Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases.

Nikola Lončar,Nataša Božić,Zoran Vujčić,Marinela Šokarda Slavić

doi:10.1038/srep15772

Abstract

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L−1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.

Highlights

IntroductionEach data point represents the mean of three independent assays (the standard errors were less than 5% of the means)
In the case of raw starch digesting amylase (RSDA), hydrophobic interactions are a property of substrate binding and high recovery is expected from a mixed mode resin
When the dry cell weight (DCW) reached a value of 15 g L−1, the post-induction phase began and the glucose feed rate was kept constant at 15 mL h−1

Summary

Introduction

Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means). The arrow indicates the point of induction. A peculiarity of raw starch digesting enzymes is their adsorption on raw starch granules via a carbohydrate binding domain or by surface binding sites[10]. In a majority of cases, surface binding sites consist of exposed tyrosine and tryptophan residues on the surface of the enzyme (Fig. 1). Hydrophobic interaction chromatography is normally destructive towards the target protein and results in lower yields. In the case of raw starch digesting amylase (RSDA), hydrophobic interactions are a property of substrate binding and high recovery is expected from a mixed mode resin. The complete workflow of overproduction of RSDA in a laboratory fermenter and proposed DSP is described

Methods

Results

Conclusion