Expression of the β-(1,3-1,4)-glucanase gene in streptococcus salivarius subsp. thermophilus

Bu çalışmada, kanatlılara yönelik rekombinant probiyotik geliştirmek amacıyla β-(1,3-1,4)-glukanaz (likenaz) geninin Streptococcus salivarius subsp. thermophilus’a aktarılması, ekspresyonu ve likenaz enziminin sıcaklığa direncinin artırılması amaçlanmıştır. β-(1,3-1,4)-glukanaz genini taşıyan rekombinant TL1R plazmidi S. salivarius subsp. thermophilus’a elektrotransformasyon yöntemi ile aktarılmıştır. β-(1,3-1,4)-glukanaz geninin S. salivarius subsp. thermophilus’da ekspresyonu likenanlı besiyeri, SDS-PAGE ve zymogram analizleri ile tespit edilmiştir. S. salivarius subsp. thermophilus’un sentezlediği β-(1,3- 1,4)-glukanaz enzimi, sıcaklığa direnci artarak 70 ºC’de 15 dakika aktivitesini korumuştur. Buna karşılık, Lactococcus lactis ve Escherichia coli tarafından sentezlenen β-(1,3-1,4)-glukanaz enzimi aynı sıcaklık uygulamasında aktivitesini kaybetmiştir. Tüm rekombinant bakterilerce sentezlenen β-(1,3-1,4)-glukanaz enzimi 37-100 ºC’lerde 15 dakika sıcaklık uygulamasından sonra denatürasyona direnerek bir şekilde suda çözünürlüğünü kaybetmemiştir.

β-(1,3-1,4)-Glukanaz geninin streptococcus salivarius subsp. thermophilus’da ekspresyonu

The purpose of this study was the transformation and expression of the β-(1,3-1,4)-glucanase (lichenase) gene in Streptococcus salivarius subsp. thermophilus to create a recombinant probiotic for poultry and improve the thermostability of the lichenase enzyme. The recombinant plasmid TL1R containing the β-(1,3-1,4)-glucanase gene was introduced into S. salivarius subsp. thermophilus by electrotransformation. The expressing of the β-(1,3-1,4)-glucanase gene in S. salivarius subsp. thermophilus was confirmed on lichenan plate, SDS-PAGE, and zymogram analysis. The β-(1,3-1,4)-glucanase enzyme expressed by S. salivarius subsp. thermophilus cells seemed to increase its capacity for thermoresistance and so it maintained its activity at 70 ºC for 15 min. In contrast, the enzyme produced by Lactococcus lactis and Escherichia coli cells easily ceased activity when exposed to the same temperature. The enzyme expressed by all the recombinant bacteria resisted denaturation and somehow remained soluble after heat treatment from 37 to 100 ºC for 15 min.

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