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Liu Y.,Zhejiang University | Pan J.,Zhejiang University | Wei P.,Zhejiang University of Science and Technology | Zhu J.,Zhejiang University | And 4 more authors.
Biotechnology and Bioprocess Engineering | Year: 2012

In order to improve the production of alcohol oxidase (AOX), a recombinant Pichia pastoris (P. pastoris) system was constructed by transformation of the plasmid pPIC9K-AOX into P. pastoris GS115. The effects of different expression conditions on alcohol oxidase activity in the culture supernatant were investigated in the shake flask scale. The results showed that the highest extracellular activity (562 U/L) of alcohol oxidase was obtained after 56 h induction with 4% methanol at OD600 1.0 in the medium containing 50 g/L maltose, which is about 4.2 folds higher than previously reported. High-purity functional recombinant AOX (>90%) was purified from the culture with the Ni-NTA affinity column and Sephadex G-100 chromatographical methods, with a total recovery rate of 68.9%. Further studies showed that the purified rAOX had similar enzymatic characteristics as the native enzyme, except that the thermal stability and resistance to H2O2 inhibition of rAOX were significantly greater compared to the previous report. The purified rAOX was well tolerant to various water-miscible organic solvents. This efficient expression and purification process will be promising for large-scale production of rAOX as an important diagnostic enzyme for alcohol detection in many areas. © The Korean Society for Biotechnology and Bioengineering and Springer 2012. Source


Zhu F.,Zhejiang University | Cai J.,Zhejiang University | Zheng Q.,Zhejiang University | Zheng Q.,Zhejiang Deqing Huining Biotechnology Corporation Ltd | And 2 more authors.
Journal of Chemical Technology and Biotechnology | Year: 2014

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is an extracellular anionic polymer with various potential applications. Renewable and economical carbon sources as the major fermentation feedstock are in need for γ-PGA production. RESULTS: Xylose has been demonstrated to support cell growth and γ-PGA biosynthesis in Bacillus subtilis HB-1. Batch fermentation using xylose could produce about 23.63 g L-1 γ-PGA in the broth with a high production rate of 0.74 g L-1 h-1. Subsequent fed-batch fermentation could further improve the concentration of γ-PGA up to 28.15 g L-1 with a high conversion rate of 0.97 g g-1 (l-glutamate). Based on these preliminary results, the multiple-sugar containing substrate (corncob fibres hydrolysate) was chosen as the alternative complex carbon source to produce γ-PGA, and one high concentration (24.92 g L-1) of γ-PGA was attained. CONCLUSIONS: Our pilot trial results suggested that the low-cost and renewable lignocellulosic biomass can be used for the environmental friendly and efficient production of γ-PGA. © 2013 Society of Chemical Industry. Source


Zhang H.,Zhejiang University | Zhu J.,Zhejiang University | Zhu J.,Zhejiang Deqing Huining Biotechnology Corporation Ltd | Zhu X.,Zhejiang University | And 6 more authors.
Bioresource Technology | Year: 2012

A new exogenous glutamic acid-independent γ-PGA producing strain was isolated and characterized as Bacillus subtilis C10. The factors influencing the endogenous glutamic acid supply and the biosynthesis of γ-PGA in this strain were investigated. The results indicated that citric acid and oxalic acid showed the significant capability to support the overproduction of γ-PGA. This stimulated increase of γ-PGA biosynthesis by citric acid or oxalic acid was further proved in the 10. L fermentor. To understand the possible mechanism contributing to the improved γ-PGA production, the activities of four key intracellular enzymes were measured, and the possible carbon fluxes were proposed. The result indicated that the enhanced level of pyruvate dehydrogenase (PDH) activity caused by oxalic acid was important for glutamic acid synthesized de novo from glucose. Moreover, isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) were the positive regulators of glutamic acid biosynthesis, while 2-oxoglutarate dehydrogenase complex (ODHC) was the negative one. © 2012 Elsevier Ltd. Source


Zhu F.,Zhejiang University | Cai J.,Zhejiang University | Wu X.,Wenzhou University | Huang J.,Zhejiang University | And 6 more authors.
Journal of Biotechnology | Year: 2013

Poly-γ-glutamic acid (γ-PGA) is an extracellular anionic polymer with many potential applications. Although microbial fermentation is the common approach to produce γ-PGA, the broth at the latter stage usually becomes very viscous and severely influences the metabolites producing pattern of target microbe. In this study, acetoin and 2,3-butanediol were confirmed to be the main byproducts of γ-PGA-producing strain B. subtilis ZJU-7 (B. subtilis CGMCC1250), and their effects on the cell growth and γ-PGA biosynthesis were further investigated in shake flasks. The outcome indicated that both acetoin and 2,3-butanediol showed clear impairment on γ-PGA production of B. subtilis ZJU-7. Moreover, the extracellular metabolites profiles of fermentation under three different pH values were acquired and the metabolic flux redistribution of pathways related to γ-PGA biosynthesis was calculated based on the collected data. As a result, the metabolic flux favored to distribute toward glycolytic pathway at pH 6.5, in which the ingestion rate of extracellular glutamic acid was higher and the subsequent γ-PGA biosynthesis was enhanced. The present work provided us a deep insight into the metabolic flux control of γ-PGA biosynthesis, which will stimulate some novel metabolic engineering strategies to improve the productivity of γ-PGA in future. © 2012 Elsevier B.V. Source


Zhu F.,Zhejiang University | Lyu Z.-L.,Zhejiang University | Yang Y.-D.,Zhejiang Deqing Huining Biotechnology Corporation Ltd | Zhu J.-Z.,Zhejiang Deqing Huining Biotechnology Corporation Ltd | And 3 more authors.
Huaxue Gongcheng/Chemical Engineering (China) | Year: 2013

The separation and purification of poly-γ-glutamtic acid (γ-PGA) from broth is very difficult due to high viscosity. The operating parameters were optimized to improve the performance of PGA isolation process containing micro-filtration, ultrafiltration and ethanol precipitation. With regards to the three parameters (permeation flux, pressure and cell removal ratio), the micro-filtration operating conditions were optimized as follows: temperature 40°C, pressure differential 0.3-0.4 MPa, the volume ratio of dilution between broth and water 1:2. Under the above optimal conditions, 92.47% of cells were removed from γ-PGA broth. The optimal concentration rate of ultrafiltration was 5: 1, while the permeation flux was 31.8 L/(m2 · h) and 81.6% of γ-PGA was retained. The effects of different pH were examined and the optimal pH was determined to be 7 for efficient PGA precipitate. The present work promised the potential large-scale production of PGA in the future. Source

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