An integrated high-throughput strategy for rapid screening of poly(γ-glutamic acid)-producing bacteria

Abstract

Poly(γ-glutamic acid) (γ-PGA) is a promising biomaterial with a wide range of unique applications. To extensively screen γ-PGA-producing bacteria with high yield and different molecular weight, we developed an integrated high-throughput strategy. Firstly, γ-PGA-producing bacteria were selected in a primary screen plate containing a basic dye (neutral red) based on the concentric zone formed through the electrostatic interaction between the dye and the secreted acidic polymer γ-PGA. Then, the isolates were cultured in 50 ml tubes instead of 250 ml flasks. A good correlation of fermentation results in 50 ml tubes and 250 ml flasks was observed. Thirdly, the γ-PGA yield and weight-average molecular weight (M (w)) were simultaneously determined by spectrophotomic assay (UV assay) and neutral red plate assay. The results showed that the diameter of the concentric zone varied among isolates and was negatively correlated with the weight-average molecular weight of γ-PGA. The accuracy of the methods was comparable to that of high-performance liquid chromatography and gel permeation chromatography assay. Lastly, γ-PGA obtained from the target isolates was rapidly identified using thin layer chromatography assay. With this strategy, 13 bacteria with high yield and various molecular weights of γ-PGA from 500 obvious single colonies on the primary screen plate were obtained.

Improvement of Bacillus subtilis for poly‐γ‐glutamic acid production by genome shuffling

“…B. subtilis GXA‐28 strain from China Center for Type Culture Collection (CCTCC M 2012347) was used in our study (Zeng et al ., ). The seed medium is composed of glucose 10.0 g l −1 , yeast extract 5.0 g l −1 , L‐glutamate 5.0 g l −1 , KH 2 PO 4 0.5 g l −1 , K 2 HPO 4 ·3H 2 O 0.5 g l −1 and MgSO 4 ·7H 2 O 0.1 g l −1 .…”

“…The seed medium is composed of glucose 10.0 g l −1 , yeast extract 5.0 g l −1 , L‐glutamate 5.0 g l −1 , KH 2 PO 4 0.5 g l −1 , K 2 HPO 4 ·3H 2 O 0.5 g l −1 and MgSO 4 ·7H 2 O 0.1 g l −1 . 0.006% (w/v) neutral red (dye) and agar (1.5%, w/v) were added into seed medium to prepare screening plate (Zeng et al ., ). In fermentation medium, glucose, yeast extract and L‐glutamate were adjusted to 30.0, 2.5 and 30.0 g l −1 respectively.…”

“…γ‐PGA yield was measured by ultraviolet spectrophotometry (UV) or high efficiency liquid chromatography (HPLC) assay (Zeng et al ., ). The molecular weight of γ‐PGA was determined by gel permeation chromatography (Zeng et al ., ).…”

An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols

“…Therefore, an accurate and rapid HTS methodology was critical to facilitate the screening procedure (Aharoni et al 2006;Mastrobattista et al 2005). Many successful HTS methods for screening enzyme mutants have been developed based on the properties of these specific enzymatic processes (Goddard and Reymond 2004;He et al 2011;Olsen et al 2000;Trollope et al 2014;Zeng et al 2013;Zheng et al 2007). Two colorimetric assays have been developed for measuring HHDH activity by determining the released halide ions and protons in dehalogenation process (Schallmey et al 2013;Tang et al 2010).…”

Degradation performance of polyglutamic acid and its application of calcium supplement

“…γ‐PGA can be obtained by chemical synthesis, pheresis, or microbial fermentation . In comparison with the former 2 methods, the latter, microbial fermentation is more suitable for industrial production due to its properties of little by‐products and low cost . Nevertheless, the molecular weight of γ‐PGA produced by microbial fermentation is usually about 100 000 to 1 million .…”