Application of Food-Grade Proteolytic Enzyme for the Hydrolysis of Regenerated Silk Fibroin fromBombyx mori

Ji An Joung,Mi Na Park,Joon Ho Choi,Ji Young You,Bong Joon Song

doi:10.1155/2018/1285823

Abstract

In vitrobiodegradation ofBombyx morisilk fibroin (SF) was studied using food-grade proteolytic enzymes to replace acid hydrolysis. Based on the residual protein quantity and yield of amino acids (AAs) after enzymatic hydrolysis, we evaluated the proteolytic enzyme process of SF. FoodPro and Alcalase that are classified as alkaline proteases are selected as two of the best candidate enzymes for hydrolysis of SF. The activity of these enzymes exhibits a broad range of pH (6.5 to 9.0) and temperature (50°C to 65°C). The single enzyme treatment of SF using FoodPro exhibited a hydrolytic efficiency of 20–25%, and >2 g/L AAs were released after reaction for 3 h. A 2-stage enzymatic treatment using a combination of FoodPro and Flavourzyme in a sequence for a reaction time of 6 h was developed to enhance the efficiency of the proteolytic process. The yield of AAs and residual protein quantity in the enzymatic hydrolysates obtained from FoodPro-treated regenerated SF in the 1st step was 2,040 ± 23.7 mg/L and 70.6%, respectively. The yield of AAs was > two-fold (4,519 ± 42.1 mg/L), whereas the residual protein quantity decreased to 55.1% after the Flavourzyme treatment (2nd step) compared to those of the single FoodPro treatment. In the mixed treatment by simultaneously using FoodPro and Flavourzyme, approximately 45% of SF was degraded and 4.5 g/L of AAs were released within 3 h of reaction time. The regenerated SF and its enzymatic hydrolysates were characterized by performing UV-visible spectra, gel electrophoresis, and size-exclusion chromatography analyses. In the 2-stage treatment using FoodPro initially and subsequently Flavourzyme, the aggregates and high molecular weight proteins of SF were dissociated and degraded into the low molecular weight proteins/peptides (10–15 kDa and 27 kDa). SF hydrolysates as functional food might be enzymatically produced using the commercial food-grade proteolytic enzymes.

Highlights

Ji An Joung, Mi Na Park, Ji Young You, Bong Joon Song, and Joon Ho Choi
A 2-stage enzymatic treatment using a combination of FoodPro and Flavourzyme in a sequence for a reaction time of 6 h was developed to enhance the efficiency of the proteolytic process. e yield of amino acids (AAs) and residual protein quantity in the enzymatic hydrolysates obtained from FoodPro-treated regenerated silk fibroin (SF) in the 1st step was 2,040 ± 23.7 mg/L and 70.6%, respectively. e yield of AAs was > two-fold (4,519 ± 42.1 mg/L), whereas the residual protein quantity decreased to 55.1% after the Flavourzyme treatment (2nd step) compared to those of the single FoodPro treatment
In the mixed treatment by simultaneously using FoodPro and Flavourzyme, approximately 45% of SF was degraded and 4.5 g/L of AAs were released within 3 h of reaction time. e regenerated SF and its enzymatic hydrolysates were characterized by performing UV-visible spectra, gel electrophoresis, and size-exclusion chromatography analyses

Summary

Introduction

Ji An Joung , Mi Na Park , Ji Young You , Bong Joon Song , and Joon Ho Choi. In vitro biodegradation of Bombyx mori silk fibroin (SF) was studied using food-grade proteolytic enzymes to replace acid hydrolysis. Based on the residual protein quantity and yield of amino acids (AAs) after enzymatic hydrolysis, we evaluated the proteolytic enzyme process of SF. E single enzyme treatment of SF using FoodPro exhibited a hydrolytic efficiency of 20–25%, and >2 g/L AAs were released after reaction for 3 h. A 2-stage enzymatic treatment using a combination of FoodPro and Flavourzyme in a sequence for a reaction time of 6 h was developed to enhance the efficiency of the proteolytic process. E yield of AAs and residual protein quantity in the enzymatic hydrolysates obtained from FoodPro-treated regenerated SF in the 1st step was 2,040 ± 23.7 mg/L and 70.6%, respectively. In the mixed treatment by simultaneously using FoodPro and Flavourzyme, approximately 45% of SF was degraded and 4.5 g/L of AAs were released within 3 h of reaction time. Silk is defined as a nondegradable material by the United States Pharmacopeia owing to a negligible loss in tensile strength in vivo, the enzymatic degradation behaviors of SF as biomaterials have been reviewed for the medical application in vivo [11, 12]

Methods

Results

Conclusion