scholarly journals The Stability Improvement of α-Amylase Enzyme from Aspergillus fumigatus by Immobilization on a Bentonite Matrix

2022 ◽  
Vol 2022 ◽  
pp. 1-7
Author(s):  
Yandri Yandri ◽  
Ezra Rheinsky Tiarsa ◽  
Tati Suhartati ◽  
Heri Satria ◽  
Bambang Irawan ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Half Life ◽  
Thermal Inactivation ◽  
Immobilized Enzyme ◽  
Residual Activity ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Optimum Temperature ◽  
The Stability

The stability of the α-amylase enzyme has been improved from Aspergillus fumigatus using the immobilization method on a bentonite matrix. Therefore, this study aims to obtain the higher stability of α-amylase enzyme from A. fumigatus; hence, it is used repeatedly to reduce industrial costs. The procedures involved enzyme production, isolation, partial purification, immobilization, and characterization. Furthermore, the soluble enzyme was immobilized using 0.1 M phosphate buffer of pH 7.5 on a bentonite matrix, after which it was characterized with the following parameters such as optimum temperature, Michaelis constant (KM), maximum velocity V max , thermal inactivation rate constant (ki), half-life (t1/2), and the change of energy due to denaturation (ΔGi). The results showed that the soluble enzyme has an optimum temperature of 55°C, KM of 3.04 mg mL−1 substrate, V max of 10.90 μmole mL−1 min−1, ki of 0.0171 min−1, t1/2 of 40.53 min, and ΔGi of 104.47 kJ mole−1, while the immobilized enzyme has an optimum temperature of 70°C, KM of 8.31 mg mL−1 substrate, V max of 1.44 μmole mL−1 min−1, ki of 0.0060 min−1, t1/2 of 115.50 min, and ΔGi of 107.37 kJ mole−1. Considering the results, the immobilized enzyme retained 42% of its residual activity after six reuse cycles. Additionally, the stability improvement of the α-amylase enzyme by immobilization on a bentonite matrix, based on the increase in half-life, was three times greater than the soluble enzyme.

scholarly journals Increasing Stability of a-amylase Obtained from Bacillus subtilis ITBCCB148 by Immobilization with Chitosan

2020 ◽  
Vol 10 (2) ◽  
pp. 155-161
Author(s):  
Yandri Yandri ◽  
Tati Suhartati ◽  
Heri Satria ◽  
Arum Widyasmara ◽  
Sutopo Hadi
Keyword(s):  
Thermal Stability ◽  
Bacillus Subtilis ◽  
Half Life ◽  
Thermodynamic Data ◽  
Immobilized Enzyme ◽  
Residual Activity ◽  
Optimum Temperature ◽  
Chitosan Matrix ◽  
T Values ◽  
Thermal Stability Of

In this research, the immobilization of α-amylase from Bacillus subtilis ITBCCB148 by crosslinking method on chitosan matrix has been performed. This research aims to know the effect of immobilization on the thermal stability of α-amylase. The results showed that the native α-amylase has an optimum temperature of  65oC, KM = 1.6 mg mL-1 substrate, and Vmax = 39.7 µmol mL-1 min-1. The immobilized α-amylase has optimum temperature of 75oC, KM = 3.5 mg mL-1 substrate, and Vmax = 7.05 µmol mL-1 min-1. The residual activity of the native and immobilized enzyme on thermal stability test at 65oC for 80 minutes was 58% and 86.15%, respectively. The immobilized enzyme can be reused up to six repeated cycles.The thermodynamic data of native enzyme was t½ = 113.6 min, ki = 6.1x10-3 min-1, and ΔGi = 107.3 kJ mol-1, while the immobilized enzyme was t½ = 433.1 min, ki= 1.6x10-3 min-1, and ΔGi 111.1 kJ mol-1. Based on the decrease of ki, and the increase of ΔGi and half-life(t½) values, the immobilization of α-amylase with chitosan can increase the thermal stability of this enzyme.

