Purification Of Invertase Research Articles

Effect of flow patterns on recovery of invertases from Saccharomyces cerevisiae using polyethylene glycol/magnesium sulfate system in microchannels

Effect of flow patterns on recovery of invertases from Saccharomyces cerevisiae using polyethylene glycol/magnesium sulfate system in microchannels

Enhanced invertase binding from baker's yeast via cryogels included boronic acids.

Invertase, an industrially significant glycoenzyme, was purified from baker's yeast using poly (2-Hydroxyethyl methacrylate) [PHema-Pba] cryogels functionalized with boronic acid. At subzero temperatures, PHema-Pba cryogels were synthesized and characterized using swelling tests, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The surface area of the PHema-Pba cryogels was 14 m2/g with a swelling ratio of 88.3% and macroporosity of 72%. The interconnected macropores of PHema-Pba cryogels were shown via scanning electron microscopy. Invertase binding capacity of PHema-Pba cryogel was evaluated by binding studies in different pH, temperature, and interaction time conditions and the maximum Invertase binding of PHema-Pba cryogel was found as 15.2mg/g. and 23.7 fold Invertase purification was achieved from baker's yeast using PHema-Pba cryogels. The results show that PHema-Pba cryogels have high Invertase binding capacity and may be used as an alternative method for enzyme purification via boronate affinity systems.

English

The single step three-phase partitioning (TPP) method was evaluated for the purification of invertase from Momordica charantia. Optimum conditions for TPP method, that is, 70% ammonium sulfate saturation, 1:0.5 t-butanol ratio and 30 min incubation time lead to 20.28% efficiency and 10.48 fold partial purification. Total protein decreased from 34.86 mg to 0.68 mg in the homogenate, and the specific activity of the enzyme increased from 21.67 to 227.02 U/mg protein. The purified enzyme showed maximum activity in 0.3 M pH 5.0 buffer at 50°C. In conclusion, the optimized single step TPP method revealed better purification. Key words: Three-phase partitioning (TPP), invertase, Momordica charantia, enzyme purification.

Isolation and partial purification of invertase from different baker's and distillery Saccharomyces cerevisiae

Isolation and partial purification of invertase from different baker's and distillery Saccharomyces cerevisiae

Monte Carlo economic analysis of Baker's yeast invertase purification using two‐ and three‐phase partitioning

AbstractBACKGROUNDInvertase use in the food industry is limited by production costs. Alternative strategies for extraction, such as aqueous two‐phase systems (ATPS) and three‐phase partitioning (TPP), could be economically feasible for yeast invertase. Economic modeling of bioprocesses makes possible the identification of critical parameters for production costs and emulation of real scenarios, moreover incorporation of uncertainty is possible. This study performed an economic analysis on the production of invertase using ATPS or TPP, also a virtual optimization of ATPS was done.RESULTSTPP provided a lower production cost than ATPS ($145 vs $59.3 per 1 million enzymatic units, respectively). The critical parameter for TPP is recovery yield as it is highly dependent on operating conditions. In contrast, ATPS is dependent on materials costs as the sample load is smaller for ATPS, requiring a larger system. Although TPP provided a lower cost, t‐butanol hinders its acceptance.CONCLUSIONVirtual optimization of ATPS found that varying sample load or system size is not enough to have a lower production cost than TPP, but provided insights for reduction of production costs and development of a safer technique. This study provides a framework for the virtual analysis of ATPS and TPP to evaluate their processes and reduce production costs. © 2018 Society of Chemical Industry

Baker’s yeast invertase purification using Aqueous Two Phase System—Modeling and optimization with PCA/LS-SVM

