Three-phase partitioning and immobilization of Bacillus methylotrophicus Y37 cellulase on organo-bentonite and its kinetic and thermodynamic properties

Clay Minerals ◽  
2020 ◽  
Vol 55 (2) ◽  
pp. 120-131
Author(s):  
Yonca Avci Duman ◽  
A. Uğur Kaya ◽  
Çiğdem Yağci
Keyword(s):  
Immobilized Enzyme ◽  
Maximum Velocity ◽  
Catalytic Performance ◽  
Covalent Immobilization ◽  
Single Step ◽  
Free Enzyme ◽  
Phase Partitioning ◽  
Bacillus Methylotrophicus ◽  
Three Phase ◽  
First Time

AbstractIn this study, for the first time Bacillus methylotrophicus Y37 cellulase was purified and recovered in a single step by three-phase partitioning (TPP). The optimal purification parameters for TPP were 40% ammonium sulfate saturation (m/v) with a 1.0:1.0 (v/v) ratio of crude extract:t-butanol, which gave 5.8-fold purification with 155% recovery of cellulase. Non-covalent immobilization of the partitioned cellulase was performed using bentonite as a support material. The activity observed in the 20th experiment was 100%. The optimal pH values and temperatures determined for the free enzyme and the immobilized enzyme were 5.0 and 6.0 and 45°C and 50°C, respectively. The Arrhenius activation energy (Ea) of the immobilized enzyme was lower than that of the free enzyme, whereas the Michaelis–Menten constant (Km) and maximum velocity (Vm) of the immobilized enzyme increased. The turnover number (kcat) and the catalytic performance (kcat/Km) demonstrated the improved catalytic properties of the immobilized enzyme compared to the free enzyme. Immobilization of cellulase is thermodynamically preferred.

scholarly journals Optimal Immobilization ofβ-Galactosidase ontoκ-Carrageenan Gel Beads Using Response Surface Methodology and Its Applications

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Magdy M. Elnashar ◽  
Ghada E. Awad ◽  
Mohamed E. Hassan ◽  
Mohamed S. Mohy Eldin ◽  
Bakry M. Haroun ◽  
...  
Keyword(s):  
Immobilized Enzyme ◽  
Covalent Binding ◽  
Maximum Velocity ◽  
Free Enzyme ◽  
Step Method ◽  
Enzyme Form ◽  
Gel Beads ◽  
Operational Temperature ◽  
Full Factorial ◽  
Central Composite

β-Galactosidase (β-gal) was immobilized by covalent binding on novelκ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 22full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55°C compared to the free enzyme. The apparentKmafter immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocityVmaxwas 131.2 μmol·min−1while it was 177.1 μmol·min−1for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of theκ-carrageenan beads loaded withβ-galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.

Three phase partitioning for simultaneous purification of aloe polysaccharide and protein using a single-step extraction

2015 ◽  
Vol 50 (3) ◽  
pp. 482-486 ◽  
Author(s):  
Zhi-Jian Tan ◽  
Chao-Yun Wang ◽  
Yong-Jian Yi ◽  
Hong-Ying Wang ◽  
Wan-Lai Zhou ◽  
...  
Keyword(s):  
Single Step ◽  
Phase Partitioning ◽  
Three Phase

scholarly journals Immobilization of Catalase on Chitosan/ZnO and Chitosan/ZnO/Fe2O3 Nanocomposites: A Comparative Study

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 820
Author(s):  
Reda M. El-Shishtawy ◽  
Nahed S. E. Ahmed ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Half Life ◽  
Immobilized Enzyme ◽  
Enzyme Stability ◽  
Catalytic Performance ◽  
Free Enzyme ◽  
Initial Activity ◽  
Reaction Systems ◽  
Optimum Ph ◽  
Immobilized Catalase ◽  
System Properties

The strong catalytic performance, eco-friendly reaction systems, and selectivity of enzyme-based biocatalysts are extremely interesting. Immobilization has been shown to be a good way to improve enzyme stability and recyclability. Chitosan-incorporated metal oxides, among other support matrices, are an intriguing class of support matrices for the immobilization of various enzymes. Herein, the cross-linked chitosan/zinc oxide nanocomposite (CS/ZnO) was synthesized and further improved by adding iron oxide (Fe2O3) nanoparticles. The final cross-linked CS/ZnO/Fe2O3 nanocomposite was used as an immobilized support for catalase and is characterized by SEM, EDS, and FTIR. The nanocomposite CS/ZnO/Fe2O3 enhanced the biocompatibility and immobilized system properties. CS/ZnO/Fe2O3 achieved a higher immobilization yield (84.32%) than CS/ZnO (37%). After 10 repeated cycles, the remaining immobilized catalase activity of CS/ZnO and CS/ZnO/Fe2O3 was 14% and 45%, respectively. After 60 days of storage at 4 °C, the remaining activity of immobilized enzyme onto CS/ZnO and CS/ZnO/Fe2O3 was found to be 32% and 47% of its initial activity. The optimum temperature was noticed to be broad at 25–30 °C for the immobilized enzyme and 25 °C for the free enzyme. Compared with the free enzyme optimum pH (7.0), the optimum pH for the immobilized enzyme was 7.5. The Km and Vmax values for the free and immobilized enzyme on CS/ZnO, and the immobilized enzyme on CS/ZnO/Fe2O3, were found to be 91.28, 225.17, and 221.59 mM, and 10.45, 15.87, and 19.92 µmole ml−1, respectively. Catalase immobilization on CS/ZnO and CS/ZnO/Fe2O3 offers better stability than free catalase due to the enzyme’s half-life. The half-life of immobilized catalase on CS/ZnO/Fe2O3 was between 31.5 and 693.2 min.

