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Frontiers of Chemical Science and Engineering

ISSN 2095-0179

ISSN 2095-0187(Online)

CN 11-5981/TQ

Postal Subscription Code 80-969

2018 Impact Factor: 2.809

Front. Chem. Sci. Eng.    2015, Vol. 9 Issue (4) : 501-510    https://doi.org/10.1007/s11705-015-1544-0
RESEARCH ARTICLE
Enhanced production of β-glucuronidase from Penicillium purpurogenum Li-3 by optimizing fermentation and downstream processes
Shen Huang1,2,Xudong Feng1,*(),Chun Li1
1. School of Life Science, Beijing Institute of Technology, Beijing 100081, China
2. College of Food and Biological Engineering, Zhengzhou University of Light Industry, Henan 450002, China
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Abstract

β-Glucuronidase from Penicillium purpurogenum Li-3 (PGUS) can efficiently hydrolyze glycyrrhizin into the more valuable glycyrrhetic acid monoglucuronide. However, a low productivity of PGUS and the lack of an effective separation strategy have significantly limited its industrial applications. Therefore, the production of PGUS has been improved by optimizing both the fermentation and purification strategies. A two-stage fermentation strategy was developed where PGUS was first grown with glucose and then PGUS was produced in the presence of glycyrrhizin as an inducer. By using this strategy, the biomass was increased 1.5 times and the PGUS activity increased 5.4 times compared to that when glycyrrhizin was used as the sole carbon source. The amount of PGUS produced was increased another 16.6% when the fermentation was expanded to a 15-L fermenter. An effective protocol was also established to purify the PGUS using a sequential combination of hydrophobic, strong anion-exchange and gel filtration chromatography. This protocol had a recovery yield of 6% and gave PGUS that was 39 times purer than the crude PGUS. The purified PGUS had a specific activity of 350 U·mg−1.

Keywords β-glucuronidase      glycyrrhetic acid monoglucuronide      cell disruption      purification      chromatography     
Corresponding Author(s): Xudong Feng   
Online First Date: 20 November 2015    Issue Date: 26 November 2015
 Cite this article:   
Shen Huang,Chun Li,Xudong Feng. Enhanced production of β-glucuronidase from Penicillium purpurogenum Li-3 by optimizing fermentation and downstream processes[J]. Front. Chem. Sci. Eng., 2015, 9(4): 501-510.
 URL:  
https://academic.hep.com.cn/fcse/EN/10.1007/s11705-015-1544-0
https://academic.hep.com.cn/fcse/EN/Y2015/V9/I4/501
Fig.1  Scheme 1 Hydrolysis of GL into GAMG and glucuronic acid catalyzed by PGUS
Fig.2  Effect of glucose concentration on (a) dry cell weight and (b) enzyme activity. Results are from triplicate fermentation experiments and the error bars represent the average±one standard deviation
Fig.3  Fermentation performance of P. purpurogenum Li-3 in a 15-L fermenter. The initial glucose concentration was 2 g·L−1
Fig.4  Effect of pH on protein concentration and PGUS activity during the disruption of cells by HPH. Results are from independent triplicate measurements and error bars represent the average±one standard deviation
Fig.5  The optical images of P. purpurogenum Li-3 cells disrupted under different conditions: (a) before disruption; (b) disrupted once under pressure of 1500 bar; (c) disrupted once under pressure of 1800 bar; (d) disrupted twice under pressure of 1800 bar; (e) disrupted three times under pressure of 1800 bar
Fig.6  Effect of the number of HPH cell disruption breaking cycles. Results are from independent triplicate measurements and error bars represent the average±one standard deviation.
Fig.7  Effect of organic solvents on the activity of PGUS during precipitation. Results are from independent triplicate measurements and error bars represent the average±one standard deviation
Fig.8  Optimization of operational parameters for the precipitation of PGUS by acetone: (a) time; (b) volume ratio of acetone to crude enzyme. Results are from independent triplicate measurements and error bars represent the average±one standard deviation
Fig.9  Purification of PGUS by butyl sepharose hydrophobic interaction chromatography
Fig.10  SDS-PAGE analysis of purified PGUS. Lane M: marker; lane 1: crude enzyme; lane 2: PGUS purified by butyl sepharose hydrophobic interaction chromatography; lane 3: PGUS purified by anion exchange chromatography; lane 4: PGUS purified by gel filtration chromatography
Fig.11  Purification of PGUS by anion exchange chromatography
Fig.12  Purification of PGUS by gel filtration chromatography
Purification steps Total protein /mg Total activity /U Specific activity /U·mg−1 Recovery yield % Purification factor
Supernatant 266 2634 9 100 1
Acetone precipitation 25 2007 80 76 9
Hydrophobic interaction 5 967 188 38 21
Strong anion exchange 3 483 308 18 34
Gel filtration 0.4 140 350 6 39
Tab.1  PGUS yields and purification data
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