Expression of soluble Saccharomyces cerevisiae alcohol dehydrogenase in Escherichia coli applicable to oxido-reduction bioconversions

Biologia ◽  
2014 ◽  
Vol 69 (6) ◽  
Author(s):  
Pavol Utekal ◽  
Csaba Tóth ◽  
Anikó Illésová ◽  
Pavol Koiš ◽  
Lucia Bocánová ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Alcohol Dehydrogenase ◽  
Kinetic Parameters ◽  
Expression System ◽  
Recombinant Yeast ◽  
Yeast Alcohol Dehydrogenase ◽  
Volatile Production ◽  
Alcohol Dehydrogenase 1 ◽  
Expression Strain

AbstractSix-carbon aldehydes and alcohols belong to flavours and fragrances with wide application in the food, feed, cosmetic, chemical and pharmaceutical sectors. In the present study, we prepared the expression system for production of recombinant yeast alcohol dehydrogenase 1 (YADH1) from Saccharomyces cerevisiae which is suitable also for catalysis of the interconversion of C-6 aldehydes and alcohols. We have demonstrated that an effective three-step strategy can overcome the insolubility problems during YADH1 production in Escherichia coli. We used trxB and gor deficient expression strain, decreased concentration of isopropyl β-D-1-thiogalactopyranoside and lowered temperature to 20°C during induction. Finally, kinetic parameters of recombinant YADH1 were determined and we concluded it is a promising enzyme also for the interconversion of C-6 alcohols/aldehydes in green note volatile production.

scholarly journals Overproduction and Biochemical Characterization of the Chryseobacterium meningosepticum BlaB Metallo-β-Lactamase

2002 ◽  
Vol 46 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Sandrine Vessillier ◽  
Jean-Denis Docquier ◽  
Sandrine Rival ◽  
Jean-Marie Frere ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ion Exchange ◽  
Kinetic Parameters ◽  
Culture Supernatant ◽  
Biochemical Characterization ◽  
Expression System ◽  
T7 Promoter ◽  
Hydrolysis Of

ABSTRACT The BlaB metallo-β-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k cat/Km ratios of >106 M−1 s−1) toward most penam and carbapenem compounds, with the exception of the 6-α-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-β-lactamase inhibitors, including β-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.

Different constructs for the expression of mammalian gamma-glutamyltransferase cDNAs in Escherichia coli and in Saccharomyces cerevisiae

1991 ◽  
Vol 37 (5) ◽  
pp. 662-666 ◽  
Author(s):  
C Angele ◽  
T Oster ◽  
A Visvikis ◽  
J M Michels ◽  
M Wellman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Clinical Chemistry ◽  
Rat Kidney ◽  
Expression System ◽  
Yeast Cells ◽  
Single Chain ◽  
Hep G2 ◽  
Gamma Glutamyltransferase ◽  
Activity Measurements

Abstract To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.

scholarly journals Expression of soluble cloned porcine pepsinogen A in Escherichia coli

1996 ◽  
Vol 315 (2) ◽  
pp. 443-446 ◽  
Author(s):  
Takuji TANAKA ◽  
Rickey Y. YADA
Keyword(s):  
Escherichia Coli ◽  
Ion Exchange ◽  
Fusion Protein ◽  
Kinetic Parameters ◽  
Expression System ◽  
Milk Clotting ◽  
Ph Dependency ◽  
Pepsinogen A ◽  
Proteolytic Activities ◽  
Intrinsic Length

A system for the production of soluble porcine pepsinogen A (EC 3.4.23.1) was developed by fusing the pepsinogen and thioredoxin genes and then expressing the fused product (Trx-PG) in Escherichia coli. The expressed fusion protein was purified using a combination of ion-exchange and hydrophobic chromatography. Trypsin digestion of the fusion protein yielded pepsinogen which was one residue longer than the intrinsic length. Acidification of either the fusion protein or pepsinogen (tryptic digestion of Trx-PG) yielded recombinant pepsin A (r-pepsin). When compared with commercial porcine pepsin A, r-pepsin had similar milk-clotting and proteolytic activities, kinetic parameters and pH dependency. The above results indicate that an expression system was developed which yielded fully active soluble pepsin(ogen) from Escherichia coli.

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