French, Carol; (1994) Production and recovery of a recombinant alpha amylase enzyme from Escherichia coli and Streptomyces lividans. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

This thesis describes the scale-up from laboratory to pilot plant, of the production and recovery of a Streptomyces thermoviolaceus alpha amylase cloned into the host organisms, Escherichia coli and S.lividans. When the enzyme was expressed in E.coli it was located intracel1ular1y, in the periplasmic space, and on expression in S.lividans the enzyme was fully secreted to the extracellular medium. A method was developed to recover the recombinant protein from the periplasm of E.coli cells. This was used to monitor the production and cellular location of alpha amylase in E.coli JM107, throughout growth in shake flask and 5L batch bioreactor cultures. The periplasmic release treatment was then scaled up, so that it was suitable for use on a pilot plant scale. Higher yields of alpha amylase were produced in E.coli shake flask cultures compared to bioreactor cultures. This was believed to be because of a higher plasmid copy number per cell, due to a lower growth rate in shake flask cultures compared to bioreactor cultures. The production and cellular location of the alpha amylase was also found to differ, depending upon the host strain of E.coli. The addition of antibiotics, ampicillin or kanamycin, were necessary to maintain stability of the plasmid throughout growth in E.coli. The production level of the alpha amylase was also examined in S.lividans TK24, in both shake flask and bioreactor cultures. Different media were studied for sporulation of the host and production of the alpha amylase in various expression vectors.

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