Due to their aesthetic value and positive health effects, anthocyanins as natural colorants have become very attractive in recent years. There is a need to develop simple and effective methods for the separation and purification of these natural pigments. An aqueous two-phase extraction (ATPE) system based on ethanol/ammonium sulfate is employed for the extraction and preliminary purification of anthocyanins from red cabbage. The influence of ethanol, ammonium sulfate, red cabbage amount and pH on the extraction of anthocyanins is investigated. The optimal extraction conditions (27% (v/v) ethanol, 22% (v/v) ammonium sulfate, 2.5% (v/v) anthocaynin extract, and pH 3.0) gave 83.7% of extraction yield for anthocyanins. The flavonoids and phenolics are concentrated at the top phase of ATPE with a yield of 78% and 82.5%, respectively. The sugars also partitioned and concentrated at the bottom phase with a yield of 79.5%. The stability studies showed that anthocyanins are very stable between the temperature range of 40−70 °C and the pH range of 3.0−6.0, respectively. At a temperature of 70 °C and at pH 6.0 nearly more than 70% of anthocyanin yield is obtained. The results indicated that ATPE technique have a great promise for the efficient recovery of anthocyanins and also removing of the impurities such as sugars.

Raffinose family oligosaccharides (RFOs) are mainly containing raffinose and stachyose. They are antinutrient compounds and the major factors responsible for the flatulence upon ingestion of soymilk. alpha-Galactosidases can hydrolyse RFOs to digestible oligosaccharides. alpha-Galactosidase was partially purified from watermelon and then immobilized on Sepabeads EC-EP with 56% of activity yield by determined best immobilization conditions. The optimum temperature and pH for both enzymes were found as 65 degrees C and pH 6.0, respectively. They showed an excellent stability between pH 3.0-7.0 and below 70 degrees C. The effect of several sugars and metal ions showed that both enzymes affected at different rates. Reusability of the immobilized enzyme with pNPG and raffinose as substrates indicated that the enzyme can be used for a long time with over 50% recovered activity. The enzymatic hydrolysis of the RFOs was realized and HPLC analysis demonstrated that 77.5% and 72.7% of the stachyose and raffinose presented in soymilk is hydrolyzed by the immobilized enzyme during 24 h incubation, respectively. This practical and alternative approach may be a potential to improve the nutritional value of soymilk by eliminating RFOs. Because of its high activity and stability properties the immobilized alpha-galactosidase may also find several applications in food and feed industries.

A new enzyme immobilization strategy is bioaffinity immobilization that using biospecific affinity interactions between the enzyme and matrix. alpha-Galactosidase was immobilized with three different methods (Method I, II and III) on chitosan functionalized with Concanavalin A (ConA). In Method I, the chitosan was first activated with glutaraldehyde, derivatized with ConA and then utilized to immobilize the alpha-galactosidase. For Method II, chitosan was functionalized with ConA by adsorption at first and then used for the immobilization of the enzyme. In Method III, by using the optimized conditions of Method I affinity layers were prepared with alpha-galactosidase. The effect of various factors on alpha-galactosidase immobilization is searched to get perfect immobilization yields. Optimal immobilization conditions were determined for each method. At these conditions, alpha-galactosidase is immobilized with 53%, 64% and 58% activity yields by method I, II and III, respectively. alpha-Galactosidase immobilized with method I, II and III could hydrolyzed 52%, 94% and 30% of raffinose in 24 h at 50 degrees C, respectively. The biochemical characterization and stability studies confirm that these immobilized enzymes promise future in industry. Especially, the immobilized enzymes could be safely used because of their potential for the hydrolysis of raffinose and raffinose type oligosaccharides in food and feed industries.

Enzymatic hydrolysis of raffinose family oligosaccharides (RFOs), mainly raffinose and stachyose, into digestible sugars improves the nutritional value of soybean and soymilk. alpha-Galactosidases have been widely employed for the degradation of RFOs in industrial food applications. In the present study, alpha-galactosidase was immobilized on Sepabeads EC-EA and Sepabeads EC-HA by direct covalent attachment and also by adsorbtion/crosslinking methods by using glutaraldehyde chemistry. The covalently immobilized enzyme on Sepabeads EC-HA completely hydrolyzed both raffinose and stachyose for 24 h. The adsorbed/ crosslinked enzyme on Sepabeads EC-HA hydrolyzed the stachyose and raffinose with 100 and 88.6% degrees at the same time, respectively. The results suggested that the covalently immobilized enzyme on Sepabeads EC-HA is more efficient in the degradation of RFOs. Therefore, this form of enzyme may be a good candidate for removing of RFOs found in many products of the food industry.

