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Expression, purification and characterization of Arabidopsis thaliana endo-beta-mannanase 7 (AtMAN7)
KTH, School of Biotechnology (BIO).
2011 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Uttryck, rening och karaktärisering av endo-beta-mannasas 7 (AtMAN7) (Swedish)
Abstract [en]

Plant endo-β-mannanases (EC catalyze the hydrolysis of mannan polysaccharides randomly through a retaining mechanism. In Arabidopsis thaliana, there are 8 mannanases identified and AtMAN7 is one of those. In the present investigation, the AtMAN7 gene was cloned and the recombinant protein was expressed in E. coli. Most of the protein yielded in inclusion bodies, but pure protein could be obtained from the protein supernatant using IMAC. The dinitrosalicylic acid (DNS) method was used to measure enzyme activity. The result showed that the optimal temperature of AtMAN7 was 40-45 °C and that the enzyme had a pH optimum at pH 5. T-DNA insertional mutant Arabidopsis seeds were used to investigate the mannanase function in situ. The germination rate of gene AtMAN6 and AtMAN7 knockouts was much higher than the wild type, while AtMAN1 knockouts germinated at a slower rate compared to wild type. Thus, this indicated that AtMAN6 and AtMAN7 had a negative effect and AtMAN1 had a positive effect on seeds germination. No significant phenotype differences on Arabidopsis seedlings were observed between the T-DNA insertional mutants and wild type.

Place, publisher, year, edition, pages
Keyword [en]
Mannan, mannanase, GH5, cell sall, Arabidopsis, recombinant expression
National Category
Engineering and Technology
URN: urn:nbn:se:kth:diva-51771OAI: diva2:464997
Subject / course
Educational program
Degree of Master
Available from: 2011-12-14 Created: 2011-12-14 Last updated: 2011-12-15Bibliographically approved

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