Piotr Biniarz1, Eugenio Spadoni Andreani2, Anna Krasowska1, Marcin Łukaszewicz1 and Francesco Secundo2*
1Faculty of Biotechnology, University of Wrocław, Wrocław, Poland
2Institute of Chemistry of Molecular Recognition, National Council of Research, Milan, Italy
Received: 09 September, 2015; Accepted: 25 September, 2015; Published: 28 September, 2015
Francesco Secundo, Institute of Chemistry of Molecular Recognition, National Council of Research, via Mario Bianco 9, 20131, Milano, Tel: ++39 0228500029; Fax: ++39 02 28901239; E-mail:
Biniarz P, Andreani ES, Krasowska A, Łukaszewicz M, Secundo F (2015) Effect of Immobilized Proteases on Bacterial Growth and Cell Adhesion on Polypropylene Surfaces. J Clin Microbiol Biochem Technol 1(1): 007-009.
© 2015 Biniarz P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Enzyme immobilization; Antibiofilm; Biocatalysis; Hydrolases; Plasma
aCT: pancreatic α-Chymotrypsin from Bos Taurus; SubC: Subtilisin Carlsberg from Bacillus licheniformis; PP: Polypropylene; DMF: N,N-Dimethylformamide; GA: Glutaraldehyde; DIC: N-N’-Diisopropylcarbodiimide; NHS: N-Hydroxysuccinimide; aCT-GA: aCT linked to PP using GA as cross-linking agent; aCT-DIC: aCT linked to PP using DIC and NHS for the cross-linking reaction; SubC-GA: SubC linked to PP using GA as cross-linking agent; SubC-DIC: SubC linked to PP using DIC and NHS for the cross-linking reaction; LB: Luria Bertani Agar; PBS: sterile Phosphate-Buffered Saline;
he bacterial planktonic growth and the removal of bacterial cells grown on polypropylene surface coated with covalently immobilized proteases (subtilisin Carlsberg or α-chymotrypsin) was investigated for Enterococcus hirae, Staphyloccocus epidermidis and Eschericha coli. Immobilization of both proteases on plasma-treated polypropylene was carried out using as cross-linking agent i) glutaraldehyde or ii) N’-diisopropylcarbodiimide and N-hydroxysuccinimide. In the presence of immobilized proteases a higher bacterial planktonic growth (up to 40 %) was observed. Instead, a different effect was observed on cell removal, and it varied according to the bacteria strain, the immobilized protease and the immobilization procedure. In particular, the presence of subtilisin in the polypropylene coating increased the cell removal of E. hirae by simple washing of the polypropylene surface and both subtilisin and α-chymotrypsin immobilized by N’-diisopropylcarbodiimide and N-hydroxysuccinimide favored the removal of S. epidermidis after sonication. No significant differences compared to the control where observed in all the other cases. In conclusion this study indicates that proteases can be an enhancer of microbial biomass (a phenomena that could be exploited for industrial fermentation) and can affect the strength of cell adhesion for some bacteria.
Among the strategies proposed to prevent or inhibit undesired and (often) pathogenic microbial biofilm, enzyme based coatings have been developed by different research groups [1,2]. In particular, coatings containing proteases might be employed to degrade i) the proteinaceous component of the self-produced polymeric substance  (a major structural constituent of the biofilm made of polysaccharides, proteins, lipids and nucleic acids) and/or ii) the proteins (e.g., adhesins) involved in adhesion processes of cells to a surface to form a biofilm.
Enzyme immobilization presents some advantages compared to the use of free enzymes. For example, in the field of biocatalysis immobilization ensures the reusability of the enzymes improving the productivity of the biocatalytic process  and it can favorably affect operational flexibility by increasing enzyme thermal stability and selectivity . This latter feature also depends on the procedure adopted for enzyme immobilization and it could be modulated for changing protease specificity toward the proteins involved in the bacterial adhesion. In addition, the confinement of enzymes on a solid surface through immobilization can be a procedure to maximize the enzyme activity just where the biofilm grows.
Herein we present how two commercial and readily available proteases (aCT and SubC), immobilized on polypropylene surface , affect bacterial planktonic growth and the removal of cells grown on the plastic surface itself. To this end, Enterococcus hirae (gram positive and it causes sepsis in humans), Staphyloccocus epidermidis (gram positive and opportunistic human pathogen) and Eschericha coli (a gram negative component of human microflora and an opportunistic human pathogen) were chosen as model bacteria.
Materials and Methods
Round PP coupons (Ø 1.3 cm) were cut from PP sheets purchased from Alfa Aesar. The enzymes aCT (54 U/mg) and SubC (8.6 U/mg) were purchased from Sigma. Analytical grade reagents were purchased from Alfa-Aesar. Bacterial strain were Enterococcus hirae (ATCC 10541), Staphyloccocus epidermidis (KTCC 1917) both Gram-positive and Eschericha coli (ATCC 25922, Gram-negative).
Protease immobilization on polypropylene coupons
Round PP coupons (Ø 1.3 cm) were preventively washed with MilliQ water and acetone and dried. Next they were exposed to oxygen plasma for 20 min using a Harrick Plasma PDC-002 plasma cleaner; 740 V, 40 mA, 29.6 W) for surface functionalization . Immediately after the plasma treatment two different immobilization procedures were applied. In the first procedure coupons were coated with 80 μL of protease (aCT or SubC) solution (5 mg/mL) in 20 mM phosphate buffer, pH 7.2 (buffer A), containing 0.005% (v/v) GA, and let dry overnight at 25 °C under vacuum . In a second procedure (for aCT-DIC or SubC-DIC preparation) coupons were dipped in 0.1 M, pH 3.5 MES buffer and dried at 40 °C under vacuum. Afterwards coupons were immersed in DMF containing 2 mM DIC and 5 mM NHS and kept shaken for 2 h at 150 rpm, then washed with buffer A. Each coupon was then covered with 80 μL of a 5 mg/mL of a-CT or SubC solution in the same buffer, which was allowed to react for 2 h. For both procedures the coupons were washed for 30 min with 3 mL of bi-deionized water for three times to get rid of the unbound enzyme. Control coupons were analogously prepared but using buffer a solution instead of protease solution (in Figure 1 they were indicated as GA or DIC).
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