Abstract Least Squares-Support Vector Machine (LS-SVM) was used to predict data of Baker’s yeast invertase purification using PEG/MgSO 4 Aqueous Two Phase-System (ATPS). Experiments were carried out changing the average molecular mass and percentage of PEG, pH, percentage of MgSO 4 as well as of raw extract in order to observe the percentage of yield (% Yield) and Purification Factor (PF) at the bottom phase. The Principal Component Analysis (PCA) was used to eliminate the less significant input variables on the % Yield as well as on the PF. The generalization capacity evaluation for these two parameters has shown that the model generated by the LS-SVM (R 2 = 0.974; 0.932) approach has given the best performance than partial least squares (R 2 = 0.960; 0.926), base radial neural network (R 2 = 0.874; 0.687) and multi-layer perceptron (R 2 = 0.911; 0.652). Also, a bi-objective optimization has been carried out using the previously adjusted models in order to obtain a set of input data producing higher % Yield for the enzymatic activity (448.34%) as well as for the PF (8.45).

Tentacle-type immobilized metal affinity cryogel for invertase purification from Saccharomyces cerevisiae

The aim of this study is to investigate the usability of cryogel columns for the purification of invertase from Saccharomyces cerevisiae. Poly(2-hydroxyethyl methacrylate) monolithic columns were produced via cryogelation. Ester groups of the poly(2-hydroxyethyl methacrylate) structure were then converted to imine groups by the reaction with poly(ethylene imine) in the presence of NaHCO3. Transition metal ions, Cu(II), Co(II), and Ni(II), were chelated on the PEI-modified cryogel columns. Purification of invertase from natural source namely S. cerevisiae was also studied, and the purification fold values were obtained as 41.350, 44.714, and 30.302 for Cu(II)-chelated, Co(II)-chelated, and Ni(II)-chelated PHEMA/PEI columns, respectively.

GA 처리 후 급 성장하는 완두콩(Pisum sativum L.) 발아체로부터 분리된 중성 invertase의 특성

Invertase (β-D-fructosfuranosidase, EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Three biochemical subgroups of invertases have been investigated in plants: vacuolar (soluble acid), cytoplasmic (soluble alkaline), and cell wall-bound (insoluble acid) invertases. An isoform of neutral invertase was purified from pea seedlings (Pisum sativum L.) and treated with gibberellic acid (GA) by sequential procedures consisting of ammonium sulfate precipitation, ion-exchange chromatography, absorption chromatography, and reactive green-19 affinity chromatography. The results of the overall insoluble invertase purification were a 430-fold increase. The purified neutral invertase was not glycosylated and had an optimum pH between neutral and alkaline (pH 6.8-7.5). It was inhibited by Tris, as well as by heavy metals, such as Hg 2+ and Cu 2+ . Typical Michaelis?Menten kinetics were observed when the activity of the purified invertase was measured, with sucrose concentrations up to 100 mM. The K m and V max values were 12.95 mM and 2.98 U/min, respectively. The molecular mass was around 20 kDa. The sucrose-cleaving enzyme activity of this enzyme is similar to that of sucrose synthase and fructosyltransferase, but its biochemical characteristics are different from those of sucrose synthase and fructosyltransferase. Based on this biochemical characterization and existing knowledge, neutral INV is an invertase isoform in plants.

Aqueous Two-Phase (PEG4000/Na2SO4) Extraction and Characterization of an Acid Invertase from Potato Tuber (Solanum tuberosum)

Invertases are key metabolic enzymes that catalyze irreversible hydrolysis of sucrose into fructose and glucose. Plant invertases have essential roles in carbohydrate metabolism, plant development, and stress responses. To study their isolation and purification from potato, an attractive system useful for the separation of biological molecules, an aqueous two-phase system, was used. The influence of various system parameters such as type of phase-forming salts, polyethylene glycol (PEG) molecular mass, salt, and polymer concentration was investigated to obtain the highest recovery of enzyme. The PEG4000 (12.5%, w/w)/Na2SO4(15%, w/w) system was found to be ideal for partitioning invertase into the bottom salt-rich phase. The addition of 3% MnSO4 (w/w) at pH 5.0 increased the purity by 5.11-fold with the recovered activity of 197%. The Km and Vmax on sucrose were 3.95 mM and 0.143 U mL−1 min−1, respectively. Our data confirmed that the PEG4000/Na2SO4 aqueous two-phase system combined with the presence of MnSO4 offers a low-cost purification of invertase from readily available potato tuber in a single step. The biochemical characteristics of temperature and pH stability for potato invertase prepared from an ATPS make the enzyme a good candidate for its potential use in many research and industrial applications.

