TY  - JOUR
A1  - Ribitsch, D.
A1  - Karl, W.
A1  - Birner-Gruenberger, R.
A1  - Gruber, K.
A1  - Eiteljoerg, I.
A1  - Remler, P.
A1  - Wieland, S.
A1  - Siegert, Petra
A1  - Maurer, Karl-Heinz
A1  - Schwab, H.
T1  - C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli
JF  - Journal of biotechnology
N2  - Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477.

This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in KM = 0.23 ± 0.01 mM and kcat = 167.5 ± 3.6 s⁻¹. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.
Y1  - 2010
U6  - http://dx.doi.org/10.1016/j.jbiotec.2010.09.947
SN  - 1873-4863 (E-Journal); 0168-1656 (Print)
VL  - 150
IS  - 3
SP  - 408
EP  - 416
PB  - Elsevier
CY  - Amsterdam
ER  - 
TY  - CHAP
A1  - Wendorff, Marion
A1  - Eggert, Thorsten
A1  - Pohl, Martina
A1  - Dresen, Carola
A1  - Müller, Michael
A1  - Jaeger, Karl-Erich
A1  - Sprenger, Georg A.
A1  - Schürmann, Melanie
A1  - Schürmann, Martin
A1  - Johnen, Sandra
A1  - Sprenger, Gerda
A1  - Sahm, Hermann
A1  - Inoue, Tomoyuki
A1  - Schörken, Ulrich
A1  - Breittaupt, Holger
A1  - Frölich, Bettina
A1  - Heim, Petra
A1  - Iding, Hans
A1  - Juchem, Bettina
A1  - Siegert, Petra
A1  - Kula, Maria-Regina
A1  - Weckbecker, Andrea
A1  - Hummel, Werner
A1  - Fessner, Wolf-Dieter
A1  - Elling, Lothar
A1  - Wolberg, Michael
A1  - Bode, Silke
A1  - Feldmann, Ralf
A1  - Geilenkirchen, Petra
A1  - Schubert, Thomas
A1  - Walter, Lydia
A1  - Dünnwald, Thomas
A1  - Demir, Ayhan S.
A1  - Kolter-Jung, Doris
A1  - Nitsche, Adam
A1  - Dünkelmann, Pascal
A1  - Cosp, Annabel
A1  - Lingen, Bettina
T1  - Catalytic asymmetric synthesis : section 2.2
T2  - Asymmetric synthesis with chemical and biological methods / ed. by Dieter Enders ...
Y1  - 2007
SN  - 978-3-527-31473-7
SP  - 298
EP  - 413
PB  - Wiley-VCH
CY  - Weinheim
ER  - 
TY  - JOUR
A1  - Siegert, Petra
A1  - McLeish, Michael J.
A1  - Baumann, Martin
A1  - Iding, Hans
A1  - Kneen, Malea M.
A1  - Kenyon, George L.
A1  - Pohl, Martina
T1  - Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida
JF  - Protein engineering, design, and selection : peds
Y1  - 2005
SN  - 1460-213X (E-Journal); 1741-0134 (E-Journal); 0269-2139 (Print); 1741-0126 (Print)
VL  - Vol. 18
IS  - Iss. 7
SP  - 345
EP  - 357
ER  - 
TY  - CHAP
A1  - Siegert, Petra
A1  - Pohl, Martina
A1  - Kneen, Malea M.
A1  - Pogozheva, Irina D.
A1  - Kenyon, George L.
A1  - McLeish, Michael J.
T1  - Exploring the substrate specificity of benzoylformate decarboxylase, pyruvate decarboxylase, and benzaldehyde lyase
T2  - Thiamine : catalytic mechanisms in normal and disease states / ed. by Frank Jordan ...
