Immobilization of ß-galactosidase from Kluyveromyces lactis on Eupergit® C and Properties of the Biocatalyst

2012 ◽  
Vol 8 (3) ◽  
Author(s):  
Graciella da Silva Campello ◽  
Renata Aguirre Trindade ◽  
Tatiane Vieira Rêgo ◽  
Janaína Fernandes de Medeiros Burkert ◽  
Carlos André Veiga Burkert
Keyword(s):  
Thermal Stability ◽  
Ionic Strength ◽  
Batch Reactor ◽  
Kluyveromyces Lactis ◽  
Immobilized Enzymes ◽  
Maximum Activity ◽  
Lactose Hydrolysis ◽  
Substrate Affinity ◽  
Burman Design ◽  
Plackett Burman Design

Abstract The main goal of this study was to investigate the immobilization of commercial ß-galactosidase from Kluyveromyces lactis (Lactozym®) on Eupergit® C. A Plackett-Burman design was proposed. The ionic strength and pH were the variables that presented significant effect (p<0.1) on immobilization. The increase in the ionic strength from 0.1 to 1.5 M and the increase in pH from 6.6 to 7.4 represented an increase of 28.56% and a reduction of 18.19% in the immobilization yield, respectively. At 25°C, pH 6.6, ionic strength of 1.5 M, immobilization for 8 h, 1 mM of divalent magnesium ion and 0.4 mL of enzyme added, reached 85% immobilization yield. The free and immobilized enzymes were characterized. pH and temperature profiles showed maximum activity at pH 6.6 and 45°C, for both free and immobilized enzymes. There was a gain in thermal stability with enzyme immobilization and there was an increase of about four times in the half-life of the immobilized derivative at 45°C (from 0.43 h to 1.78 h). This greater thermal stability was also made clear through the calculation of thermodynamic parameters (ΔH, ΔG and ΔS). Km values, 30.33 mM and 104.00 mM for free and immobilized enzymes, respectively, represented a reduction in substrate affinity after immobilization, possibly owing to stereo-conformational factors. In a batch reactor for lactose hydrolysis from cheese whey, an increase in lactose conversion with immobilization was observed at 40°C and 45°C (90.43% and 65.36%, respectively) in relation to the free enzyme (84.17% and 39.58%, respectively).

scholarly journals Purification and characterization of the heat-stable serine proteinase from Thermomonospora fusca YX

1987 ◽  
Vol 246 (2) ◽  
pp. 511-517 ◽  
Author(s):  
T W Gusek ◽  
J E Kinsella
Keyword(s):  
Thermal Stability ◽  
Ionic Strength ◽  
Serine Proteinase ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Purification And Characterization ◽  
Thermomonospora Fusca ◽  
Heat Stable

The proteinase secreted from Thermomonospora fusca YX grown on cellulose was purified by (NH4)2SO4 fractionation and cation-exchange chromatography. The isolated proteinase readily hydrolysed several proteins and demonstrated activity towards casein from 35 to 95 degrees C (at pH 8.0) with maximum activity at 80 degrees C. It exhibited broad pH and ionic-strength optima centered at pH 9.0 and 0.2 M-NaCl respectively, and it retained high activity in the presence of 2% (w/v) SDS, 20 mM-dithiothreitol and 1.0 M-NaCl. The proteinase, which was fully inhibited by phenylmethanesulphonyl fluoride, had an Mr of 14,500 and an isoelectric point at 9.21. A measurement of proteinase thermal stability demonstrated a T50% (15 min) of 85 degrees C at pH 4.5.

Parameter Screening for the Important Factors Influencing the As(V) Adsorption Using a Plackett-Burman Design

2014 ◽  
Vol 931-932 ◽  
pp. 178-182
Author(s):  
Nusavadee Pojananukij ◽  
Kitirote Wantala ◽  
Sutasinee Neramittagapong ◽  
Arthit Neramittagapong
Keyword(s):  
Aqueous Solution ◽  
Batch Reactor ◽  
Operating Parameters ◽  
Adsorption Efficiency ◽  
Initial Concentration ◽  
Factors Influencing ◽  
Adsorption Temperature ◽  
Burman Design ◽  
Plackett Burman Design ◽  
Adsorbent Dose

