A new and efficient method for purification of poly-γ- glutamic acid from high-viscosity fermentation broth

Fengqing Wang,Wei Xiao,Wei Wang,Jinzhong Liang,Dawei Fu

doi:10.4314/tjpr.v16i6.9

Abstract

Purpose: To devise an efficient strategy for the separation and recovery of high-quality γ-PGA by investigation of the physical properties, pigment properties and microfiltration mode of high-viscosity fermentation broth. Methods: The bacterial strain, Bacillus subtilis 115, was used in this study. The viscosity of the fermentation broth was determined by digital viscometer with spindle SP-2 at 25 o C. The concentrations of glucose and L-glutamate were analyzed with a biosensor equipped with both glucose oxidase and Lglutamate oxidase electrodes. The pigment in the fermentation liquid was scanned with a UV spectrophotometer at wavelength range of 200 - 500 nm and was removed using activated carbon. Measurement of IR spectrum was performed using an IR spectrophotometer with KBr pellet. Results: The results showed that the γ-PGA yield was 35 g/L. The viscosity of the fermentation broth was 1600 mPa.s at the end of the batch fermentation. After 3-fold dilution, the viscosity was reduced to one-fortieth of the original value at 65 °C for 30 min., which allowed effective removal of Bacillus subtilis 115 from the broth. Maximum UV absorption of the pigment was occurred at 260 nm. The pigment was removed by shaking with 0.6 % activated carbon powder at 50 rpm for 20 min, resulting in 88 % decolorization. Concentration with hollow-fiber membrane (MWCO 500,000) resulted in complete removal of residual glucose and glutamic acid from the aqueous solution of γ-PGA. The molecular weight of the γ-PGA was 1095 kDa, and its UV scanning spectrum showed an absorption peak at 216 nm. The decomposition temperature (Td) of the γ-PGA was 312.92 o C. Its IR spectrum was consistent with the presence of carboxyl, hydroxyl, carbonyl and amide groups. Conclusion: An efficient method for the extraction and purification of high-quality γ-PGA from highviscosity fermentation broth. Keywords: Bacillus subtilis 115, γ-Polyglutamic acid, De-pigmentation, Activated carbon, Ultra-filtration, High-viscosity fermentation broth

Highlights

Γ - Polyglutamic acid (γ - PGA), a homo polyamide of D- and L-glutamic acid units, was first discovered as a component of capsules of Bacillus anthracis by Ivonovics and Bruckner [1]
The portion of activated carbon granules retained on the screen was washed three times with excess volume of 70 % aqueous ethanol to remove any powdered carbon adhering to the surface of the carbon granules
PH played an important role in the production of γ - PGA by Bacillus subtilis 115, results from the experiments in this study indicated that strict control of pH was not necessary

Summary

Introduction

Γ - Polyglutamic acid (γ - PGA), a homo polyamide of D- and L-glutamic acid units, was first discovered as a component of capsules of Bacillus anthracis by Ivonovics and Bruckner [1]. The polymer is environmentally friendly, nontoxic and non immunogenic, and can be used safely in a variety of rapidly increasing applications Because of these potential applications, the development of processes for improved production and recovery of γ-PGA from the fermentation broth is great importance. The negative surface charges confer high stability on γ - PGA - encapsulated cells in the culture broth. The stability and high viscosity of the broth create difficulties in sedimentation of the cells during the separation process [2]. Saccharification of corn starch was catalyzed by the double-enzyme method In this process, a 1:3 (w:v) mixture of corn powder and water (in a tank) was liquefied at 85 oC for 30 min at pH 6.7 7.0 using commercial thermo - stable α amylase (20 KU/g). Complete starch hydrolysis required more than 7 h of pre saccharification

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