Studies on cloning of thermostable amylase genes

ABSTRACTPhD THESISSTUDIES ON CLONING OF THERMOSTABLEAMYLASE GENESBahri Devrim ÖZCANDEPARTMENT OF ANIMAL SCIENCEINSTITUTE OF NATURAL AND APPLIED SCIENCESUNIVERSITY OF ÇUKUROVASupervisor : Prof.Dr. Numan ÖZCANYear: 2005, Pages: 122Jury : Prof.Dr. Numan ÖZCANProf.Dr. Hasan Rüştü KUTLUProf.Dr. Halil KASAPProf.Dr. Mehmet TOPAKTAŞDoç.Dr. Mehmet Sait EKİNCİIn this study, the recombinant pC194α vector carrying thermostable α-amylase gene from Bacillusstearothermophilus was transformed into Bacillus subtilis YB886 and Bacillus subtilis BR151 by electroporationtechnique. It was showed on LB/Agar plates and zymogram analyses on SDS-Starch-PAGE that recombinantstrains synthesized thermostable α-amylase extracellularly. Hind III digestion of recombinant plasmids yielded5.25 kbp fragment of DNA carrying the gene encoding thermostable α-amylase and 2.9 kbp fragment of pC194vector on agarose gel electrophoresis. Furthermore, extracellular proteins of recombinant strains were treated atemperature of 80 ºC for 15 minutes and thus mesophilic proteins origined from Bacillus subtilis weredenaturated and thermostable α-amylase enzyme showed on both SDS-PAGE and SDS-Starch-PAGE.On the other hand thermostable β-amylase gene from Thermoanaerobacterium thermosulfurogenes wascloned in Escherichia coli using pBluescript II KS/SK, pL2 and pNW33N, in Bacillus subtilis BR151 andBacillus amyloliquefaciens using pUB110 by PCR technique. Digestion of plasmids isolated from recombinantstrains by Bam HI was showed the 1.935 kbp β-amylase gene band following agarose gel electrophoresis. OnLB/Starch/Agar plates, all recombinant Escherichia coli colonies showed positive zones with I2 staining.Thermostable β-amylase gene was isolated from recombinant plasmids by PCR and visualized by agarose gelelectrophoresis. In addition, 1.5 ve 0.5 kbp fragments produced by digestion of PCR products with Hind IIIvisualized by agarose gel electrophoresis supported the results. Although the thermostable β-amylase cloned in B.subtilis BR151 and B. amyloliquefaciens, these microorganisms didn?t show enzymatic activity onLB/Starch/Agar plates. But after denaturing and electrophoresis of extracellular proteins on SDS-Starch-PAGEand hydrolysis of substrat by renaturated protein, enzymatic activity have been seen by zymogram analysis.Key Words: Bacillus stearothermophilus, Thermoanaerobacterium thermosulfurogenes, α-amylase, β-amylase,CloningII