Cyclodextrin glycosyltransferase (CGTase) enzyme is used industrially to obtain cyclodextrins. In this study, a newly isolated alkaliphilic CGTase producing Bacillus sp. SD5 was used as the source organism for the gene encoding for CGTase. The gene was amplified and sequenced in preparation for cloning into Pichia pastoris for methanol‐induced expression and extracellular secretion. Three strategies were employed in designing the amplification primers for obtaining the fragments to be used in transformation. Hence, three P. pastoris X33 clones were obtained: N2 (with natural gene sequence), S3 (also encoding the c‐myc epitope and polyhistidine tail), and M5 (encoding the polyhistidine tail). Extracellular CGTase activity was monitored throughout fermentation for each clone. The molecular weights of the enzymes were in the range of 85–90 kDa and all three clones showed maximum CGTase activity upon 24 h of induced fermentation. The CGTase enzyme obtained from the M5 clone had Km and Vmax values 13.59 mg/mL and 1153 µmol/(mg min), respectively. The enzyme was purified and displayed optimum activity at 50°C and pH 6 and pH 9. HPLC analysis of reaction products following 10 min of incubation in a reaction solution containing 4% starch showed 852.5 mg/L β‐cyclodextrin and 89.6 mg/L γ‐cyclodextrin being produced.

The aim of this study was to purify and characterize the peptidase of Bacillus amyloliquefaciens (Fukumoto) (strain FE-K1) isolated from ropey bread. Peptidases were purified from crude enzyme solution by affinity chromatography with an efficiency of 25 % and a purification coefficient of 1.53. The optimum pH of partially purified peptidase (PPPase) solution was determined as 7.5 and the peptidases retained approximately 90 % of their initial activity in the pH range 7.0-8.5 following incubation at 37°C for 2 h. The optimum temperature for the PPPase was 60°C. The approximate molecular weight of the PPPase was determined as 36 kDa. Inactivation of the PPPase in the presence of O-FEN and EDTA showed them to be metallopeptidases and 5 mM of K +1 and 5 mM of Mn +2 ions increased the enzyme activity by 4 % and 6.15 %, respectively. The presence of Hg +2 , Fe +3 and SDS (0.1-1.0 % w/v) caused inactivation whereas the enzyme retained most of its activity in the presence of 0.1-1.0 % (v/v) Triton X-100, Tween 20 and Tween 80 and 1-20 % (v/v) xylene, ethanol, acetone and acetonitrile. Characterization of the PPPase revealed the enzyme as a neutral serine metallopeptidase compatible with some organic solvents and surfactants.Özet: Bu çalışmanın amacı, sünmüş ekmeklerden izole edilen Bacillus amyloliquefaciens (Fukumoto) (suş FE-K1) ile elde edilen peptidazı saflaştırmak ve karakterize etmektir. Peptidazlar ham enzim çözeltisinden afinite kromatografisi ile % 25 verim ve 1,53 saflaştırma katsayısı ile saflaştırılmıştır. Kısmi olarak saflaştırılmış peptidaz (PPPaz) çözeltisinin optimum pH değeri 7,5 olarak tespit edilmiş olup, pH 7,5-8,0 aralığında peptidaz, 37°C'de 2 saat inkübasyonun ardından başlangıç aktivitesini yaklaşık % 90 oranında korumuştur. PPPaz'ın optimum sıcaklığı 60°C'dir. PPPaz'ın yaklaşık molekül ağırlığı 36 kDa olarak belirlenmiştir. PPPaz'ın O-FEN ve EDTA varlığında inaktive olması, enzimin metallopeptidaz olduğunu göstermiştir. Ayrıca 5 mM K +1 ve 5 mM Mn +2 , enzimin aktivitesini sırasıyla % 4 ve % 6,15 oranında artırmıştır. Hg +2 , Fe +3 ve SDS (% 0,1-1,0 w/v) varlığı enzimin inaktivasyonuna neden olurken, % 0,1-1,0 (v/v) Triton X-100, Tween 20 ve Tween 80; ve % 1-20 (v/v) ksilen, etanol, aseton ve asetonitril varlığında enzim aktivitesini büyük ölçüde korumuştur. PPPaz'ın karakterizasyonu, enzimin bazı organik çözücü ve yüzey aktif maddelerle ile uyumlu nötr bir serin metalopeptidaz olduğunu ortaya çıkarmıştır.

BACKGROUND: Salep is a highly valued food product, susceptible to adulteration with less costly additives such as starch. The aim of this study is to develop a real-time polymerase chain reaction (PCR) method for the detection and quantification of authentic salep using primers designed from the nr-ITS2 region. RESULTS: The designed primers were shown to amplify selectively with salep DNA and cloning/sequencing of end-point PCR products obtained with authentic salep (Orchideceae) confirmed amplimers to be copies of the intended target region. Detection of salep DNA using the designed primer set was possible at levels as low as 20 pg. After initial evaluation of the real-time PCR method with binary DNA mixtures of salep and cornflour, quantification of salep was conducted in binary mixtures of potato starch (a more likely adulterant) and salep, showing that quantification within the range 25%-100% could be achieved. The real-time assay was performed using the 18S rDNA region co-amplified as an internal standard and evaluated using the Ct method. The calibration curves obtained as a result of real-time PCR runs showed linearity, with R 2 values above 99%. CONCLUSION: A real-time PCR method using SybreGreen intercalating dye was established for salep detection and quantification based on the specific amplification of salep DNA. Method validation was performed utilizing blind and commercial samples.

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In this study, gluten degradation during prolonged tarhana fermentation was monitored in a traditional dough as well as in dough containing sourdough starter, peptidase additives and combination of both. Gluten content, determined using the sandwich ELISA assay method, was reduced significantly in the first 5 days of fermentation for all formulations. After 30 days into fermentation, approximately 15% and 20% reduction of gluten was observed in the traditional dough and sourdough starter added dough, respectively. In enzyme supplemented formulations, the mean gluten levels were below 20 ppm by 20 days of fermentation. Results showed that proteolytic enzyme addition to the dough mix can potentially render long fermented tarhana a food which conforms to regulations set forth for gluten-free foods, even though wheat flour is one of the main production ingredients. Sensory evaluation of the 30 day fermented tarhana samples revealed differences in sourness and odour properties of the products.

Production of many confectionery products such as sweetmeats and Turkish delights are based on mixture of starch, sugar and water, heated for pasting of the starch which is then poured in a mold to set as a gel. The main ingredients, sugar and starch are components which are determinants for the typical texture of these gels. Pullulan is a non-digestible fungal polysaccharide which can potentially be used as an ingredient in lokum production. The purpose of this study was partial substitution of starch used in the formulation of sugar-starch gels with pullulan and to evaluate the effects of cooking time and storage on gel texture. Starch fraction in a base formula was substituted with pullulan at three different levels. First two levels involved substitution of 10 and 20% of the starch with pullulan at a 1:1 ratio and the third level was performed by substituting 20% of starch at a starch/pullulan ratio of 2:3. Firmness and springiness values were determined to assess textural properties. Increased duration of cooking and storage increased mean firmness values however substitution at 20% pullulan did not result in significantly different mean values from the control formulation. Springiness was affected by cooking, storage and pullulan content and reduced loss in springiness was observed in stored samples with increasing pullulan content. Results obtained in this study show that pullulan may be added to lokum formulations for modification of retrogradation behavior however more research is needed on the subject with regard to optimization of product texture for satisfying consumer preference requirements.