Production and properties of invertase from a Cladosporium cladosporioides in SmF using pomegranate peel waste as substrate

Abstract Objective To use suitable fungal strain for production of invertase and to study the effect of external substances and metal ions that may enhances the production of invertase. Methods The culture and nutrient requirements of Cladosporium cladosporioides ( C. cladosporioides ) for production of invertase in a medium at different pH, temperature, incubation period, carbon source and nitrogen source were quantified in present study. Results The optimum pH, temperature and incubation period for enzyme production were 4, 30 °C and 4th day respectively. Among the carbon source pomegranate peel was recorded to be the best carbon source for enzyme production and yeast extract at 1% was ideal nitrogen source for the invertase production. The enzyme was purified by ammonium sulphate precipitation, dialysis and DEAE-Cellulose column chromatography. A trial for the purification of invertase resulted in an enzyme with specific activity of 197.50 Units/mL with 6.35 folds of purification. The purified invertase had a maximum activity at pH 6 and the K m value for pomegranate was 0.26 mg/mL. Analysis of this enzyme for molecular mass was carried out by SDS-PAGE electrophoresis which revealed one band 61 kDa. Conclusion From this study, it can be concluded that the pomegranate peel waste can be more effectively used as a substrate for the production of enzyme under optimized culture conditions.

Aqueous two phase extraction of invertase from baker's yeast: Effect of process parameters on partitioning

Invertase (�-d-fructofuronoside fructohydrolase) is an industrially important enzyme useful for the hydrolysis of sucrose. The potential of aqueous two phase extraction for the isolation and purification of invertase from crude baker’s yeast is explored. Influence of the process parameters such as type of phase forming salts, PEG molecular weight, concentration of salt and polymer, tie line length and volume ratio on partitioning of invertase was studied. PEG 3350/magnesium sulphate system was found most suitable for the extraction which has resulted in favorable pH (5±0.2) for the enzyme extraction. Polymer and salt concentration were found to significantly affect the degree of purification and enzyme recovery of invertase. The purity of ∼8.81 fold was obtained compared to crude extract with recovery of 77% at the standardized process conditions. Overall results demonstrated the feasibility of aqueous two phase extraction for the isolation and purification of invertase without the need of multiple steps.

Partitioning of invertase from tomato in poly(ethylene glycol)/sodium sulfate aqueous two-phase systems

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) Na 2 SO 4 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.

Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato

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 20 kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases.

Production and partial purification of invertase using Cympopogan caecius leaf powder as substrate

The present study investigates the efficiency of Aspergillus niger to produce invertase, an industrially important enzyme by using powdered stem of Cympopogan caecius (Lemon grass) as sole substrate and sole carbon source for the microorganism. The molecular weight of invertase was estimated to be 66-70 kDa by sodium do decyl sulphate poly acrylamide gel electrophoresis (SDS PAGE). The production of the enzyme was studied at different pH scales ranging from pH 4.0 to 7.0 at a constant temperature of 30°C and 2% substrate concentration. The maximum production of invertase (specific activity -0.0516 μk/mg protein) was obtained at pH 5.5 at 30°C temperature, and incubation for 48 h. The activity was found to be stable at pH 5.5 for 30 min. The enzyme was found to be stable in the temperature range of 20-55°C. The effect of divalent metal ions Cu(2+), Fe(2+), Co(2+) on the activity of the enzyme invertase showed that these ions affected the activity by a certain factor. The study can be further industrially exploited in a country-like India where lemon grass is found in plenty and can be used as substrate for enzyme production. Moreover, the preparation of the substrate is also a simple process.