Steroid Δ4-Reductases of Pig Liver: Partial Purification of Testosterone 5β-Reductase

1971 ◽  
Vol 49 (3) ◽  
pp. 385-392 ◽  
Author(s):  
J. C. Nduaguba ◽  
A. F. Clark
Keyword(s):  
Enzyme Activity ◽  
Soluble Fraction ◽  
Reductase Activity ◽  
Maximum Velocity ◽  
Supernatant Fraction ◽  
Partial Purification ◽  
Pig Liver ◽  
Total Enzyme Activity ◽  
Distribution Studies ◽  
The Stability

Intracellular distribution studies of steroid Δ4-reductase activity in female pig liver were done using testosterone as substrate. About 60% of the total enzyme activity was found in the 100 000 × g soluble fraction. Labelled 17β-hydroxy-5β-androstan-3-one but not 17β-hydroxy-5α-androstan-3-one was isolated from the incubation of 1,2-3H-testosterone with the 100 000 × g supernatant fraction, indicating the presence of 5β-reductase activity. 5β-Reduction may play an important role in the inactivation of some Δ4-3-ketosteroids in the pig liver.Evidence that 5β-reductases differing in substrate specificity are present in the soluble fraction includes (a) variation in the ratios of enzyme activities for several Δ4-3-ketosteroids in different (NH4)2SO4 fractions obtained from the 100 000 × g soluble fraction, (b) kinetic data showing that the maximum velocity for an equimolar mixture of testosterone and hydrocortisone is the sum of the maximum velocities for the substrates when used singly, and (c) separation of the enzyme activity specific for testosterone from that specific for hydrocortisone by use of Sephadex G-100 and hydroxylapatite chromatography.Utilizing (NH4)2SO4 precipitation and chromatography on Sephadex G-100 and hydroxylapatite, a 105-fold purification of testosterone Δ4-5β-reductase from the 100 000 × g supernatant fraction has been attained. The presence of 5 mM β-mercaptoethanolamine increased the stability of the enzyme.

scholarly journals PEMANFAATAN ZEOLIT ALAM SEBAGAI MEDIA PENDUKUNG AMOBILISASI ENZIM α-AMILASE

2015 ◽  
Vol 5 (1) ◽  
pp. 79
Author(s):  
Upita Septiani ◽  
Agrina Lisma
Keyword(s):  
Surface Morphology ◽  
Unit Activity ◽  
Incubation Time ◽  
Natural Zeolite ◽  
Immobilized Enzyme ◽  
Natural Zeolites ◽  
Optimum Temperature ◽  
Optimal Activity ◽  
Immobilization Enzyme ◽  
The Stability

 ABSTRACT The utilization of natural zeolite as supporting media of α-amylase enzyme has been done. Natural zeolite which is activated with 3M HCl can remove impurities in the surface natural zeolite, uncover and widen the pores of the zeolite and activate functional groups to interact with α-amylase enzyme in the process of immobilization enzyme process. The mass of activated natural zeolite which is used as a material immobilized to get the optimal activity of α-amylase enzyme was 0.3 gram. Based on the measurement result of optimization of α-amylase enzyme were obtained optimum temperature of 35 oC, pH 5.6 and incubation time of 35 minutes with 0.04845 units/mL of the unit activity. And for α-amylase immobilized enzyme will be stable at the optimum temperature of 50 oC, pH 5.6 and incubation time of 45 minutes with 0.030 units/mL of the unit activity. SEM-EDX pattern shows the differences in surface morphology between natural zeolite and activated natural zeolites which contain α-amylase enzyme. A mobilization technique can increase the stability utilized in a α-amylase enzyme. Keywords : Zeolite, amobilization, α-amylase enzyme

scholarly journals An accomplished procedure of horseradish peroxidase immobilization for removal of acid yellow 11 in aqueous solutions

