Optimization of protease immobilization by covalent binding using glutaraldehyde

Chae, Hee Jeong; In, Man-Jin; Kim, Eui Yong

doi:10.1007/bf02785655

Cited by 44 publications

(25 citation statements)

References 7 publications

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“…Glutaraldehyde can be used either to alter enzymes after immobilisation or to activate the support for enzyme immobilisation. In addition, the use of glutaraldehyde can increase protein stability, thus avoiding protease autolysis [2,12].…”

Section: Introductionmentioning

confidence: 99%

The immobilisation of proteases produced by SSF onto functionalized magnetic nanoparticles: Application in the hydrolysis of different protein sources

Yazid

Barrena

Sánchez

2016

Journal of Molecular Catalysis B: Enzymatic

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Alkaline proteases produced from protein-rich waste (hair waste and soya residues) by solid state fermentation (SSF) were immobilised onto functionalized magnetic iron oxide nanoparticles (MNPs) using glutaraldehyde as a crosslinking agent. The covalent binding method had a better immobilisation yield compared to simple adsorption, retaining 93%-96% (459±106 U/mg nanoparticles, 319±34 U/mg nanoparticles) of hair waste and soya residues proteases, respectively after crosslinking with 5% glutaraldehyde for 6 h. However, the adsorption immobilisation yield was 47%-54% after 8 h for both proteases. MNPs and immobilised proteases were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and electron diffraction. Our results indicated successful crosslinking between the proteases and amino-functionalized MNPs. The operational stability (pH and temperature) and storage stability of free and immobilised enzyme were also analysed. Despite the fact that the optimum pH of free and immobilised proteases was identical in the alkaline region, the immobilised proteases reached their optimum condition at higher temperatures (40ºC-60ºC 40 ºC-60 ºC). After 2 months of storage at 4ºC 4 ºC, the immobilised proteases showed good stability, retaining more than 85% of their initial activity. The high magnetic response of MNPs render an ease of separation and reusability, which contributes to the residual activity of both immobilised proteases on MNPs remained retaining more than 60% of their initial values after seven hydrolytic cycles. These results resulted showed the enhancement of the stability of the crosslinking interactions between the proteases and nanoparticles. The immobilised proteases were capable of hydrolysing select selected proteins (casein, oat bran protein isolate, and egg white albumin). However, differences in the degree of hydrolysis were observed, depending on the combination of the protease and type of substrate used.

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Section: Introductionmentioning

confidence: 99%

The immobilisation of proteases produced by SSF onto functionalized magnetic nanoparticles: Application in the hydrolysis of different protein sources

Yazid

Barrena

Sánchez

2016

Journal of Molecular Catalysis B: Enzymatic

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“…The bifunctional agents can enhance the efficiency of favourable materials, such as glutaraldehyde, cyanogen bromide, and carbodiimide which are essential to stimulate the covalent binding between enzyme molecules and NFs (Chae 1998). Among these agents, glutaraldehyde is one of the most preferred agent commonly used as intermolecular crosslinking in proteins or to modify adsorbed proteins on aminated supports (Hwang 2004).…”

Section: Discussionmentioning

confidence: 99%

IMMOBILIZATION OF Bacillus subtilis E6-5 PROTEASE AND COMMERCIAL PROTEASE IN NANOFIBRILS CONTAINING DIFFERENT AMINO ACIDS

