Title

Siderophore Production in Methanotrophy of Methylosinus Trichosporium Ob3b During Cultivation Optimization

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Date of Award

Summer 2016

Abstract

Methanobactin (mb) is a small chromopeptide with strong copper binding affinity. In addition to copper binding and trafficking, mb has catalytic activity including superoxide dismutase and hydrogen peroxide reductase. Because of the copper binding and antioxidant activities, mb has attracted therapeutic and commercial interest. The mb isolation from Methylosinus trichosporium (mb-OB3b; C45H58N10O16S5; Mw 1154.26) has been extensively studied as the archetype molecule. However, our results indicated that mb-OB3b used for previous studies was heavily contaminated with a minimum 40% of methanotrophic siderophores. Siderophores are iron binding compounds secreted by many microorganisms as a part of their iron trafficking system. It chelates water insoluble ferric ions (Fe3+) and makes them bioavailable. M. trichosporium OB3b growing at low copper concentration requires a large quantity of ferric ion because the key enzyme in methane fixation (soluble methane monooxygenase) is an enzyme with a di-iron center. However, the same low copper concentration condition has been a golden standard of methanobactin overproduction favorable for methanobactin preparation. As a result, excessive amounts of siderophore acts as a contaminating agent. Now, the validity of earlier mb-OB3b studies is questionable. The present study is focused on the purification of mb-OB3b using step wise elution to reduce siderophore contamination. M. trichosporium OB3b was cultivated in Bioflo110 bioreactor, where agitation speed, gas flow, temperature, and copper availability were controlled in semi-continuous batch mode. Dissolved oxygen, pH, and cell density were monitored as potential indicators of in situ mb-OB3b productivity levels. Secreted mb-OB3b was isolated by tangential flow filtration followed by step-wise elution of reverse phase HP20 Diaion column over different concentrations of acetonitrile. Eluted fractions of different acetonitrile concentrations were analyzed by Waters Synaptic IM-MS.

Advisor

Dong Won Choi

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Biology | Life Sciences

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