The adsorption efficiency of As (V) ions from an aqueous solution was investigated. The adsorption experiments were carried out in a batch reactor. The effects of operating parameters; i.e. the absorbent dose (g/L), the pH, the adsorption temperature (°C), the As (V) initial concentration (ppb), and type of adsorbents, on the adsorption efficiencies were studied. A PlackettBurman experimental design was used to screen for the important factors that influence As (V) adsorption. It has been found that the most important effect on the As (V) adsorption capacity was the type of adsorbent. The factor importance could be written in descending order as follows: the type of adsorbents, the adsorbent dose, the As (V) initial concentration, the adsorption temperature, and the pH.

Assessment of liquid chromatographic method robustness by use of Plackett-Burman design

2010 ◽  
Vol 22 (2) ◽  
pp. 281-296 ◽  
Author(s):  
M. Mašković ◽  
B. Jančić-Stojanović ◽  
A. Malenović ◽  
D. Ivanović ◽  
M. Medenica
Keyword(s):  
Chromatographic Method ◽  
Liquid Chromatographic Method ◽  
Burman Design ◽  
Liquid Chromatographic ◽  
Plackett Burman Design

Using a Plackett–Burman design to maximise yield of rosemary essential oil by distillation

2021 ◽  
Vol 166 ◽  
pp. 113488
Author(s):  
Agnese Spadi ◽  
Giulia Angeloni ◽  
Lorenzo Guerrini ◽  
Ferdinando Corti ◽  
Marco Michelozzi ◽  
...  
Keyword(s):  
Essential Oil ◽  
Burman Design ◽  
Plackett Burman Design ◽  
Rosemary Essential Oil

scholarly journals Analysis of insecticide residues in honey by liquid chromatography tandem mass spectrometry using QuEChERS optimized by the Plackett Burman design

2021 ◽  
Vol 19 (1) ◽  
pp. 326-332
Author(s):  
Ana Paula Ferreirade Souza ◽  
Mateus Henrique Petrarca ◽  
Patrícia Aparecida de Campos Braga ◽  
Nadia Regina Rodrigues ◽  
Felix Guillermo Reyes Reyes
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Tandem Mass ◽  
Insecticide Residues ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Burman Design ◽  
Plackett Burman Design

scholarly journals Intracellular Ionic Strength Sensing Using NanoLuc

2021 ◽  
Vol 22 (2) ◽  
pp. 677
Author(s):  
Tausif Altamash ◽  
Wesam Ahmed ◽  
Saad Rasool ◽  
Kabir H. Biswas
Keyword(s):  
Thermal Stability ◽  
Phase Separation ◽  
Drug Discovery ◽  
Ionic Strength ◽  
Cellular Signaling ◽  
Live Cells ◽  
Protein Activity ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Wide Range

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

scholarly journals Use of chemometric techniques to design a microbiological method for sulfonamide detection in milk

2013 ◽  
Vol 31 (No. 6) ◽  
pp. 627-632 ◽  
Author(s):  
O.G. Nagel ◽  
M.P. Molina ◽  
R.L. Althaus
Keyword(s):  
Experimental Design ◽  
Logistic Model ◽  
Logistic Regression Model ◽  
Culture Medium ◽  
Spore Concentration ◽  
Microbiological Method ◽  
Burman Design ◽  
Detection Capabilities ◽  
Sulfonamide Residues ◽  
Plackett Burman Design

We proposed an experimental design of a microbial bioassay of dichotomous response (positive or negative) using Bacillus subtilis BGA for the detection of sulfonamide residues. In the first stage, the bioassay response time was reduced to 6 h by increasing the spore concentration of B. subtilis. Then, the effects of spore, indicator, trimethoprim (TMP) concentration, and volume of the culture medium were examined with a Plackett Burman design (2<sup>4-1</sup>). Finally, the effect of TMP concentration on the method detection capabilities and specificity was analysed using a logistic model with interaction. The detection capabilities of sulfonamides in milk are close to the MRLs when using 500 mg/l of TMP in the culture medium of the bioassay. It is concluded that the experimental design techniques and a logistic regression model can be used to design successfully a dichotomous response bioassay.

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