Cloning and expression of thermostable beta-amylase gene of thermoanaerobacterium thermosulfurogenes in escherichia coli and bacillus subtilis BR151
Citation
Ozcan, B. D., Ozcan, N., (2010). Cloning and expression of thermostable beta-amylase gene of thermoanaerobacterium thermosulfurogenes in escherichia coli and bacillus subtilis BR151. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 16(6), 1011-1016. DOI: 10/9775/kvfd.2010.2290Abstract
DNA fragment encoding thermostable beta-amylase gene from Thermoanaerobacterium thermosulfurogenes was amplified by PCR and then cloned into pBluescript II KS/SK, pBT10, pNW33N and pUB110 plasmids. Recombinant plasmids were designated as pBluescript beta, pBT10 beta, pNW33N beta and pUB110 beta respectively. pBluescript beta, pBT10 beta and pNW33N beta recombinant plasmids were transferred into Escherichia coli: and pUB110 beta was electrotransformed into Bacillus subtilis BR151. Insert and PCR analysis of recombinant plasmids from E. coli and B. subtilis confirmed the 1935 bp beta-amylase gene fragment on agarose gel electrophoresis. On LB-starch-agar plates, all recombinant E. coli colonies showed positive zones with 12 staining. Although thermostable beta-amylase gene was cloned in B. subtilis BR151. the enzyme activity was not detected on LB-starch-agar plate. But after renaturation of extracellular proteins from B. subtilis on SDS-Starch-PAGE, beta-amylase enzyme regained enzymatic activity by zymogram technique and thereby confirmed that enzyme was not folding properly in B. subtilis host. Thermoanaerobacterium thermosulfurogenes DNA’sından sıcaklığa dirençli β-amilaz genini kodlayan DNA parçası PCR ile
amplifiye edilerek pBluescript II KS/SK, pBT10, pNW33N ve pUB110 plazmidlerine klonlanmıştır. Rekombinant plazmidler sırasıyla
pBluescriptβ, pBT10β, pNW33Nβ ve pUB110β olarak isimlendirilmişlerdir. pBluescriptβ, pBT10β ve pNW33Nβ rekombinant
plazmidleri Escherichia coli, pUB110β plazmidi ise Bacillus subtilis BR151 bakterisine transfer edilmişlerdir. E. coli ve B. subtilis
bakterilerinden izole edilen rekombinant plazmidlerin insört ve PCR analizleri, agaroz jel elektroforezde 1935 bç’lik β-amilaz genini
doğrulamıştır. LB-nişasta-agar plaklarında, tüm rekombinant E. coli kolonileri I2 boyaması ile pozitif zon vermişlerdir. Sıcaklığa dirençli
β-amilaz geni B. subtilis BR151 bakterisinde klonlanmasına karşın, LB-nişasta-agar plağında enzimatik aktivite gözlenememiştir.
B. subtilis kökenli hücre dışı proteinlerin renatürasyonundan sonra, SDS-Nişasta-PAGE’de β-amilaz enzimatik aktivitesini tekrar
kazanmış ve bu sonuç B. subtilis konukçusunda enzimin uygun katlanmayı yapamadığı görüşünü desteklemiştir.