In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The different areas where enzymes are used and their economic value of biotechnological products further increases their importance. There are hundreds of different types of cheese but each is made by coagulating milk using rennet to give curds. Today, researchers have begun to develop alternative systems in the cheese industry related to milk-clotting enzymes. In this study, the nucleic acid sequence encoding the optimized chymosin enzyme was used and cloned by Not I and Mlu I restriction enzymes into pTOLT vector system. Then using this construct, the enzyme as a fusion with Tol-A-III protein was produced in Escherichia coli BL21 (DE3) cells. After disrupting the E. coli cell and separating from the constituents by high speed centrifugation, the enzyme was purified by affinity chromatography and fractions were analyzed by SDS-PAGE. Purified enzyme has shown its activity. Optimum temperature and pH of CHY-Tol-A-III protein were 40°C and 6.5, respectively.

In this study, Bacillus subtilis akky1 strain was isolated from the soil of beech forest in Akkuş City, Ordu Province, Turkey. akky1 strain was identified by 16S rRNA analysis. The full-length 16S rRNA sequence of akky1 strain showed the 100% similarity with Bacillus subtilis strain B7 (KC310823.1). A 642 bp DNA fragment was obtained from genomic DNA using primers designed based on the gene sequence of Bacillus subtilis xylanase given in GenBank. The gene encoding xylanase was cloned into pET28b (+) plasmid vector, sequenced and expressed in Escherichia coli BL21 (DE3). The hexahistidine (6xHis) tagged fusion protein was purified using nickel affinity chromatography and the xylanase activity was measured. The molecular mass of the purified xylanase was approximately 26 kDa as estimated by SDS-PAGE. The xylanase had optimal activity at pH 6.0 and 60°C. The Km values of the recombinant enzyme towards beechwood was 3.33 mg/ml.

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ÖzAquamarine proteini siyan (mavi) floresan protein ailesinin bir üyesi olup ve Aequorea victoria'dan elde edilen GFP (yeşil floresan protein) türevi floresan bir proteindir. Aquamarine benzeri floresan proteinler genetik mühendisliği teknikleri ile geliştirilmiş özellikleri sayesinde canlı hücrelerin biyolojik olarak görüntülenmesi ve hücre içerisinde lokalizasyon çalışmaları için sıklıkla kullanılmaktadır. Bu çalışmada Escherichia coli BL21(AI) hücreleri pROEX Aqua plasmid DNA'sı ile transforme edilmiş ve %0.04 konsantrasyonda arabinoz ilavesi ile protein ekspresyonu indüklenmiştir. 3L kültür hacimli biyoreaktörde yüksek ekspresyon seviyesinde üretilen Histidin etiketli hedef protein Ni +2 afinite kromotografisi ile saflaştırılmıştır. Saflaştırılan protein konsantrasyonu bir litre bakteri kültürü için 80 mg olarak belirlenmiştir. Rekombinant Aquamarine'ne ait fotolüminesans özellikler florometre ile analiz edilmiştir. SDS-PAGE analizi rekombinant Aquamarine proteine ait tek bir bandın olduğunu ve saflaştırılan proteinin ileri çalışmalarda kullanılmak üzere yüksek verimde ve saflıkta (>%95) elde edildiğini göstermektedir.

Among the vascular endothelial growth factor (VEGF) family variants, the 165-amino acid isoform (VEGF165) is the best characterized and most potent endothelial cell mitogenic factor. It is known that VEGF165 mediates angiogenesis and has the potential for the therapeutic applications. In this study, the expression system of Kluyveromyces lactis that produces the recombinant human VEGF165 has been evaluated. The gene encoding human VEGF165 was successfully cloned in the pKLAC2 expression vector containing a strong LAC4 promoter, after which a pKLAC2-VEGF165 plasmid was constructed. After the transformation, the recombinant human vascular endothelial growth factor 165 (rhVEGF165) was expressed in K. lactis GG799 cells (~ 5.7 mg/L) confirmed by SDS-PAGE and Western blotting and downstream purification processed comprising ammonium sulphate precipitation and affinity chromatography. The biological activity of the purified rhVEGF165 was confirmed by the proliferation of the human umbilical vein-derived endothelial cells (HUVEC) in a dose- and time-dependent manner. The K. lactis-derived rhVEGF165 exhibited a higher proliferative activity compared with a commercially available rhVEGF165 with a half-maximal effective concentration of 3.0. Cell migration analysis was conducted to evaluate the in vitro wound healing effect of the produced rhVEGF165 via a scratch assay. These findings indicate that K. lactis could be a suitable host for secreting bioactive human VEGF165 for therapeutic use.