Immobilization of Subtilisin on Polycaprolactam for Antimicrobial Food Packaging Applications

Prabhawathi, Veluchamy; Sivakumar, Ponnurengam Malliappan; Doble, Mukesh

doi:10.1021/jf201124v

Cited by 32 publications

(33 citation statements)

References 50 publications

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“…Nevertheless, especially in the case of S. epidermidis, the presence of a protease appears to be crucial for a higher removal by washing and sonication. These results confirm the utility of proteases as biofilm dispersal, as highlighted by other research groups [9,10] and highlight the importance of the immobilization procedure to affect the enzyme capacity to weaken bacterial adhesion (Figure 1c). …”

Section: Resultssupporting

confidence: 88%

Effect of Immobilized Proteases on Bacterial Growth and Cell Adhesion on Polypropylene Surfaces

Secundo¹

2015

J Clin Microbiol Biochem Technol

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The 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.

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Section: Resultssupporting

confidence: 88%

Effect of Immobilized Proteases on Bacterial Growth and Cell Adhesion on Polypropylene Surfaces

Secundo¹

2015

J Clin Microbiol Biochem Technol

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“…Multipoint covalent immobilization was said to improve the stability by preventing aggregation and proteolysis. Similar observations were made while monitoring the storage stability of immobilized subtilisin [8]. LIP retained 65% of its activity at the end of one year.…”

Section: Resultssupporting

confidence: 74%

“…Subtilisin, an enzyme, when immobilized on polycaprolactam exhibits antimicrobial activity against both Gram positive as well as negative microbes [8]. Silver nanocomposites also exhibit such properties [9].…”

Section: Introductionmentioning

confidence: 99%

Antibiofilm Properties of Interfacially Active Lipase Immobilized Porous Polycaprolactam Prepared by LB Technique

et al. 2014

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Porous biomaterial is the preferred implant due to the interconnectivity of the pores. Chances of infection due to biofilm are also high in these biomaterials because of the presence of pores. Although biofilm in implants contributes to 80% of human infections [1], there are no commercially available natural therapeutics against it. In the current study, glutaraldehyde cross linked lipase was transferred onto a activated porous polycaprolactam surface using Langmuir-Blodgett deposition technique, and its thermostability, slimicidal, antibacterial, biocompatibility and surface properties were studied. There was a 20% increase in the activity of the covalently crosslinked lipase when compared to its free form. This immobilized surface was thermostable and retained activity and stability until 100°C. There was a 2 and 7 times reduction in carbohydrate and 9 and 5 times reduction in biofilm protein of Staphylococcus aureus and Escherichia coli respectively on lipase immobilized polycaprolactam (LIP) when compared to uncoated polycaprolactam (UP). The number of live bacterial colonies on LIP was four times less than on UP. Lipase acted on the cell wall of the bacteria leading to its death, which was confirmed from AFM, fluorescence microscopic images and amount of lactate dehydrogenase released. LIP allowed proliferation of more than 90% of 3T3 cells indicating that it was biocompatible. The fact that LIP exhibits antimicrobial property at the air-water interface to hydrophobic as well as hydrophilic bacteria along with lack of cytotoxicity makes it an ideal biomaterial for biofilm prevention in implants.

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“…21, 48 In an another study, polycaprolactam crosslinked with subtilisin was developed for food packaging applications. 49 Subtilisin was also entrapped in sol−gel matrices for biocatalytic applications, and the entrapped enzyme was sufficiently stable to allow reuse of the catalyst for several cycles. 50−52 We present the first attempt to immobilize subtilisin by physical entrapment using the sol−gel method for antifouling applications.…”

Section: Discussionmentioning

confidence: 99%

Entrapment of Subtilisin in Ceramic Sol–Gel Coating for Antifouling Applications

Regina

Søhoel

Lokanathan

et al. 2012

ACS Appl. Mater. Interfaces

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Enzymes with antifouling properties are of great interest in developing nontoxic antifouling coatings. A bottleneck in developing enzyme-based antifouling coatings is to immobilize the enzyme in a suitable coating matrix without compromising its activity and stability. Entrapment of enzymes in ceramics using the sol-gel method is known to have several advantages over other immobilization methods. The sol-gel method can be used to make robust coatings, and the aim of this study was to explore if sol-gel technology can be used to develop robust coatings harboring active enzymes for antifouling applications. We successfully entrapped a protease, subtilisin (Savinase, Novozymes), in a ceramic coating using a sol-gel method. The sol-gel formulation, when coated on a stainless steel surface, adhered strongly and cured at room temperature in less than 8 h. The resultant coating was smoother and less hydrophobic than stainless steel. Changes in the coating's surface structure, thickness and chemistry indicate that the coating undergoes gradual erosion in aqueous medium, which results in release of subtilisin. Subtilisin activity in the coating increased initially, and then gradually decreased. After 9 months, 13% of the initial enzyme activity remained. Compared to stainless steel, the sol-gel-coated surfaces with active subtilisin were able to reduce bacterial attachment of both Gram positive and Gram negative bacteria by 2 orders of magnitude. Together, our results demonstrate that the sol-gel method is a promising coating technology for entrapping active enzymes, presenting an interesting avenue for enzyme-based antifouling solutions.

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Immobilization of Subtilisin on Polycaprolactam for Antimicrobial Food Packaging Applications

Cited by 32 publications

References 50 publications

Effect of Immobilized Proteases on Bacterial Growth and Cell Adhesion on Polypropylene Surfaces

Effect of Immobilized Proteases on Bacterial Growth and Cell Adhesion on Polypropylene Surfaces

Antibiofilm Properties of Interfacially Active Lipase Immobilized Porous Polycaprolactam Prepared by LB Technique

Entrapment of Subtilisin in Ceramic Sol–Gel Coating for Antifouling Applications

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