Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses

Çalık, Pınar
Ozdamar, TH
Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. For this aim, subC gene was cloned into pHV1431 Escherichia coli-Bacillus shuttle vector, and transferred into nine host Bacillus species, i.e. B. alvei, B. amyloliquefaciens, B. badius, B. cereus, B. coagulans, B. firmus, B. licheniformis, B. sphaericus and B. subtilis. The influence of the host Bacillus species on SAP production on a defined medium with glucose was investigated in bioreactor systems. For each of the recombinant (r-) Bacillus species, effects of initial glucose concentration on cell growth and SAP production were investigated; and, physiological differences and similarities between the wild-type and r-Bacillus species are discussed. The highest biomass concentration was obtained with r-B. coagulans as 3.8 kg m(-3) at the initial glucose concentration of C-Go = 20 kg m(-3) and the highest volumetric SAP activity was obtained with r-B. amyloliquefaciens as 1650 U cm(-3) at C-Go = 20 kg m(-3). Overall SAP activity per amount of substrate consumed was the highest for r-B. sphaericus (137 U g(-1) cm(-3)) and r-B. licheniformis (130 U g(-1) cm(-3)). Among the r-Bacillus species the highest activity increase compared to the wild types was obtained with r-B. sphaericus while the lowest increase was obtained with r-B. amyloliquefaciens and r-B. licheniformis due to high SAP production potential of the wild-type strains. During storage of the host microorganisms, r-B. alvei and r-B. amyloliquefaciens were not able to bear the recombinant plasm id, probably, due to the restriction enzymes synthesized. Due to the highest stable volumetric activities r-B. licheniformis (950 U cm(-3)) and r-B. sphaericus (820 U cm(-3)) appear to be the favorable hosts for the production of SAP. All the r-Bacillus species excreted organic acids oxaloacetic and succinic acids, but, none excreted the amino acid valine. The variations in by-product distributions with each recombinant organism were also discussed.


Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network
Çalık, Pınar; Oliver, SG; Ozdamar, TH (Elsevier BV, 2003-05-20)
This work reports on cloning of serine alkaline protease (SAP) encoding gene subC to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the subC gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined bioreactor-operation conditions. Significant physiological changes wer...
Determination of metabolic bottlenecks using reaction engineering principles in serine alkaline protease production by recombinant bacillus sp.
Telli, İlkin Ece; Çalık, Pınar; Department of Chemical Engineering (2004)
In this study, firstly, bioprocess characteristics for Serine Alkaline Protease (SAP) production, using recombinant Bacillus subtilis carrying pHV1431::subC, were examined. The cell concentration, substrate concentration, SAP activity and SAP synthesis rate profiles demonstrated that the system reaches to a steady state in terms of cell growth and SAP synthesis between t=15-25 h, therefore, this time interval is appropriate to employ both metabolic flux analysis and metabolic control analysis, which apply s...
Influence of controlled-pH and uncontrolled-pH operations on recombinant benzaldehyde lyase production by Escherichia coli
Çalık, Pınar; Demir, AS (Elsevier BV, 2006-03-02)
To select the host microorganism having the highest benzaldehyde lyase (BAL) production capacity, pUC 18::bal gene was transferred into four Escherichia coli strains. As the highest enzyme activity was obtained with E. coli K12 (ATCC 10798) carrying pUC18::bal gene, BAL production medium was designed for K 12. Using the designed medium containing 8.0 kg m(-3) glucose, 5.0 kg m(-3) (NH4)(2)HPO4 and the salt solution, the effects of uncontrolled-pH and controlled-pH operations were investigated at uncontrolle...
Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis
Okay, S.; Tefon, B. E.; ÖZKAN, MELEK; Özcengiz, Gülay (Wiley, 2008-01-01)
Aims: The present study focused on cloning and expression of chiA gene from a highly chitinolytic local isolate of Serratia marcescens in an anti-Coleopteran Bacillus thuringiensis and comparison of the characteristics of the native and recombinant ChiAs.
Vector construction for transformation of tobacco with a NAC-type transcription factor, TaNAC69-1
Eroğlu, Ayten; BALOĞLU, MEHMET CENGİZ; Yucel, Meral (2016-08-10)
NAC type transcription factors are a large family of plant transcription factors related to defense, development, biotic and abiotic responses of plants. To construct vectors for tobacco transformation, traditional and Gateway cloning technology were used. For traditional cloning, firstly TaNAC69-1 gene was isolated from Triticum aestivum L. cv. Yuregir-89. After cDNA synthesis, TaNAC69-1 gene was cloned into pJET1.2 cloning vector. To transfer the gene into dicot expression vector, pORE-E3, both pJET1.2 a...
Citation Formats
P. Çalık and T. Ozdamar, “Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses,” ENZYME AND MICROBIAL TECHNOLOGY, pp. 967–974, 2003, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/44978.