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  - JOUR
A1  - Niehaus, F.
A1  - Gabor, E.
A1  - Wieland, S.
A1  - Siegert, Petra
A1  - Maurer, Karl-Heinz
A1  - Eck, J.
T1  - Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases
JF  - Microbial biotechnology
Y1  - 2011
SN  - 1432-0614 (E-Journal); 0171-1741 (Print); 0175-7598 (Print); 0340-2118 (Print)
VL  - Vol. 4
IS  - Iss. 6
SP  - 767
EP  - 776
PB  - Springer
CY  - Berlin
ER  -