C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli.
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 K(M)=0.23 ± 0.01 mM and k(cat)=167.5 ± 3.6s(-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.
Ribitsch
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D
ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria. doris.ribitsch@acib.at
Karl
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W
Birner-Gruenberger
R
R
Gruber
K
K
Eiteljoerg
I
I
Remler
P
P
Wieland
S
S
Siegert
P
P
Maurer
K H
KH
Schwab
H
H
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003902:Detergents / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D057057:Serine Proteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020615:Stenotrophomonas maltophilia; D013696:Temperature; D016959:Xanthomonas campestris
D003902:Detergents; D011994:Recombinant Proteins; D057057:Serine Proteases