2020 ◽  
Vol 81 (12) ◽  
pp. 2664-2673 ◽  
Author(s):  
Melda Altikatoglu Yapaoz ◽  
Azade Attar
Keyword(s):  
Horseradish Peroxidase ◽  
Storage Stability ◽  
Dye Degradation ◽  
Residual Activity ◽  
Immobilized Enzymes ◽  
The Novel ◽  
Optimum Temperature ◽  
Short Term ◽  
Waste Effluents ◽  
The Stability

Abstract Horseradish peroxidase (HRP) characteristics were improved by two techniques, Na-alginate entrapment and glutaraldehyde crosslinking prior to alginate entrapment, in order to enhance the stability, functionality and removal of dyes in waste water. Free, entrapped and crosslinked-entrapped enzymes were compared by activity assays, which indicated the optimum temperature is 25 °C and pH 4.0–5.0. Kinetics results showed that alginate entrapment and crosslinking prior to entrapment increased Vmax and did not cause any significant decrease in Km. The thermal resistance of the free enzyme was short-term, zero residual activity after 250 min, while the immobilized enzymes preserved more than 50% of their activity for 5 h at 60 °C. Immobilized HRP was resistant to methanol, ethanol, DMSO and THF. The storage stability of free HRP ended in 35 days whereas entrapped and crosslinked-entrapped HRPs had 87 and 92% residual activity at the 60th day, respectively. HRP was used in the decolorization of azo dye Acid yellow 11 and total decolorization (>99%) was obtained using crosslinked-entrapped HRP. Reusability studies presented the improvement that crosslinked-entrapped HRP reached 74% decolorization after 10 batches. The results demonstrated that the novel immobilized HRP can be used as an effective catalyst for dye degradation of industrial waste effluents.

scholarly journals Immobilization of the Peroxygenase from Agrocybe aegerita. The Effect of the Immobilization pH on the Features of an Ionically Exchanged Dimeric Peroxygenase

Catalysts ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 560
Author(s):  
Diego Carballares ◽  
Roberto Morellon-Sterling ◽  
Xiaomin Xu ◽  
Frank Hollmann ◽  
Roberto Fernandez-Lafuente
Keyword(s):  
Anion Exchange ◽  
Immobilized Enzyme ◽  
Residual Activity ◽  
Free Enzyme ◽  
The Other ◽  
Vinyl Sulfone ◽  
Agrocybe Aegerita ◽  
Inactive Form ◽  
The Stability

This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47 °C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance.

scholarly journals Immobilization of α - amylase from locale bacteria isolate Bacillus subtilis ITBCCB148 with diethylaminoethyl cellulose (DEAE-Cellulose)

2010 ◽  
Vol 7 (1) ◽  
pp. 123-128 ◽  
Author(s):  
Yandri. Yandri ◽  
Tati Suhartati ◽  
Sutopo Hadipo Hadi
Keyword(s):  
Thermal Stability ◽  
Bacillus Subtilis ◽  
Immobilized Enzyme ◽  
Storage Temperature ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Ionic Exchange ◽  
Optimum Temperature ◽  
Diethylaminoethyl Cellulose ◽  
Exchange Matrix

The thermal stability increase of a-amylase obtained from locale bacteria isolate Bacillus subtilis ITBCCB148 was achieved by immobilization process using an ionic exchange matrix of DEAE-Cellulose. The result showed that the immobilized enzyme has an optimum temperature of 60°C; KM 14.8 mL substrate and Vmax 42.4 U/mL. The thermal stability storage temperature of 60°C, pH 9.0 and 60 minutes demonstrated the immobilized enzyme has residual activity of 28.1%; ki = 0.0224 min.-1; and ΔGi = 103.7 kJ mol-1. Although the immobilized enzyme’s thermal stability was only increased 1.5 times, at higher temperatures, it was much more stable than the native enzyme.