scholarly journals Preparation and characterization of penicillin acylase immobilized on sepabeads EC-EP carrier

2007 ◽  
Vol 13 (4) ◽  
pp. 205-210 ◽  
Author(s):  
Milena Zuza ◽  
Slavica Siler-Marinkovic ◽  
Zorica Knezevic
Keyword(s):  
Thermal Stability ◽  
Immobilized Enzyme ◽  
Conformational Stability ◽  
Penicillin Acylase ◽  
Covalent Immobilization ◽  
Free Enzyme ◽  
Penicillin G ◽  
High Catalytic Activity ◽  
Decimal Reduction Time ◽  
Promising Solution

This paper reports the covalent immobilization of penicillin G acylase from E. coli on Sepabeads EC-EP, an epoxy-activated polymethacrylic carrier, and describes the properties of the immobilized enzyme. Due to its versatility to mediate hydrolysis of penicillins and semi-synthetic B-lactam antibiotics synthesis reactions, the selected enzyme belongs to a class of biocatalysts of great industrial interest. The immobilized enzyme was characterized in its pH and thermal stability and reaction kinetics. The immobilization of penicillin acylase resulted in a slightly different pH activity profile and temperature optima, indicating that the immobilization by this method imparted the structural and conformational stability to this enzyme. The immobilized enzyme also retained a high catalytic activity and showed the increased thermal stability compared with a free enzyme. By comparison of decimal reduction time values obtained at 50?C, it can be concluded that the immobilized enzyme was approximately 5-fold more stable than a free enzyme. The immobilization procedure developed is quite simple and easily reproduced, and provides a promising solution for the application of penicillin acylase for the purpose of 6-aminopenicillanic acid production.

scholarly journals Three phase partitioning as a rapid and efficient method for purification of plant-esterase from wheat flour

2020 ◽  
Vol 22 (2) ◽  
pp. 42-49
Author(s):  
Liang Dong ◽  
Linxin He ◽  
Danqun Huo
Keyword(s):  
Wheat Flour ◽  
Single Step ◽  
Phase Partitioning ◽  
Surface Method ◽  
Three Phase ◽  
High Efficient ◽  
New Type ◽  
Purification Plant ◽  
Very High

AbstractThree-phase partitioning (TPP) was used to purify plant-esterase from wheat flour. Effect of various process parameters has been evaluated and plant-esterase was purified to 11.35-fold by optimized single step TPP system (50%, (w/v) (NH4)2SO4 saturation, 1:1 (v/v) ratio of crude extract: t-butanol at pH 4).The enzyme was found to be exclusively partitioned in the aqueous phase. Using TPP system, plant-esterase quickly purified to homogeneity with very high purity and activity. On the basis of single factor research, purification process was optimized by using response surface method, established a new type of high efficient purification plant-esterase method. To the best of our knowledge, this is the first report for purification and characterization of plant-esterase by using three phase partitioning (TPP). The results indicated that, TPP is a simple, quick, economical and very attractive process for purification of plant-esterase compared to conventional chromatographic protocols.

scholarly journals Covalent Immobilization of β-Glucosidase into Mesoporous Silica Nanoparticles from Anhydrous Acetone Enhances Its Catalytic Performance

Nanomaterials ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 108 ◽  
Author(s):  
Filomena Sannino ◽  
Aniello Costantini ◽  
Francesco Ruffo ◽  
Antonio Aronne ◽  
Virginia Venezia ◽  
...  
Keyword(s):  
Silica Nanoparticles ◽  
Conformational Changes ◽  
Mesoporous Silica Nanoparticles ◽  
Covalent Binding ◽  
Cellulose Hydrolysis ◽  
Catalytic Performance ◽  
Covalent Immobilization ◽  
Free Enzyme ◽  
Anhydrous Acetone ◽  
Km Value

An immobilization protocol of a model enzyme into silica nanoparticles was applied. This protocol exploited the use of the bifunctional molecule triethoxysilylpropylisocyanate (TEPI) for covalent binding through a linker of suitable length. The enzyme β-glucosidase (BG) was anchored onto wrinkled silica nanoparticles (WSNs). BG represents a bottleneck in the conversion of lignocellulosic biomass into biofuels through cellulose hydrolysis and fermentation. The key aspect of the procedure was the use of an organic solvent (anhydrous acetone) in which the enzyme was not soluble. This aimed to restrict its conformational changes and thus preserve its native structure. This approach led to a biocatalyst with improved thermal stability, characterized by high immobilization efficiency and yield. It was found that the apparent KM value was about half of that of the free enzyme. The Vmax was about the same than that of the free enzyme. The biocatalyst showed a high operational stability, losing only 30% of its activity after seven reuses.