Y1  - 2004
SN  - 0-8247-4062-9
SP  - 275
EP  - 290
PB  - Dekker
CY  - New York, NY
ER  - 
TY  - JOUR
A1  - Dünkelmann, Pascal
A1  - Kolter-Jung, Doris
A1  - Nitsche, Adam
A1  - Demir, Ayhan S.
A1  - Siegert, Petra
A1  - Lingen, Bettina
A1  - Baumann, Martin
A1  - Pohl, Martina
A1  - Müller, Michael
T1  - Development of a donor-acceptor concept for enzymatic cross-coupling reactions of adehydes : the first asymmetric cross-benzoin condensation
JF  - Journal of the American Chemical Society
Y1  - 2002
SN  - 1520-5126 (E-Journal); 0002-7863 (Print)
VL  - Vol. 124
SP  - 12084
EP  - 12085
ER  - 
TY  - JOUR
A1  - Dünnwald, Thomas
A1  - Demir, Ayhan S.
A1  - Siegert, Petra
A1  - Pohl, Martina
A1  - Müller, Michael
T1  - ChemInform Abstract: Enantioselective synthesis of (S)-2-Hydroxypropanone derivatives by Benzoylformate Decarboxylase Catalyzed C—C Bond Formation
JF  - Cheminform
Y1  - 2001
SN  - 1522-2667 (E-Journal); 0931-7597 (Print)
VL  - Vol. 32
IS  - Iss. 4
SP  - Publ. online
ER  - 
TY  - JOUR
A1  - Dünnwald, Thomas
A1  - Demir, Ayhan S.
A1  - Siegert, Petra
A1  - Pohl, Martina
A1  - Müller, Michael
T1  - Enantioselective Synthesis of (S)-2-Hydroxypropanone Derivatives by Benzoylformate Decarboxylase Catalyzed C−C Bond Formation
JF  - European journal of organic chemistry
Y1  - 2000
SN  - 0365-5490 (E-Journal); 1099-0690 (E-Journal); 0075-4617 (Print); 0170-2041 (Print); 0947-3440 (Print); 1434-193X (Print); 1434-243X (Print)
VL  - Vol. 2000
IS  - Iss. 11
SP  - 2161
EP  - 2170
ER  - 
TY  - CHAP
A1  - Siegert, Petra
A1  - Iding, Hans
A1  - Baumann, Martin
A1  - McLeish, Michael J.
A1  - Kenyon, George L.
A1  - Pohl, Martina
T1  - Broadening of the substrate spectra of two ThDP-dependent decarboxylases using site-directed-mutagenesis
T2  - Proceedings of the 4th International Congress on Biochemical Engineering : 17 and 18 February 2000, Stuttgart
Y1  - 2000
SN  - 3-8167-5570-4
SP  - 38
EP  - 42
ER  - 
TY  - JOUR
A1  - Iding, Hans
A1  - Dünnwald, Thomas
A1  - Greiner, Lasse
A1  - Liese, Andreas
A1  - Müller, Michael
A1  - Siegert, Petra
A1  - Grötzinger, Joachim
A1  - Demir, Ayhan S.
A1  - Pohl, Martina
T1  - Benzoylformate Decarboxylase from Pseudomonas putida as Stable Catalyst for the Synthesis of Chiral 2-Hydroxy Ketones
JF  - Chemistry - a European journal
Y1  - 2000
SN  - 1521-3765 (E-Journal); 0947-6539 (Print)
VL  - Vol. 6
IS  - Iss. 8
SP  - 1483
EP  - 1495
ER  - 
TY  - JOUR
A1  - Iding, Hans
A1  - Siegert, Petra
A1  - Mesch, K.
A1  - Pohl, Martina
T1  - Application of α-keto acid decarboxylases in biotransformations
JF  - Biochimica et biophysica acta (BBA) - Protein structure and molecular enzymology
Y1  - 1998
SN  - 1879-2588 (E-Journal); 0167-4838 (Print)
VL  - Vol. 1385
IS  - Iss. 2
SP  - 307
EP  - 322
ER  -