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Purification and Characterization of an alpha-Mannosidase from the Tropical Fruit Babaco (Vasconcellea x Heilbornii Cv. Babaco)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
2008 (English)In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 56, no 22, 10872-10878 p.Article in journal (Refereed) Published
Abstract [en]

An alpha-mannosidase (EC 3.2.1.24) present in the lyophilized latex of babaco (Vasconcellea heilbornii) has been purified to apparent homogeneity by native PAGE. The purification involves a three-step procedure with successive anion exchange with 0 Sepharose HP, lectin affinity chromatography using ConA Sepharose 4B, and gel filtration using Superdex 200 prep grade. The molecular mass was determined to be in the range of 260-280 kDa by Superdex 200 prep grade gel filtration, and isoelectric focusing showed a pI range between 5.85 and 6.55, suggesting different glycosylated isoforms. The optimal temperature for the alpha-mannosidase was determined to lie between 50 and 60 degrees C, and the optimal pH was 4.5 at 50 degrees C. The K-m value for p-nitrophenyl alpha-mannopyranoside (pNPM) was found to be 1.25 mM and the V-max, 2.4 mu kat mg(-1) at 50 degrees C and 1.94 mu kat mg(-1) at 40 degrees C. The pure alpha-mannosidase was specific for mannose and did not display activity for any other tested synthetic substrates.

Place, publisher, year, edition, pages
2008. Vol. 56, no 22, 10872-10878 p.
Keyword [en]
alpha-Mannosidase, babaco, latex, purification, characterization, subunit composition
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-107031DOI: 10.1021/jf800857kISI: 000261056700065OAI: oai:DiVA.org:uu-107031DiVA: diva2:227583
Available from: 2009-07-15 Created: 2009-07-15 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Purification Processes for Complex Biomacromolecules
Open this publication in new window or tab >>Purification Processes for Complex Biomacromolecules
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis details various techniques and considerations for the purification of complex biomacromolecules.

 

Initially an α-mannosidase from babaco fruit was purified using anion exchange-, lectin affinity- and size exclusion chromatography.  The enzyme was approximately 260-280 kDa in size with an apparent an unusual octagonal stoichiometry and displayed properties similar to other known plant α-mannosidases.

 

Mucins were fractionated by ion exchange and size exclusion chromatography to assess the properties that govern the mucin surface coating interactions in biomaterial research.  Commercially available mucins, of bovine and porcine origin, as wells as crude human mucin were tested. All showed to consist of a population of molecules which differ in size, charge and composition.

 

The third part of the thesis concerns different aspects of plasmid DNA purification processes.

A two-step method for analysis of plasmid DNA consisting of size exclusion followed by thiophilic adsorption chromatography was evaluated. It allowed determination of the supercoiled plasmid DNA concentration in all process steps without requirement for extensive sample preparation. This method was shown to be fully comparable in terms of accuracy to capillary gel electrophoresis, considered as the industry standard.

Purification of plasmid DNA generally involves bacterial cell alkaline lysis, which creates a solution with flocculate material which needs to be removed prior to further processing. The addition of ammonium hydrogen carbonate to the suspension was evaluated to clarify the solution. The released carbon dioxide and ammonium lifts the flocculate to the surface and allows draining of a clear solution. The method is fully scalable, does not affect the plasmid DNA quality and requires no special equipment.

Thiophilic adsorption chromatography was evaluated for simplification of an existing commercial large scale purification process and was shown to increase both product purity and yields of several tested plasmids. Also, implementation of this step significantly reduced overall production process time.

 

 

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 928
Keyword
Chromatography, enzymes, mucins, fractionation, plasmid DNA, analysis, clarification, process design
National Category
Chemical Sciences
Research subject
Chemistry with specialization in Surface Biotechnology
Identifiers
urn:nbn:se:uu:diva-172892 (URN)978-91-554-8353-1 (ISBN)
Public defence
2012-05-25, BMC B7:101, Husargatan 3, Uppsala, 13:15 (English)
Supervisors
Available from: 2012-05-04 Created: 2012-04-17 Last updated: 2012-08-01Bibliographically approved

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