Purification and characterisation of Saccharomyces cerevisiae external invertase isoforms

Abstract Four external invertase isoforms (EINV1, EINV2, EINV3 and EINV4) from Saccharomyces cerevisiae were highly purified by isoelectric precipitation, ethanol precipitation, ion-exchange on QAE-Sephadex and gel filtration using Sephacryl S-200. Unlike previously published procedures for external invertase purification, a specially designed step elution was applied on QAE-Sephadex which enabled the separation of four isoforms. The isoforms have the same molecular mass and catalytic properties: K m for sucrose (25.6 mM), pH optimum (3.5–5.0) and temperature optimum (60 °C), but they exhibit significant difference in p I values, thermal stability and chemical reactivity. Deglycosylation studies showed that the observed differences between isoforms arise from posttranslational modifications. Results showed that external invertase is a mixture of at least four isoforms, but in order to improve the efficiency of food industry processes, only the most stable isoform (EINV1) should be purified and utilised. Substantially different chemical reactivity of the isoforms could be used to improve the yield of covalent immobilization procedures.

Poly(glycidylmethacrylate) brushes generated on poly(VBC) beads by SI-ATRP technique: Hydrazine and amino groups functionalized for invertase adsorption and purification

Crosslinked-poly(vinylbenzylchloride), poly(VBC), beads were prepared by suspension polymerization and poly(glycidylmethacrylate) was grafted by surface-initiated-atom radical polymerization (SI-ATRP) technique. Epoxy groups of the grafted poly(GMA) were reacted with hydrazine and ammonia to create an affinity binding sites. The hydrazine and amine functionalized poly(VBC-g-GMA) beads were used as an affinity support for adsorption of invertase from solution and yeast crude extract. The influence of pH, equilibrium time, ionic strength and initial invertase concentration on the adsorption capacities of both hydrazine and amine functionalized beads has been investigated. Maximum invertase adsorptions onto hydrazine and amine functionalized beads, were 86.7 and 30.4 mg/g at pH 4.0 and 5.5, respectively. The experimental equilibrium data fitted well to the Temkin isotherm model. Finally, the hydrazine functionalized poly(VBC-g-GMA) beads were used for the purification of invertase from crude yeast extract in a batch system and the purity of the eluted invertase from the hydrazine functionalized beads was determined as 92% by HPLC from single step purification protocol.

Concanavalin A carrying reactive beads for yeast invertase purification

Abstract A novel polymeric carrier using an immobilized form of Concanavalin A (Con A) was proposed for the purification of invertase from Saccharomyces cerevisiae. For the synthesis of the carrier, cross-linked, spherical poly(p-chloromethylstyrene), (PCMS) beads with an average diameter of 186 μm were obtained by suspension polymerization. The selected ligand, Con A was covalently attached onto the bead surface via a direct chemical reaction between the chloromethyl groups of the support and the primary amine groups of Con A. Invertase was adsorbed onto the Con A-carrying PCMS beads. The desorption of invertase from the carrier was achieved by the use of methyl-α- d -mannopyranoside as the counter-ligand. The kinetic parameters of purified invertase (i.e. VM and KM) were determined according to the Michealis–Menten model and compared with the crude one. The specific activity of purified invertase (VM=2061 U/mg protein) was determined as 4.2 times higher than the activity of crude invertase mixture (VM=495 U/mg protein). KM value of the purified invertase (% 1.06, w/w) was found to be approximately half of the KM (% 2.00, w/w) of the crude one. The activity half-life of purified invertase was found to be approximately 1.4 times higher. The activation energy of purified invertase (3428 cal/mol) was higher than that of the crude one (2829 cal/mol). These results showed that the carrier developed based on a reactive polymer (i.e. PCMS) could be efficiently used for invertase purification.

An invertase inhibitory protein from Pteris deflexa link fronds.