Quality Analysis of Fresh Cheese Prepared Using Wrightia tinctoria Proteases

2020 ◽  
Vol 16 (9) ◽  
pp. 1309-1317
Author(s):  
Anusha Rajagopalan ◽  
Bindhu O. Sukumaran
Keyword(s):  
Textural Properties ◽  
Artemia Salina ◽  
Quality Analysis ◽  
Human Consumption ◽  
Cheese Making ◽  
Phase Partitioning ◽  
Milk Clotting ◽  
Three Phase ◽  
Fresh Cheese ◽  
Wrightia Tinctoria

Background: Aqueous leaf extract of Wrightia tinctoria has been in use for artisanal cheese preparation in diverse parts of India. Scientific validation behind the milk clotting potential of W. tinctoria stem proteases purified through three-phase partitioning (TPP) has been attempted. However, its contribution to cheese quality standards has not been tried yet. Objective: To evaluate the suitability of three-phase partitioned W. tinctoria (WT) stem proteases in fresh cheese making. Methods: Fresh cheese was prepared using W. tinctoria TPP proteases and its characteristics were compared with that of commercial plant milk coagulant, Enzeco® (CEz) and rennin (CRn). Yield, organoleptic and textural properties, total fat, protein and moisture content of all cheese were determined according to FSSAI standards. Toxicity of the TPP proteases was assessed on the shrimp model (Artemia salina egg and Litopenaeus vannamei post-larval shrimps). Results: TPP proteases were found to be non-toxic and safe for human consumption with no change in egg hatchability and survival of the shrimps in comparison to that of control. Cheese analysis results indicated perceptible resemblance in nutritional characteristics of WT cheese with control cheese. Yield, textural properties and organoleptic acceptance of WT cheese resembled more closely with that of CEz cheese. Conclusion: Observation from the study paves the way for the acceptance of W. tinctoria proteases as a suitable vegetable rennet for fresh cheese making.

Immobilized Nanoparticles-Mediated Enzymatic Hydrolysis of Cellulose for Clean Sugar Production: A Novel Approach

2019 ◽  
Vol 15 (3) ◽  
pp. 296-303 ◽  
Author(s):  
Swapnil Gaikwad ◽  
Avinash P. Ingle ◽  
Silvio Silverio da Silva ◽  
Mahendra Rai
Keyword(s):  
Catalytic Activity ◽  
Enzymatic Hydrolysis ◽  
Immobilized Enzyme ◽  
Free Enzyme ◽  
Magnetic Nanomaterials ◽  
Sugar Production ◽  
Cellulase Enzyme ◽  
Enzymatic Hydrolysis Of Cellulose ◽  
Hydrolysis Of Cellulose ◽  
Hydrolysis Of

Background: Enzymatic hydrolysis of cellulose is an expensive approach due to the high cost of an enzyme involved in the process. The goal of the current study was to apply magnetic nanomaterials as a support for immobilization of enzyme, which helps in the repeated use of immobilized enzyme for hydrolysis to make the process cost-effective. In addition, it will also provide stability to enzyme and increase its catalytic activity. Objective: The main aim of the present study is to immobilize cellulase enzyme on Magnetic Nanoparticles (MNPs) in order to enable the enzyme to be re-used for clean sugar production from cellulose. Methods: MNPs were synthesized using chemical precipitation methods and characterized by different techniques. Further, cellulase enzyme was immobilized on MNPs and efficacy of free and immobilized cellulase for hydrolysis of cellulose was evaluated. Results: Enzymatic hydrolysis of cellulose by immobilized enzyme showed enhanced catalytic activity after 48 hours compared to free enzyme. In first cycle of hydrolysis, immobilized enzyme hydrolyzed the cellulose and produced 19.5 ± 0.15 gm/L of glucose after 48 hours. On the contrary, free enzyme produced only 13.7 ± 0.25 gm/L of glucose in 48 hours. Immobilized enzyme maintained its stability and produced 6.15 ± 0.15 and 3.03 ± 0.25 gm/L of glucose in second and third cycle, respectively after 48 hours. Conclusion: This study will be very useful for sugar production because of enzyme binding efficiency and admirable reusability of immobilized enzyme, which leads to the significant increase in production of sugar from cellulosic materials.

scholarly journals Broad scope gold(i)-catalysed polyenyne cyclisations for the formation of up to four carbon–carbon bonds

2017 ◽  
Vol 15 (10) ◽  
pp. 2163-2167 ◽  
Author(s):  
Zhouting Rong ◽  
Antonio M. Echavarren
Keyword(s):  
Single Step ◽  
Simultaneous Formation ◽  
Broad Scope ◽  
Carbon Carbon Bonds ◽  
Carbon Bonds ◽  
First Time ◽  
High Stereoselectivity

The polycyclisation of polyeneynes catalyzed by gold(i) has been extended for the first time to the simultaneous formation of up to four carbon–carbon bonds, leading to steroid-like molecules with high stereoselectivity in a single step with low catalyst loadings.

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