Purification of Trichoderma reesei cellulase by three-phase partitioning (TPP) technique and encapsulation of the enzyme in calcium alginate gel were studied. These two basic experiments were optimized with all variables and finally optimum conditions for both purification and encapsulation studies were determined. Ideal TPP condition for the purification of T. reesei cellulase was determined as; 50% (w/v) ammonium sulphate saturation, 1:1 (v/v) enzyme: t-butanol ratio, pH 4.5 and 0.1 mg of the protein. By using this condition, the cellulase enzyme was successfully purified with 113% activity yield and 1.42 purification fold. As a result of the encapsulation studies, optimum immobilization condition for cellulase was determined as 0.5 mg enzyme (protein amount) and 2% (w/v) alginate, 0.5% (w/v) chitosan in 0.15 M CaCl2, 3 h for immobilization, 2.0% (v/v) glutaraldehyde and 2 h for cross-linking. The enzyme was immobilized with 82% activity yield by using this conditions. The cellulase, which was concentrated in the middle phase of TPP, could also immobilized with this condition. The optimal temperature of the free and immobilized cellulase were determined as 65 degrees C and the optimum pHs were found as pH 4.0 and pH 5.0, respectively. The immobilized cellulase was also used three times with high activity yields. The biochemical characterization and stability studies confirm that this immobilized enzyme show promise for industry that could be safely used especially both in the feed and food industry and food processing.

Phytases catalyze the hydrolysis of phytic acid into myo-inositol phosphates and inorganic phosphates and are used as animal feed additives. The main objective of this study is to discover a new phytase, which has high thermal stability, protease resistance, and pH stability for its usability in the feed industry. For this purpose, this study focused on purifying and characterizing the thermostable phytase derived from thermotolerant Aspergillus tubingensis TEM 37 strain at first time, which was isolated from a hot spring soil in the Gediz geothermal field (Turkey). The optimum pH and temperature of the phytase were determined as pH 2.0-5.5 and 45 degrees C, respectively. The molecular weight of the enzyme was determined as 48 kDa. The phytase preserved almost 100% of its activity at 80 degrees C for 3 h. The enzyme showed high resistance against K+, Ba2+, Cu2+, Mg2+, Zn2+ and Tween 20, CTAB, and isooctane among tested compounds. The enzyme also showed high stability against proteases and retained its activity as 88% for pepsin and 98% for trypsin for 120 minutes. In conclusion, it was demonstrated that the thermotolerant A. tubingensis TEM 37 strain naturally produces the phytase that was thermostable and resistant to proteases as required by feed industry.

Üçlü-faz ayırma (TPP) tekniği α-galaktozidazın Aspergillus lentulus’dan tek adımda kısmi saflaştırılması için ilk kez başarıyla kullanıldı. Yüksek aktivite ve saflaştırma katı elde etmek için enzimin ekstraksiyon etkinliğine amonyum sülfat konsantrasyonu, ekstrakt t-butanol oranı ve pH etkisi araştırıldı. Sistemin optimum saflaştırma parametreleri %55(w/v) amonyum sülfat konsantrasyonu, 1:1 (v/v) ham ekstrakt t-butanol oranı ve pH 5.5 olarak belirlendi. Bu optimize TPP sistemi α-galaktosidaz için 5.3 saflaştırma katı ile %178 aktivite verimi oluşturdu. pH 6.5 ve 50oC’de maksimum aktivite gözlendi. α-Galaktozidaz 25-60°C sıcaklık aralığında ve pH 2.6-5.5 aralığında oldukça iyi bir kararlılık gösterdi. KM ve Vmax değerleri sırasıyla 0.365 mM ve 0.093 U olarak belirlendi. Metal iyonları ve şekerler arasında Na2CO3 ve galaktoz enzim aktivitesi üzerinde kuvvetli inhibitor etkisi gösterdi. TPP ile A. lentulus’dan farklı biyokimyasal özelliklere sahip yeni bir α-galaktozidazın elde edilmesi onun çeşitli biyoteknolojik uygulamaları açısından ilgi çekici olacaktır.