Plant invertases play important roles in sucrose metabolism. Cell wall invertase was reported to participate in phloem loading and unloading. Soluble invertases would be involved in hexose level regulation in mature tissues and in stored sucrose utilization within vacuoles. Invertase inhibitory proteins were described as one of the possible mechanisms for invertase activity regulation in some plant species; nevertheless, these proteins were found only in sink tissues, suggesting that this mechanism would not be relevant in the sucrose turnover of leaves. This report describes the purification of invertase from Pteris deflexa fronds and the occurrence of an invertase inhibitory protein in this fern organ, as well as its purification and invertase-inhibitor interactions. The Mr of the invertase and of its inhibitory protein were 90,000 and 18,000, respectively. SDS-PAGE in the presence of 2-mercaptoetanol gave two subunits for the enzyme (Mr=66,000 and 30,000) and only one for the inhibitor. The inhibitor protein is a glycoprotein (12% w/w of neutral sugars) that did not show agglutinating activity like some others, and also showed a high heat stability at pH 5.0. The optimum pH of invertase activity is 5.0, while invertase inhibitory protein caused maximal inhibition at the same pH value. Invertase-inhibitor complex formation occurs in an immediate manner and a protease activity was discarded. The inhibition is non-competitive (Ki=1.5 x 10(-6) M) without interactions among the binding sites. The complex is slightly dissociable and sucrose was able to partially reduce the inhibitory effect. Up to the present, invertase inhibitory proteins have been found solely in heterotrophic tissues. In this work we demonstrate that this protein is also present in an autotrophic tissue of a lower vascular plant.

Affinity chromatography of invertase on concanavalin A-bead cellulose matrix: the case of an extraordinary strong binding glycoenzyme.

This work presents confirmation of the biospecific character of the interaction of Concanavalin A (Con A) immobilized on bead cellulose with invertase. In spite of the extraordinary strong binding of invertase to this Con A conjugate (KD = 5 x 10(-9) M), conditions have been found to use Con A-bead cellulose as an affinity chromatography medium. The effective factor in the release of the bound invertase by the counter ligand (alpha-methyl-D-mannopyranoside) is the time of incubation. This phenomenon was demonstrated in both batch and flow-through experiments. A concentration of 1.5 mg Con A per ml of gel was found to be suitable with regard to the maximal invertase/Con A binding ratio and the optimal invertase recovery (94%). As a result of the strong biospecific interaction the purification of invertase was very effective (above ten-fold). Verification by FPLC and PAGE of the product purity revealed only one significant protein band.

CHARACTERIZATION OF THREE SOLUBLE INVERTASES FROM LILIUM LONGIFLORUM FLOWER BUDS

Three soluble invertases (EC 3.2.1.26) previously identified in developing flower buds of Lilium longiflorum Thunb (HortSci. 25:1076, 1990) have been further investigated. These enzymes are fully separable on DEAE-Sephacel and differ substantially in enzymological properties. Each enzyme was further purified by consecutive use of Sephacryl S-200 gel filtration, Con A Sepharose affinity chromatography and Phenyl-Agarose hydrophobic interaction chromatography. This produced 135, 189 and 202 fold purification of invertase I, II and III, respectively. Each was an acid invertase showing pH optima between 4.0 and 5.0. The molecular weight of each invertase was estimated to be 75,000 Da by gel filtration. Invertase I, II and III showed temperature optimum at 40, 50 and 45°C, respectively. A temperature stability study revealed that Invertase III was the most stable followed by II and I. Invertase I, II and III had Km values of 1.0, 6.4 and 6.6 mM for sucrose, respectively. Invertase II and III had lower affinity to raffinose and stachyose than Invertase I. All three invertases were completely inhibited by Hg2+ and Ag+ ions at 1.7 mM concentration. At this concentration Cu2+ inhibited 45% of activity of Invertase I, but only 30% of activity of Invertase II and III.