β-(1,3-1,4)-Glukanaz Geninin Streptococcus salivarius subsp. thermophilus’da Ekspresyonu

Bu çal›flmada, kanatl›lara yönelik rekombinant probiyotik gelifltirmek amac›yla β-(1,3-1,4)-glukanaz (likenaz) geninin Streptococcus salivarius subsp. thermophilus’a aktar›lmas›, ekspresyonu ve likenaz enziminin s›cakl›¤a direncinin art›r›lmas› amaçlanm›flt›r. β-(1,3-1,4)-glukanaz genini tafl›yan rekombinant TL1R plazmidi S. salivarius subsp. thermophilus’a elektrotransformasyon yöntemi ile aktar›lm›flt›r. β-(1,3-1,4)-glukanaz geninin S. salivarius subsp. thermophilus’da ekspresyonu likenanl› besiyeri, SDS-PAGE ve zymogram analizleri ile tespit edilmifltir. S. salivarius subsp. thermophilus’un sentezledi¤i β-(1,3- 1,4)-glukanaz enzimi, s›cakl›¤a direnci artarak 70 ºC’de 15 dakika aktivitesini korumufltur. Buna karfl›l›k, Lactococcus lactis ve Escherichia coli taraf›ndan sentezlenen β-(1,3-1,4)-glukanaz enzimi ayn› s›cakl›k uygulamas›nda aktivitesini kaybetmifltir. Tüm rekombinant bakterilerce sentezlenen β-(1,3-1,4)-glukanaz enzimi 37-100 ºC’lerde 15 dakika s›cakl›k uygulamas›ndan sonra denatürasyona direnerek bir flekilde suda çözünürlü¤ünü kaybetmemifltir.

Anahtar Kelimeler:

Streptococcus salivarius subsp. thermophilus, β-(1, 3-1, 4)-glukanaz, ekspresyon, s›cakl›¤a dirençlilik

Expression of the β-(1,3-1,4)-Glucanase Gene in Streptococcus salivarius subsp. thermophilus

The purpose of this study was the transformation and expression of the b -(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 b -(1,3-1,4)-glucanase gene was introduced into S. salivarius subsp. thermophilus by electrotransformation. The expressing of the b -(1,3-1,4)-glucanase gene in S. salivarius subsp. thermophilus was confirmed on lichenan plate, SDS-PAGE, and zymogram analysis. The b -(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.

Keywords:

Streptococcus salivarius subsp. thermophilus, b -(1, 3-1, 4)-glucanase, expression, thermostability,

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