Covalent immobilization of an alkaline protease from Bacillus licheniformis

Yakup Aslan,Derya Ömerosmanoğlu,Eda Öndül Koç

doi:10.1515/tjb-2017-0155

Yakup Aslan, Derya Ömerosmanoğlu + Show 1 more

Open Access

https://doi.org/10.1515/tjb-2017-0155

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Abstract

Abstract Objective Since the soluble enzymes can not be used in repeated reactions and are not stable in operational conditions and not suitable for continuous processes, this study aimed the covalent immobilization of Bacillus licheniformis protease (BLP) onto Eupergit CM. Methods Optimum conditions for immobilization were determined by changing the conditions individually. The proteins and L-tyrosine were determined by UV/VIS spectrophotometer. Results The immobilization resulted in 100% immobilization and 107.7% activity yields. The optimum pH (7–8) and the optimum temperature (70°C) have not changed after immobilization. The Km values for free and immobilized enzyme were 26.53 and 37.59 g/L, while the Vmax values were 2.84 and 3.31 g L-Tyrosine/L·min, respectively. The immobilized enzyme has not lost its initial activity during the repeated 20 uses and 20 days of storage. The milk proteins were hydrolyzed in 2 h by using immobilized enzyme. The pH of the milk dropped from 6.89 to 6.53, the color was clearer but there was no change in the smell or the taste. Conclusion Consequently, it can be said that the immobilized BLP obtained can be used for industrial purposes.

Highlights

Cow’s milk protein (CMP) allergy is the immunological reactions that occur in the body against cow’s milk proteins [1]
Considering that more than 138 million babies came into the world each year [3], the CMP allergy which is seen as a world widespread disease can be greatly reduced by consumption of protein-free cow’s milk
Data was analyzed by using Microsoft Windows Excell. bTwo hundred micro liter free Bacillus licheniformis protease (BLP) having 78.51 IU activity was reacted with different amounts of support in 5 mL of sodium phosphate buffer (0.5 M, pH 755) and room temperature with shaking at 0.25155 g in an incubator for 16 h. cTwo hundred micro liter free BLP having 78.51 IU activity or 0.286 g Eupergit CM carrying 84.06 IU BLP activity were reacted with 5 mL of 1% (w/v) casein solutions at 0.25155 g and at 70°C in an incubator for 60 min

Summary

Objective

Since the soluble enzymes can not be used in repeated reactions and are not stable in operational conditions and not suitable for continuous processes, this study aimed the covalent immobilization of Bacillus licheniformis protease (BLP) onto Eupergit CM. Methods: Optimum conditions for immobilization were determined by changing the conditions individually. The optimum pH (7–8) and the optimum temperature (70°C) have not changed after immobilization. The Km values for free and immobilized enzyme were 26.53 and 37.59 g/L, while the Vmax values were 2.84 and 3.31 g L-Tyrosine/L · min, respectively. The immobilized enzyme has not lost its initial activity during the repeated 20 uses and 20 days of storage. The milk proteins were hydrolyzed in 2 h by using immobilized enzyme. Amaç: İmmobilize enzimler, tekrarlanan reaksiyonlarda kullanılamadığı ve kullanım şartlarında kararlı olmadığı ve endüstriyel sürekli süreçler için uygun olmadığından, bu çalışma, BLP’nin Eupergit CM’ye kovalent immobilizasyonunu amaçlamıştır. Serbest ve immobilize enzim için Km değerleri sırasıyla 26.53 ve 37.59 g/L iken, Vmax değerleri sırasıyla 2.84 ve 3.31 g L-Tirozin/L · dk idi. Anahtar Kelimeler: Eupergit CM; Immobilizasyon; Inek sütü; Proteaz: Protein; Protein alerjisi

Introduction

Materials and methods

Immobilization procedure

Results