Güler

Demirkan

Sevgi

2020

Trakya University Journal of Natural Sciences

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In this study, polyamide 6 polymer surfaces that have a high surface area were produced by electrospinning method with the participation of Glycine, Tyrosine and Glutamic acid amino acids, and lyophilized Bacillus subtilis E6-5 protease and commercial protease enzymes were immobilized on nanofibrils. Enzyme reusability were investigated. The immobilization efficiencies of the enzymes were approximately between 50-55 %. In studies with lyophilized Bacillus protease, glutaraldehyde activated PA6 nanofibrils and glutaraldehyde unactivated PA6 nanofibrils were found to be more immobilized in the presence of Glutamic acid. Although the lyophilized protease enzyme immobilized on non-glutaraldehyde activated and activated surfaces has been used 4 times, the best functional stability has been achieved with 2 times use. In pure PA6/Glutamic acid nanofibrils, the immobilization yield of the two times used enzymes was found to be 38 %. In glutaraldehyde-activated PA6 nanofibrils, the PA6/Glutamic acid nanofibril surfaces were found to have 65 % immobilization yield of the two repetitive used enzymes. The enzyme immobilization efficiency has been doubled by glutaraldehyde activation of the nanofibrils. In studies with commercial protease, the most functional stability was obtained for 3 repeated uses, although the enzyme was used 6 times on the non-glutaraldehyde activated nanofibril surfaces. The most successful immobilization was found in 58 % of PA6 nanofibrils. In glutaraldehyde-activated PA6 nanofibrils, the enzyme was found to be used 6 times, but the functional stability was maintained as much as 4 times of repeated use. Özet: Bu çalışmada, elektrospin yöntemiyle yüksek yüzey alanına sahip, glisin, tirozin ve glutamik asit aminoasitleri ile oluşturulmuş poliamid 6 polimer yüzeyler üretilmiş ve liyofilize Bacillus subtilis E6-5 proteaz ve ticari proteaz enzimleri nanofibriller üzerinde immobilize edilmiştir. Enzimlerin yeniden kullanılabilirliği araştırıldı. Enzimlerin immobilizasyon verimlilikleri yaklaşık olarak % 50-55 arasındaydı. Liyofilize Bacillus proteazı ile yapılan çalışmalarda glutaraldehitle aktifleştirilmiş PA6 nanolifler ve glutaraldehitle aktifleştirilmeyen PA6 nanoliflerde glutamik asit aminoasidi varlığında immobilizasyonun daha başarılı olduğu saptanmıştır. Glutaraldehit ile aktifleştirilmemiş ve aktifleştirilmiş yüzeylerde immobilize edilen liyofilize proteaz enziminin 4 kez kullanımı olmasına rağmen, en iyi işlevsel stabilite 2 kez kullanım ile elde edilmiştir. Saf PA6/glutamik asit nanoliflerinde iki tekrarlı kullanım sonucu enzimin immobilizasyon verimi % 38 olarak bulunmuştur. Glutaraldehitle aktifleştirilmiş PA6 nanoliflerde de PA6/glutamik asit nanolif yüzeyleri iki tekrarlı kullanım sonucu enzimin immobilizasyon verimi % 65 olarak bulunmuştur. Nanoliflerin glutaraldehitle aktifleştirmesi sonucu enzim immobilizasyon verimi iki kat arttırılmıştır. Ticari proteaz ile yapılan çalışmalarda ise glutaraldehitle aktifleştirilmemiş nanolif yüzeylerde enzimin 6 kez kullanımı olmasına rağmen en işlevsel ...

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“…The epoxy-activated supports are ideal matrixes to perform a simple immobilization of proteins and have been used to immobilize various commer-cial enzymes via multipoint covalent attachment 16 . It was reported that enzymes could be easily inactivated by a neighboring unbound activated group (aldehyde or epoxy) on the carrier in the process of immobilization 36 . There is minimal literature on the stabilization of enzymes by additives during immobilization 37 .…”

Section: Immobilization Of Cca In the Presence Of Inhibitorsmentioning

confidence: 99%

Effects of Inhibitors on the Catalysis and Immobilization of Cephalosporin C Acylase

Luo

Han

Chang

et al. 2018

Chem.Biochem.Eng.Q.

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Numerous compounds, including weak bases (e.g., glucosamine, ethylenediamine, and pyridine) and weak acids (e.g., bicarbonate, acetate, propionate, and butyrate), were found to inhibit the catalysis of cephalosporin C acylase (CCA), which is a recombinant enzyme expressed in E. coli. Additionally, the protective effect of the inhibitors on free and immobilized CCA against heat treatment was investigated. The inhibitors were added to increase recovery of the activity of the enzyme immobilized by covalent attachment to an epoxy support. The activities of immobilized CCA obtained in the presence of acetate or bicarbonate were 99.2±2.5 U g-1 and 94.1±3.0 U g-1 , respectively, which were 31.7 % and 25 % higher, respectively, than that of the control. In addition, the immobilized CCA exhibited improved thermostability. The half-life of immobilized CCA obtained in the presence of acetate or bicarbonate increased by 190 % and 120 %, respectively, compared to that of immobilized CCA obtained in the absence of an inhibitor.

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Optimization of protease immobilization by covalent binding using glutaraldehyde

Cited by 44 publications

References 7 publications

The immobilisation of proteases produced by SSF onto functionalized magnetic nanoparticles: Application in the hydrolysis of different protein sources

The immobilisation of proteases produced by SSF onto functionalized magnetic nanoparticles: Application in the hydrolysis of different protein sources

IMMOBILIZATION OF Bacillus subtilis E6-5 PROTEASE AND COMMERCIAL PROTEASE IN NANOFIBRILS CONTAINING DIFFERENT AMINO ACIDS

Effects of Inhibitors on the Catalysis and Immobilization of Cephalosporin C Acylase

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