WEKO3
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Studies on characterization of a novel hemicellulase Abf62A-Axe6A from Ruminiclostridium josui and development of its host-vector system
http://hdl.handle.net/10076/00017925
http://hdl.handle.net/10076/00017925899c3113-537e-4be6-b451-d82de6b00703
名前 / ファイル | ライセンス | アクション |
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2018DB0906 (3.1 MB)
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||||||||
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公開日 | 2018-11-15 | |||||||||||
タイトル | ||||||||||||
言語 | en | |||||||||||
タイトル | Studies on characterization of a novel hemicellulase Abf62A-Axe6A from Ruminiclostridium josui and development of its host-vector system | |||||||||||
言語 | ||||||||||||
言語 | eng | |||||||||||
資源タイプ | ||||||||||||
資源タイプ識別子 | http://purl.org/coar/resource_type/c_db06 | |||||||||||
資源タイプ | doctoral thesis | |||||||||||
アクセス権 | ||||||||||||
アクセス権 | open access | |||||||||||
アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||||||||
著者 |
汪, 亜運
× 汪, 亜運
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抄録 | ||||||||||||
内容記述タイプ | Abstract | |||||||||||
内容記述 | From the viewpoint of reduction of the global warming, the utilization and application of the renewable energies are required. Among various renewable energies, plant biomass is one of the most important energy resources. In the current process of bioconversion of plant biomass to value-added products, plant biomass is hydrolyzed by cellulolytic enzymes from fungi such as Trichoderma reesei and the resultant fermentable monosaccharides are fermented to biofuels etc. by yeasts or bacteria. On the other hand, “consolidated bioprocessing (CBP)” is widely recognized as the efficient strategy for simultaneous hydrolysis and fermentation of biomass using cellulolytic anaerobic bacteria. A novel hemicellulase Abf62A-Axe6A from Ruminiclostridium josui was biochemically characterized and its host-vector system was established for better use of this bacterium for CBP. R. josui Abf62A-Axe6A is a modular enzyme consisting of an N-terminal signal peptide, a catalytic module of glycoside hydrolase family 62 (GH62), a family 6 carbohydrate binding module (CBM6), a dockerin module and another catalytic module of carbohydrate esterase family 6 (CE6) in order from the N-terminus. Therefore, three recombinant enzymes, RjAbf62A-Axe6A consisting of 4 modules devoid of the signal peptide, RjAbf62A-CBM6 consisting of GH62 and CBM6 and RjAxe6 consisting of CE6 only, were produced by Escherichia coli recombinants and biochemically characterized. RjAbf62A-Axe6 was highly active toward arabinoxylan and moderately active toward sugar beet arabinan, releasing mainly arabinose. Analysis of the hydrolysis products of arabinoxylooligosaccharides indicated that RjAbf62A-Axe6 released not only α-1,2- and α-1,3-linked arabinofuranoses from singly substituted xylosyl residues but also from doubly substituted xylosyl residues. RjAbf62A-Axe6A efficiently hydrolyzed 32-α-L-arabinofuranosyl xylobiose (A3X) to release arabinose, and the activity toward A3X was stronger than toward 33-α-L-arabinofuranosyl xylotetraose (XA3XX), 23-α-L-arabinofuranosyl xylotriose (A2XX) and (23,33-di-α-L-arabinofuranosyl xylotriose) A2,3XX. RjAbf62A-Axe6A seemed to prefer Araf of substituted xylosyl residues located at non-reducing end and the 1,3-linked Araf residues in doubly substituted xylosyl residues. The enzyme showed a weak activity toward linear 1,5-α-L-arabinan and arabinooligosaccharides, indicating that it had an exo-α-1,5-L-arabinofuranosidase activity. Surprisingly, RjAbf62A-Axe6 demonstrated an endoxylanase activity toward birchwood and beechwood xylans and xylooligosaccharides. RjAbf62A-Axe6 is the first GH62 enzyme having arabinofuranosidase and endoxylanase activities. Both RjAbf62A-Axe6 and RjAxe6 had an acetylxylan esterase activity toward insoluble wheat arabinoxylan. R. josui host-vector system has not yet been established because of the existence of a restriction and modification system in R. josui. A restriction endonuclease RjoI was purified from R. josui cell extract using column chromatography and characterized. The results showed that RjoI is an isoschizomer of DpnI, recognizing the sequence 5’-GmetATC-3’, where the A nucleotide is Dam-methylated. RjoI cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. Plasmids prepared from E. coli C2925 (dam−/dcm−) could be introduced into R. josui by electroporation. The highest transformation efficiency of 6.6 × 103 transformants/μg of DNA was obtained using a square wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui, a truncated form of Cel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of the fundamental technology for molecular breeding of R. josui. | |||||||||||
言語 | en | |||||||||||
内容記述 | ||||||||||||
内容記述タイプ | Other | |||||||||||
内容記述 | 本文 / Graduate School of Bioresources Mie University | |||||||||||
内容記述 | ||||||||||||
内容記述タイプ | Other | |||||||||||
内容記述 | 114p | |||||||||||
書誌情報 |
発行日 2018-09-19 |
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フォーマット | ||||||||||||
内容記述タイプ | Other | |||||||||||
内容記述 | application/pdf | |||||||||||
著者版フラグ | ||||||||||||
出版タイプ | VoR | |||||||||||
出版タイプResource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||||||
その他の言語のタイトル | ||||||||||||
その他のタイトル | Ruminiclostridium josuiの新規ヘミセルラーゼAbf62A-Axe6Aの酵素特性と宿主・ベクター系の開発に関する研究 | |||||||||||
言語 | ja | |||||||||||
出版者 | ||||||||||||
出版者 | 三重大学 | |||||||||||
出版者(ヨミ) | ||||||||||||
ミエダイガク | ||||||||||||
学位名 | ||||||||||||
学位名 | 博士(学術) | |||||||||||
学位授与機関 | ||||||||||||
学位授与機関識別子Scheme | kakenhi | |||||||||||
学位授与機関識別子 | 14101 | |||||||||||
学位授与機関名 | 三重大学 | |||||||||||
学位授与年月日 | ||||||||||||
学位授与年月日 | 2018-09-19 | |||||||||||
学位授与番号 | ||||||||||||
学位授与番号 | 甲学術第1922号 | |||||||||||
資源タイプ(三重大) | ||||||||||||
Doctoral Dissertation / 博士論文 |