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Silk degumming time controls horseradish peroxidase-catalyzed hydrogel properties.

ABSTRACT: Hydrogels provide promising applications in tissue engineering and regenerative medicine, with silk fibroin (SF) offering biocompatibility, biodegradability and tunable mechanical properties. The molecular weight (MW) distribution of SF chains varies from ∼80 to 400 kDa depending on the extraction and purification process utilized to prepare the protein polymer. Here, we report a fundamental study on the effect of different silk degumming (extraction) time (DT) on biomaterial properties of enzymatically crosslinked hydrogels, including secondary structure, mechanical stiffness, in vitro degradation, swelling/contraction, optical transparency and cell behaviour. The results indicate that DT plays a crucial role in determining material properties of the hydrogel; decrease in DT increases β-sheet (crystal) formation and mechanical stiffness while decreasing degradation rate and optical transparency. The findings on the relationships between properties of silk hydrogels and DT should facilitate the more rational design of silk-based hydrogel biomaterials to match properties needed for diverse purpose in biomedical engineering.

SUBMITTER: Sahoo JK

PROVIDER: S-EPMC7390697 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

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Silk degumming time controls horseradish peroxidase-catalyzed hydrogel properties.

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