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Recombinant therapeutic protease production by Bacillus sp.

Korkmaz, Nuriye
The first aim of this study is the development of extracellular recombinant therapeutic protease streptokinase producing Bacillus sp., and the second aim is to determine fermentation characteristics for streptokinase production. In this context, the signal (pre-) DNA sequence of B.licheniformis (DSM1969) extracellular serine alkaline protease enzyme gene (subC: Acc. No. X03341) was ligated to 5’ end of the streptokinase gene (skc: Acc. No. S46536) by SOE (Gene Splicing by Overlap Extension) method through PCR. The resulting hybrid gene pre(subC)::skc was cloned into the pUC19 plasmid. Then, the hybrid gene was sub-cloned to pMK4 plasmid which is an E. coli-Bacillus shuttle vector with high copy number and high stability. Recombinant plasmid pMK4::pre(subC)::skc was finally transferred into B. subtilis (npr- apr-) and B. licheniformis 749/C (ATCC 25972) species. Streptokinase production capacities of these two recombinant Bacillus species were compared. The highest production was observed in recombinant B. lichenifomis 749/C (ATCC 25972) strain in a defined medium which was optimized in terms of carbon and nitrogen sources by a statistical approach, namely Response Surface Methodology (RSM). RSM evaluated the streptokinase concentration as the response and the medium components as the independent variables. The highest recombinant streptokinase concentration was found as 0.0237 kgm-3 at glucose and (NH4)2HPO4 concentrations of 4.530 and 4.838 kgm-3 respectively. The fermentation and oxygen transfer characteristics of the streptokinase production were investigated in a 3 dm3 pilot scale batch bioreactor (Braun CT2-2) equipped with temperature, pH, foam, air inlet and agitation rate controls having a working volume of VR=1.65 dm3 using the production medium optimized for the recombinant B. lichenifomis 749/C (ATCC 25972) strain. Streptokinase and β-lactamase activities, cell, glucose and organic acid concentrations, dissolved oxygen, pH, oxygen uptake rate, overall liquid phase mass transfer coefficient for oxygen, maintenance coefficient for oxygen, specific cell growth rate and yield coefficients were determined through the bioprocess. The bioprocess of recombinant streptokinase production was performed at uncontrolled pH of these bioreactor operation conditions: air inlet rate of Q0/VR=0.5 vvm, and the agitation rate of N=400min-1. The resulting streptokinase volumetric activity reached its maximum as 1.16 PUml-1 (0.0026 g/l streptokinase) at t=20 h.