Title page for ETD etd-06192006-125711

Type of Document Dissertation
Author Yan, Run-Tao
URN etd-06192006-125711
Title Enzymology of butanol formation in Clostridium Beijerinckii
Degree PhD
Department Anaerobic Microbiology
Advisory Committee
Advisor Name Title
Chen, J. S. Committee Chair
Dean, D. R. Committee Member
Gregory, E. M. Committee Member
Johnson, John L. Committee Member
Krieg, N. R. Committee Member
  • Clostridium Research.
  • Butanol Research.
Date of Defense 1991-05-05
Availability restricted
The present study encompasses an investigation of the expression of solvent forming enzymes and purification and characterization of butanol-forming enzymes. More sensitive and accurate procedures for the determination of acids and solvents in cultures have been developed, which led to the recognition of the onset of solvent production at the mid-exponential phase, about two h earlier than previously reported. Activities of solvent-forming enzymes started to increase about one h before the onset of measurable solvent production and the

activities of solvent-forming enzymes did not increase simultaneously. CoA-acylating aldehyde dehydrogenase (ALDH) was purified to near homogeneity. The ALDH showed a native M.. of 100,000, and a subunit Mr of 55,000. ALDH

could use either NAD(H) or NADP(H) as the coenzyme. ALDH was oxygenlabile.

The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA. Both NADH- and NADPH-dependent alcohol dehydrogenase activities were present in crude extracts. The ratio of NADPH-dependent activity to NADH-dependent activity (the PID ratio) varied in crude extracts. The PID ratio was affected by O~ ionic strength, pH, growth stage of cell, Fe in culture medium and temperature. Two ADHs have been identified in crude extracts. The NADPH-dependent ADH (P-ADH) could be separated from the NADH/NADPH-dependent ADH (D/P-ADH). The D/P-ADH has been extensively purified. The D/P-ADH showed a native Mr of 70,000 and subunits with Mr of 45,300 and 40,000. The D/P-ADH activity could be inactivated by a,a' -dipyridyl and restored by Fe2+.

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