BAHRİ DEVRİM ÖZCAN, Numan ÖZCAN, MAKBULE BAYLAN, Ali İrfan GÜZEL

Cloning and expression of cellulosimicrobium cellulans β-1,3-glucanase gene in lactobacillus plantarum to create new silage inoculant for aerobic stability

Bu çalışmada, rekombinant pTEG5 plazmit DNAsına (pUC18 + Cellulosimicrobium cellulansın β-1,3-glukanaz geni) pLP3537 plazmitinin p353-2 kriptik plazmit bölgesinin aktarılması ile rekombinant pTE353-βG plazmit DNAsı oluşturulmuştur. Rekombinant pTE353-βG plazmiti Lactobacillus plantaruma elektroporasyon tekniği ile transfer edilmiştir. SacI enzimi ile kesilmiş pTE353-βGnin agaroz jelde 1.9 kbç büyüklüğünde gen bandı üretmesinin yanı sıra, β-1,3-glukanaz genini şifreleyen 1.9 kbç büyüklüğündeki DNA parçasının PCR ile rekombinant vektörden amplifiye edilmesi genin plazmite entegrasyonunu göstermektedir. pTE353-βG içeren rekombinant L. plantarum kolonileri MRS-laminarin-agar plağında Congo-red boyaması ile β-1,3-glukanaz geninin eksprese olduğunu gösteren açık renkli pozitif zon vermişlerdir. Rekombinant suş tarafından üretilen enzim, C. cellulans enzimi ile aynı moleküler ağırlıkta aktivite bandı üretmiş ve böylece enzimin herhangi bir proteolitik parçalanmaya maruz kalmadığı anlaşılmıştır. Enzimatik analizler sonucunda L. plantarum tarafından üretilen β-1,3-glukanaz enziminin optimum sıcaklık ve pH değerleri sırasıyla 40°C ve 6.0 olarak bulunmuştur. Bu sonuçlar, rekombinant L. plantarumun silajın aerobik bozulmasını önlemek için inokülant olarak düşünülebileceğini ortaya çıkarmıştır.

Aerobik stabilite için yeni silaj inokülantı oluşturmak amacıyla cellulosimicrobium cellulans β-1,3-glukanaz geninin lactobacillus plantarumda klonlanması ve ekspresyonu

In this study, the recombinant plasmid pTE353-βG was created by inserting the p353-2 cryptic plasmid region of pLP3537 into pTEG5 recombinant plasmid contains pUC18 and β-1,3-glucanase gene of Cellulosimicrobium cellulans. The recombinant plasmid pTE353-βG was then introduced into Lactobacillus plantarum by electroporation. Insert analysis of pTE353-βG digested with SacI produced 1.9 kbp β-1,3-glucanase gene band on agarose gel as well as 1.9 kbp DNA encoding β-1,3-glucanase gene insert amplified on the recombinant vector via PCR indicated the integration of the gene into the plasmid. Recombinant L. plantarum colonies with pTE353-βG on MRS-laminarin-agar plate showed clear positive zones by Congo-red staining that revealed the expression of β-1,3-glucanase encoding gene. The β-1,3-glucanase enzyme of recombinant strain produced the same activity band with C. cellulans enzyme in terms of molecular weight, which showed the activity of secreted protein without any proteolytic degradation. Optimal temperature and pH values of L. plantarum β-1,3-glucanase have been determined 40ºC and 6.0 respectively, by enzymatic analysis. These results revealed that recombinant L. plantarum could be considered as a silage inoculant for aerobic spoilage of silage.

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