BACKGROUND:Cross-linked enzyme aggregates (CLEAs) of α-galactosidase, partially purified from maize (Zea mays) flour, were prepared. The impact of various parameters on enzyme activity was examined to optimize the immobilization procedure. Biochemical characterization of the free and immobilized enzyme was carried out. Stability (thermal, pH, storage and operational stability) and reusability tests were performed. The potential use of the free enzyme and the CLEAs in hydrolysis processes of raffinose-type oligosaccharides present in soymilk was investigated.RESULTS:α-galactosidase CLEAs were prepared with 47% activity recovery under optimum conditions [1:5 (v/v) enzyme solution:saturated ammonium sulfate solution ratio; 7.5 mg protein and 0.1% (v/v) glutaraldehyde, 6 h, 4 °C, 150 rpm]. α-galactosidase CLEAs exhibited increased stability in comparison to the free enzyme. The CLEAs and the free enzyme showed a maximum activity at 40°C and their optimal pH values were5.5 and 6.0, respectively. Kinetic constants (KM , Vmax and kcat ) were calculated for the free enzyme and the CLEAs in the presence of p-nitrophenyl-α-d-galactopyranoside, stachyose, melibiose and raffinose. The effect of various chemicals and sugars on enzyme activity showed that both enzyme forms were significantly inhibited by HgCl2 and galactose. The CLEAs hydrolyzed 85% of raffinose and 96% of stachyose.CONCLUSION:The α-galactosidase CLEAs, with their satisfactory enzymatic characteristics, have much potential for use in the food and feed industry. © 2019 Society of Chemical Industry.

α-Glucosidase was first purified from corn by three-phase partitioning(TPP) and then characterized. Ammonium sulfate and t-butanol were used in order to obtain three phases. Effect of different process parameters such as; ammonium sulfate concentration, enzyme to t-butanol ratio and pH required for efficient purification of the corn α-glucosidase was studied to get highest purification fold and activity recovery. Optimum purification parameters of the TPP system were determined as 50% (w/v) ammonium sulfate saturation with 1.0:0.75 (v/v) ratio of crude extract: t-butanol at pH 4.5. Under optimized conditions α-glucosidase was purified with 3.8 purification fold and 60% activity recovery. The molecular weight of α-glucosidase was determined approximately as 29 kDa by using sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE). Characterization studies showed that, optimum pH and temperature of α-glucosidase were pH 4.5 and 55°C, respectively. The purified enzyme was found to be very stable at a temperature range of 25-60°C and a pH range of 3.5-5.5. Kinetic constants (KM and Vmax) were calculated from Lineweaver-Burk plot as 0.64 mM and 0.45 U, respectively. With 20 mM of CaCl2 concentration α-glucosidase activity was significantly increased to 130%. TPP is a useful strategy to concentrate and purify α-glucosidase for its applications.

Enzyme immobilization with affinity binding which is based on the specific affinity interactions have an important advantage as; high selectivity. In the present study, chitosan and Sepabeads EC-EA were derivatized with aminophenylboronicacid(APBA) for the affinity immobilization of alpha-galactosidase. The influence of various process parameters on immobilization of the enzyme is investigated to get high immobilization yields. Under optimized immobilization conditions, the chitosan and Sepabeads EC-EA immobilized enzymes exhibited activity yield of 89.5% and 72%, respectively. The maximum activities were detected at 40 degrees C for free and Sepabeads EC-EA immobilized enzyme and 55 degrees C for chitosan immobilized enzyme. The optimum pH was found as pH 5.0 for free and Sepabeads EC-EA immobilized enzyme and pH 5.5 for chitosan immobilized enzyme. Both immobilized enzymes were very stable at temperature ranged from 4 to 55 degrees C and also in a pH range of 2.6-7.0. The immobilized alpha-galactosidases were also used in the hydrolysis of raffinose. The chitosan and Sepabeads EC-EA immobilized enzymes hydrolysed 55% and 42% of raffinose in 32 hat 50 degrees C, respectively. The obtained results shed light for the useability of these immobilized enzymes in the hydrolysis of raffinose in food industry and make these immobilized enzymes good candidates for their various biotechnological applications.

Three-phase partitioning (TPP) was used to concentrate and purify alpha-galactosidase from watermelon (Citrullus vulgaris). The various process parameters required for efficient purification of alpha-galactosidase were optimized to get highest purity fold and yield. The best alpha-galactosidase yield (76.7%) with a nearly 2.7-fold purification was obtained in the interphase of the TPP system, which consisted of the crude extract to t-butanol ratio of 1:1 (v/v) in the presence of 50% (w/v) (NH4)(2)SO4 at pH 5.5. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a substantial level of purification of a-galactosidase from watermelon. The molecular weight of the enzyme was determined approximately as 45 kDa. The purified enzyme was characterized with respect to its activity and stability. Various parameters (temperature, pH and substrate concentration) affecting to the enzyme activity and stability were studied. Optimum pH and temperature of a-galactosidase were determined as pH 6.0 and 60 degrees C, respectively. The purified enzyme was also very stable at a temperature range of 4-50 degrees C and a pH range of 4.0-6.5. The K-m and V-max, values were calculated from Lineweaver-Burk plot as 0.14 mM and 0.12 U, respectively. The results indicated that, TPP is a simple, quick, economical and very attractive process for primary purification of alpha-galactosidases compared to conventional chromatographic protocols. (C) 2013 Elsevier B.V. All rights reserved.