Peningkatan Stabilitas Termal Enzim α-Amilase dari Bacillus subtilis ITBCCB148 dengan Amobilisasi Menggunakan Zeolit

2019 ◽  
Vol 2 (2) ◽  
Author(s):  
Yandri ◽  
Fathaniah Sejati ◽  
Tati Suhartati
Keyword(s):  
Thermal Stability ◽  
Bacillus Subtilis ◽  
Immobilized Enzyme ◽  
Specific Activity ◽  
Residual Activity ◽  
Stability Test ◽  
Optimum Temperature ◽  
Before And After ◽  
Cation Exchange Column ◽  
Thermal Stability Of

The objective of the research is to increase the thermal stability of -amylase from Bacillus subtilis ITBCCB148 by immobilization using zeolite. For that reason, firstly we need to produce, isolate, and purify the enzyme. The purification of the enzyme was conducted by the following steps: fractionation with ammonium sulphate, dialysis, and CM-cellulose cation exchange column chromatography. The purified enzyme was immobilized using zeolite. The success in immobilization of the enzyme was evaluated by comparing the thermal stability of the enzyme before and after immobilization. Activity of α-amylase was determined by the Mandels and Fuwa method. The protein content was determined based on the method by Lowry. The results showed that the specific activity of purified enzyme was 2473.7 U / mg, increased 19 times compared to crude extract of enzyme having specific activity of 1285.9 U / mg. The purified enzyme has the optimum temperature at 65ºC, while the immobilized enzyme has the optimum temperature at 75ºC. The thermal stability test of the purified enzyme at 65ºC for 100 minutes showed the purified enzyme having residual activity of 20%; t 1 / 2 = 30 min, k i = 0.023 min -1 and ΔGi = 103.65 kJ mol -1 . The thermal stability test of the immobilized enzyme at 65ºC for 100 minutes showed that the immobilized enzyme had residual activity of 40%; t 1/ 2 = 49 min, k i = 0.014 min -1 and ΔGi = 105.03 kJ mol -1 . Immobilization using zeolite has succeeded in increasing the thermal stability of enzyme by 1.64 times compared to the purified enzyme, which is indicated by the decreasing of k i value, the increase of half-life and denaturation energy change (ΔGi).

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

scholarly journals Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Erin N Asleson ◽  
Dennis M Livingston
Keyword(s):  
Half Life ◽  
Translational Regulation ◽  
Cellular Level ◽  
Missense Mutations ◽  
Wild Type ◽  
Mutant Proteins ◽  
Gal1 Promoter ◽  
Rad52 Protein ◽  
The Stability ◽  
Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

scholarly journals Entrapment of the Fastest Known Carbonic Anhydrase with Biomimetic Silica and Its Application for CO2 Sequestration

Polymers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 2452
Author(s):  
Chia-Jung Hsieh ◽  
Ju-Chuan Cheng ◽  
Chia-Jung Hu ◽  
Chi-Yang Yu
Keyword(s):  
Carbonic Anhydrase ◽  
High Activity ◽  
Co2 Sequestration ◽  
Residual Activity ◽  
Entrapment Efficiency ◽  
Free Form ◽  
Uncatalyzed Reaction ◽  
The Stability ◽  
Time Required

Capturing and storing CO2 is of prime importance. The rate of CO2 sequestration is often limited by the hydration of CO2, which can be greatly accelerated by using carbonic anhydrase (CA, EC 4.2.1.1) as a catalyst. In order to improve the stability and reusability of CA, a silica-condensing peptide (R5) was fused with the fastest known CA from Sulfurihydrogenibium azorense (SazCA) to form R5-SazCA; the fusion protein successfully performed in vitro silicification. The entrapment efficiency reached 100% and the silicified form (R5-SazCA-SP) showed a high activity recovery of 91%. The residual activity of R5-SazCA-SP was two-fold higher than that of the free form when stored at 25 °C for 35 days; R5-SazCA-SP still retained 86% of its activity after 10 cycles of reuse. Comparing with an uncatalyzed reaction, the time required for the onset of CaCO3 formation was shortened by 43% and 33% with the addition of R5-SazCA and R5-SazCA-SP, respectively. R5-SazCA-SP shows great potential as a robust and efficient biocatalyst for CO2 sequestration because of its high activity, high stability, and reusability.

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