylooligosaccharides.

著者

Maki Ishiguro Tomonobu Hori Takuya Ishida Makoto Yoshida Koji Takabatake Satoshi Kaneko Kiyohiko Igarashi Masahiro Samejima

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.27, no.3, pp.273-281, 2010-06-25 (Released:2010-07-15)

参考文献数

26

被引用文献数

5 4

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The basidiomycete Flammulina velutipes is one of the most popular edible mushrooms in Japan, and has the ability to grow on cellulosic biomass as a carbon source. In this study, we have isolated two enzymes belonging to glycoside hydrolase (GH) family 7 (FvCel7A and FvCel7B) from the cellulose-grown culture of the fungus, and cloned cDNAs encoding these enzymes by utilizing a transcriptomic database of this fungus. Although both enzymes contain a catalytic domain belonging to GH family 7, only FvCel7A has the family 1 carbohydrate-binding module at the C-terminal. Sequence comparison indicated that FvCel7A and FvCel7B have a similar pattern of disulfide bonds and similar active site architecture to other fungal GH family 7 enzymes, but show small differences at loop regions covering the active site, which may affect the reactivity of cellulosic substrates.