Molecularly imprinted polymers were the new, simple and unexpensive materials that can be used in several clinical applications. Phenylboronic acid has been frequently used as functional monomer for the covalent imprinting of diols. In this study, the phenylboronic acid esters of fructose, galactose, glucose and raffinose were synthesized and then used as template analytes. The adsorption capacities of fructose, galactose and glucose–phenylboronic acid imprinted polymers were 75, 10 and 30%, respectively. The batch rebinding studies and Scatchard analysis were done for all sugar imprinted polymer. Glucose is one of the mostly found sugar in the urine. The glucose:phenylboronic acid imprinted polymer was used for the analysis of glucose, fructose, galactose, sucrose, maltose, lactose and raffinose in spiked urine. The selectivity of glucose:phenylboronic acid imprinted polymer to urine monosaccharides was found as nearly 45–55% and to di- and polysaccharides was found as 30–35%, respectively.

Three-phase partitioning (TPP) is a bioseparation technique used for the extraction, concentration and purification of biomolecules. The technique consists of simultaneous addition of a salt (generally ammonium sulfate) to the crude extract followed by the addition of an organic solvent (generally t-butanol). In the present study, α-galactosidase was purified from pepino (Solanum muricatum) fruit by TPP. The influence of various process parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) on α-galactosidase partitioning is investigated to get highest purity fold and yield. The results showed that, 50% (w/v) ammonium sulfate saturation with 1:1.5 crude extract to t-butanol ratio at pH 5.25 gave 6.2-fold purification with 127% activity recovery of α-galactosidase. Characterization and determination of biochemical properties of the partitioned α-galactosidase were also aimed. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis showed considerable purification and the molecular weight of the enzyme was nearly found to be as 38kDa. Various parameters (temperature, pH and substrate concentration) affecting to the enzyme activity and stability were studied. The optimum temperature and pH of the partitioned α-galactosidase were found to be 50°C and pH 5.5, respectively. The enzyme was very stable at the temperature ranged from 37 to 45°C and also in a pH range of 4.5–7.0. The kinetic constants; Km and Vmax were determined to be 0.37mM and 0.46U, respectively. The results indicated that, TPP technique is very attractive process for purification of pepino α-galactosidase and the characteristic properties of the enzyme partitioned by TPP make the enzyme good candidates for its several industrial applications.

α-Galactosidase from tomato has been immobilized on Sepabead EC-EA and Sepabead EC-HA, which were activated with ethylendiamino and hexamethylenediamino groups, respectively. Two strategy was used for the covalent immobilization of α-galactosidase on the aminated Sepabeads: covalent immobilization of enzyme on glutaraldehyde activated support and cross-linking of the adsorbed enzymes on to the support with glutaraldehyde. By using these two methods, all the immobilized enzymes retained very high activity and the stability of the enzyme was also improved. The obtained results showed that, the most stable immobilized α-galactosidase was obtained with the second strategy. The immobilized enzymes were characterized with respect to free counterpart. Some parameters effecting to the enzyme activity and stability were also analyzed. The optimum temperature and pH were found as 60°C and pH 5.5 for all immobilized enzymes, respectively. All the immobilized α-galactosidases were more thermostable than the free enzyme at 50°C. The stabilities of the Sepabead EC-EA and EC-HA adsorbed enzymes treated with glutaraldehyde compared to the stability of the free enzyme were a factor of 6 for Sepabead EC-EA and 5.3 for Sepabead EC-HA. Both the free and immobilized enzymes were very stable between pH 3.0 and 6.0 and more than 85% of the initial activities were recovered. Under the identical storage conditions the free enzyme lost its initial activity more quickly than the immobilized enzymes at the same period of time. The immobilized α-galactosidase seems to fulfill the requirements for different industrial applications.

