Increase in
Hydrophobicity of Signal Peptide Enhances Secretion of Penicillin G Acylase
YANG Yun-Gui1, XU Jing-Ning2, HU
Tai-Shan1, QIAN You-Cun1, YANG Sheng-Li1, GONG
Yi1
( 1Shanghai Research Center of Biotechnology, Institute of
Biological Science, Chinese Academy of Sciences and Shanghai Center of Life
Sciences, Shanghai 200233, China； 2The Sate Key Laboratory of Bioreactor
Engineering, East China University of Science and Technology, shanghai 200237, China )
Abstract An
artificial strong hydrophobic signal peptide (ASP), containing ten leucines in
tandem in the hydrophobic core, was utilized to replace the wild type signal
peptide (WTSP) of penicillin G acylase (PAC), by the fusion to its －4 pro site. PAC expression
plasmids, including pKKpacΔSP, pKKpacWTSP, pKKpacASP, pETpacWTSP and pETpacASP,
were constructed. The length and the amino- and carboxyl-terminus amino acid
composition of ASP and WTSP were kept identical. The activity assay and
Western-blotting analysis were used to study the effect of ASP and WTSP on the
secretion of PAC in tac, T7 and dissolved-oxygen regulation expression systems,
respectively. Lack of signal peptide in pKKpacΔSP resulted in the accumulation of
91 kD PAC precursor (without signal peptide, but with the space peptide between
α-subunit and β-subunit) in the cytosol, indicating that the secretion of PAC
depends on the signal peptide. In BL21(pKKpacASP) cells, the PAC activity and
proprecursor (with signal peptide and space peptide) processing capacity were
increased by about 54% and 38.5%, respectively, in comparison with
BL21(pKKpacWTSP) cells. Compared with BL21(DE3) (pETpacWTSP), however, the PAC
activity and proprecursor processing capacity in BL21(DE3) (pETpacASP) were
enhanced by about 69% and 43.5%, respectively. The PAC activity expressed from
pETpacASP was about 67% more than that from pETpacWTSP in the
dissolved-oxygen-regulated expression system GJ100. Resulting from the strong
hydrophobicity of ASP, therefore, the PAC activity and proprecursor processing
capacity were increased by about 63% and 41% on average, respectively, in
comparison with WTSP. In conclusion, the increase in hydrophobicity of the
signal peptide hydrophobic core enhanced the secretion of penicillin G acylase.
Key words Penicillin G acylase； signal peptide； hydrophobic core； hydrophobicity
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