• Bulletin of the Korean Chemical Society
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• v.32 no.spc8
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• pp.3113-3116
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• 2011

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.1-7
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• 1998

• Bulletin of the Korean Chemical Society
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• v.25 no.12
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• pp.1917-1923
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• 2004

Although the proteolytic activity of the Cu(II) complex of cyclen (Cyc) is greatly enhanced upon attachment to a cross-linked polystyrene (PS), the Cu(II)Cyc-containing PS derivatives reported previously hydrolyzed only a very limited number of proteins. The PS-based artificial metalloproteases can overcome thermal, mechanical, and chemical instabilities of natural proteases, but the narrow substrate selectivity of the artificial metalloproteases limits their industrial application. In the present study, artificial metalloproteases exhibiting broad substrate selectivity were synthesized by attaching Cu(II)Cyc to a PS derivative using linkers with various structures in an attempt to facilitate the interaction of various protein substrates with the PS surface. The new artificial metalloproteases hydrolyzed all of the four protein substrates (albumin, myoglobin, ${\gamma}$-globulin, and lysozyme) examined, manifesting $k_{cat}/K_m$ values of 28-1500 $h_{-1}M_{-1}$ at 50 $^{\circ}C$. The improvement in substrate selectivity is attributed to steric and/or polar interaction between the bound protein and the PS surface as well as the hydrophobicity of the microenvironment of the catalytic centers.

• Journal of Microbiology and Biotechnology
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• v.31 no.11
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• pp.1591-1600
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• 2021

Streptomyces coelicolor is a filamentous soil bacterium producing several kinds of antibiotics. S. coelicolor abs8752 is an abs (antibiotic synthesis deficient)-type mutation at the absR locus; it is characterized by an incapacity to produce any of the four antibiotics synthesized by its parental strain J1501. A chromosomal DNA fragment from S. coelicolor J1501, capable of complementing the abs- phenotype of the abs8752 mutant, was cloned and analyzed. DNA sequencing revealed that two complete ORFs (SCO6992 and SCO6993) were present in opposite directions in the clone. Introduction of SCO6992 in the mutant strain resulted in a remarkable increase in the production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, in S. coelicolor J1501 and abs8752. However, introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is primarily involved in stimulating the biosynthesis of antibiotics in S. coelicolor. In silico analysis of SCO6992 (359 aa, 39.5 kDa) revealed that sequences homologous to SCO6992 were all annotated as hypothetical proteins. Although a metalloprotease domain with a conserved metal-binding motif was found in SCO6992, the recombinant rSCO6992 did not show any protease activity. Instead, it showed very strong β-glucuronidase activity in an API ZYM assay and toward two artificial substrates, p-nitrophenyl-β-D-glucuronide and AS-BI-β-D-glucuronide. The binding between rSCO6992 and Zn²⁺ was confirmed by circular dichroism spectroscopy. We report for the first time that SCO6992 is a novel protein with β-glucuronidase activity, that has a distinct primary structure and physiological role from those of previously reported β-glucuronidases.