The goal of this study was to determine some characteristics of Baker's yeast invertase partitioned with poly(ethylene glycol)/MgSO4 aqueous two-phase system (ATPS). Under optimized conditions [PEG-3000 (15%, w/w) and MgSO4 (23%, w/w) with 5% (w/w) MnCl2 at pH 5.0] yeast invertase was partitioned by using an ATPS with purification factor of 6.2-fold and activity recovery of 217.7%, respectively. The yeast invertase was characterised with respect to its activity and stability at various pH and temperature ranges. Optimum pH and temperature were determined at pH 5.5 and 60°C, respectively. The enzyme was very stable in the range of pH 4.0–7.0 and more than 95% of its initial activity was recovered. The yeast invertase was also stable at the temperature range of 4–50°C and retained nearly about 98% of its initial activity at 50°C. Kinetic parameters, Km and Vmax using sucrose as substrate were measured as 24.1mM and 35.5U, respectively. MnSO4 and MnCl2 efficiently enhanced the activity and also showed an activator effect for invertase. Relative activities were found as 151% and 156% for MnSO4 and MnCl2, respectively. The biochemical properties of the yeast invertase partitioned in an ATPS make the enzyme good candidates for several industrial applications.

The aim of this study is to extract and purify tomato (Lycopersicon esculentum) invertase using an aqueous two-phase system (ATPS). The ATPSs are formed by mixing the polymer with a salt and a protein solution. Invertase was extracted by partitioning in ATPS composed of polyethylene glycol (PEG) in the presence of sodium sulfate. The partitioning of the enzyme in ATPSs was studied at 25°C. The effects of phase composition, molecular mass of the PEG, PEG concentration, salt concentration, pH and neutral salt (KCl) concentration on enzyme partitioning and purification were investigated. The best optimal ATPS condition for the partitioning and purification of invertase was 15% (w/w) PEG 3000, 12% (w/w) Na2SO4 and 5% (w/v) KCl (pH 4.5) which increased the purity by 5.5-fold with the recovered activity of 90%. Therefore, ATPS can be effectively used to recover and purify invertase from tomato fruit.

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37°C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37°C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60°C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37°C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t1/2 was determined as 6.72h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The Km and Vmax values were measured as 1.07mM and 0.01U/mg for free enzyme and 0.89mM and 0.1U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.

Three-phase partitioning (TPP), a technique used in protein purification, was used to purify invertase from tomato (Lycopersicon esculentum). The method consists of simultaneous addition of ammonium sulfate and t-butanol to the crude enzyme extract in order to obtain the three phases. Different parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) essential for the extraction and purification of invertase were optimized to get highest purity fold and yield. It was seen that, 50% (w/v) ammonium sulfate saturation with 1:1 (v/v) ratio of crude extract to t-butanol at pH 4.5 gave 8.6-fold purification with 190% activity recovery of invertase in a single step. Finally, the purified enzyme was also characterized and the general biochemical properties were determined. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was nearly found to be as 20kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases.

Three-phase partitioning (TPP) was used to purify invertase from Baker's yeast (Saccharomyces cerevisiae) in a single step. The influence of various process parameters like ammonium sulfate concentration, crude extract to t-butanol ratio and pH on invertase partitioning is investigated in order to enhance the purification fold degree and activity recovery of invertase. Under optimized conditions (50%, w/v, ammonium sulfate saturation, 1:0.5 crude extract to t-butanol ratio and pH 4.0), the invertase was purified to 15-fold with 363% activity recovery. The enzyme obtained from TPP showed considerable purification on sodium dodecyl polyacrylamide gel electrophoresis with a molecular weight of 52kDa. The optimum temperature and pH were determined as 60°C and pH 6.0, respectively. The purified enzyme was also very stable at a pH range of 4.0–6.5 and temperature range of 4–50°C. The Km and Vmax values were calculated from Lineweaver–Burk plot as 0.19M and 29.8U, respectively.

BACKGROUND:Type 2 diabetes is a complex disease that still requires a great deal of work to be carried out to understand the pathophysiology. Recently, researchers have focused on studying the organs and tissues known to be involved in the development of the type 2 phenotype using a proteomic approach. Little work has been reported on plasma of type 2 diabetics in whom the clinical status has been well characterized. In this study, changes in plasma proteins of type 2 diabetics were investigated by proteomic analysis in well-characterized individuals with type 2 diabetes (early and late stage) and control groups (with or without a family history of diabetes).METHODS:Samples were analysed by two-dimensional gel electrophoresis and significantly differentiated proteins were identified by nano-LC-ESI-MS.RESULTS:A total of 12 protein signatures that were differentially displayed with high significance compared with controls were selected. Four of the differentially displayed proteins were identified as haptoglobin alpha2, haptoglobin Hp2(fragment) and transthyretin and Chain A (formerly prealbumin), and all were up-regulated. Thiol-specific antioxidant protein, Chain A, tertiary structures of three amyloidogenic transthretin variants and haptoglobin-related protein precursor were all down-regulated in controls with a family history of diabetes, early and late diabetic patients in comparison with the control.CONCLUSION:A proteomic-based approach was used to discover and identify the differentially expressed proteins in various states of type 2 diabetes.