Continuous production of restriction endonucleases: continuous two-stage cultivation with E. coli JM103; continuous cell disintegration and purification by affinity chromatography

1992 ◽  
Vol 38 (2) ◽  
Author(s):  
H.-D. Beer ◽  
H.-E. Maschke ◽  
K. Sch�gerl
Keyword(s):  
Affinity Chromatography ◽  
Continuous Production ◽  
Restriction Endonucleases ◽  
Two Stage ◽  
E Coli ◽  
Cell Disintegration

scholarly journals Continuous production of biohythane from hydrothermal liquefied cornstalk biomass via two-stage high-rate anaerobic reactors

2016 ◽  
Vol 9 (1) ◽  
Author(s):  
Bu-Chun Si ◽  
Jia-Ming Li ◽  
Zhang-Bing Zhu ◽  
Yuan-Hui Zhang ◽  
Jian-Wen Lu ◽  
...  
Keyword(s):  
Continuous Production ◽  
High Rate ◽  
Two Stage ◽  
Anaerobic Reactors

scholarly journals Heterologous Expression and Purification of ? Galactosidase Protein Using Affinity Chromatography

2013 ◽  
Vol 5 (3) ◽  
pp. 499-513
Author(s):  
M. Z. Alam ◽  
L. Ragionieri ◽  
M. A. S. Santos ◽  
A. Iqbal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Affinity Chromatography ◽  
Recombinant Dna ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Lacz Gene ◽  
Recombinant Dna Technology ◽  
E Coli ◽  
Elution Buffer

Enzymes and other protein purification using recombinant DNA technology have become popular due to scarcity of natural protein. Saccharomyces cerevisiae is a demanding host, since it facilitates protein expression by its relative simplicity, safe organisms, inexpensive and has many properties of eukaryotic expression system. As an alternative host we express E. coli lacZ gene with GST tag in Saccharomyces cerevisiae and successfully purified from soluble extracts. The concentration of soluble GST-? galactosidase protein was approximately 0.57 mg/ml of elution buffer yielded from 50 ml yeast cell culture. The ?-galactosidase protein from insoluble extract was low due to the increasing solubility of GST tag. Keywords: ?-galactosidase; Heterologous expression; GST tag; Affinity chromatography. © 2013 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi: http://dx.doi.org/10.3329/jsr.v5i3.13820 J. Sci. Res. 5 (3), 499-513 (2013)  

Enhanced bio-energy recovery in a two-stage hydrogen/methane fermentation process

2009 ◽  
Vol 59 (11) ◽  
pp. 2137-2143 ◽  
Author(s):  
M. J. Lee ◽  
J. H. Song ◽  
S. J. Hwang
Keyword(s):  
Hydrogen Production ◽  
Fermentation Process ◽  
Continuous Production ◽  
Strong Acid ◽  
Treatment Method ◽  
Engineering System ◽  
Production Rates ◽  
Two Stage ◽  
Methane Fermentation ◽  
Pre Treatment

A two-stage hydrogen/methane fermentation process has emerged as a feasible engineering system to recover bio-energy from wastewater. Hydrogen-producing bacteria (HPB) generate hydrogen from readily available carbohydrates, and organic acids produced during the hydrogen fermentation step can be degraded to generate methane in the following step. Three strong acids, HCl, H2SO4, and HNO3, were tested to determine the appropriate pre-treatment method for enhanced hydrogen production. The hydrogen production rates of 230, 290, and 20 L/kg-glucose/day was observed for the sludge treated with HCl, H2SO4, and HNO3, respectively, indicating that the acid pre-treatment using either HCl or H2SO4 resulted in a significant increase in hydrogen production. The fluorescent in situ hybridization method indicated that the acid pre-treatment selectively enriched HPB including Clostridium sp. of cluster I from inoculum sludge. After hydrogen fermentation was terminated, the sludge was introduced to a methane fermentation reactor. This experiment showed methane production rates of 100, 30, and 13 L/kg-glucose/day for the sludge pre-treated with HCl, H2SO4, and HNO3, respectively, implying that both sulfate and nitrate inhibited the activity of methane-producing bacteria. Consequently, the acid pre-treatment might be a feasible option to enhance biogas recovery in the two-stage fermentation process, and HCl was selected as the optimal strong acid for the enrichment of HPB and the continuous production of methane.

scholarly journals pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

2001 ◽  
Vol 67 (3) ◽  
pp. 1262-1267 ◽  
Author(s):  
Shuhei Fujimoto ◽  
Yasuyoshi Ike
Keyword(s):  
Affinity Chromatography ◽  
Protein Purification ◽  
Enterococcus Faecalis ◽  
Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Chromatography Method ◽  
Shuttle Vectors ◽  
E Coli ◽  
One Step

ABSTRACT Two novel Enterococcus faecalis-Escherichia colishuttle vectors that utilize the promoter and ribosome binding site ofbacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli andE. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coliand E. faecalis at levels of 103 and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.

Continuous hydrogen and methane production in a two-stage cheese whey fermentation system

2011 ◽  
Vol 64 (2) ◽  
pp. 367-374 ◽  
Author(s):  
C. B. Cota-Navarro ◽  
J. Carrillo-Reyes ◽  
G. Davila-Vazquez ◽  
F. Alatriste-Mondragón ◽  
E. Razo-Flores
Keyword(s):  
Methane Production ◽  
Cheese Whey ◽  
Continuous Production ◽  
Uasb Reactor ◽  
Organic Loading Rate ◽  
Stable Operation ◽  
Operational Parameters ◽  
Two Stage ◽  
Production Of Hydrogen ◽  
Stage Process

The feasibility of integrating biological hydrogen and methane production in a two-stage process using mixed cultures and cheese whey powder (CWP) as substrate was studied. The effect of operational parameters such as hydraulic retention time (HRT) and organic loading rate (OLR) on the volumetric hydrogen (VHPR) and methane (VMPR) production rates was assessed. The highest VHPR was 28 L H2/L/d, obtained during stable operation in a CSTR at HRT and OLR of 6 h and 142 g lactose/L/d, respectively. Moreover, hydrogen (13 L/L/d) was produced even at HRT as low as 3.5 h and OLR of 163 g lactose/L/d, nonetheless, the reactor operation was not stable. Regarding methane production in an UASB reactor, the acidified effluent from the hydrogen-producing bioreactor was efficiently treated obtaining COD removals above 90% at OLR and HRT of 20 g COD/L/d and 6 h, respectively. The two-stage process for continuous production of hydrogen and methane recovered over 70% of the energy present in the substrate. This study demonstrated that hydrogen production can be efficiently coupled to methane production in a two-stage system and that CWP is an adequate substrate for energy production.

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