| Keratins make up the largest subgroup of intermediate filament proteins and represent the most abundant proteins in epithelial cells. On the basis of the structural features of keratin filaments, they exhibit high mechanical resilience and play an important role in maintaining physiological functions of normal epidermis. It has been proved that abnormality or deficiency of keratin expression and functions can lead to a variety of skin disorders. For some skin disorders with hyperkeratosis, it has been claimed that they are associated with keratins abnormality. In addition, keratins abnormality may result in hyperkeratosis, which decrease permeability of skin and bring great difficulties for topical drugs applications. Keratinase exhibit specific activities of hydrolyzing keratin into soluble polypeptides and amino acid and secreted by some micro-organisms, especially fungi. In this way, fungi obtained their essential nitrogen and carbon source and are capable of growing and proliferation. In view of important roles in growth and proliferation, keratinase is also regarded as an invasive factor within fungi. With efficient activity of hydrolyzing keratins, it isthought that keratinase has a potential application in different fields, such as medicine, cosmetics, waste byconversion and animal feedstuff production.Objectives: The present study aimed to isolate keratinase with good application potential in bio-medicine, cosmetics. For further investigations and clinical application, the hydrolyzing activity of keratinase was also thoroughly identified. We attempted to explore the probability of combination of keratinase with topical drugs in improving permeability and increasing efficacy. Additionally, our study will lay a foundation for clinical application of keratinase in psoriasis, skin disorders with hyperkeratosis and nail disorders.Methods: All works are divided into three sections. The experimental procedures are described as follows:1. Keratinase producfon in dermatophytes induced by purified keratinUsing purified keratins as substrate, keratinase was induced in three common fungi including Trichophyton mentagrophytes, Trochiphyton rubrum and Micrisporum canis. The hydrolytic activity, optimum pH value and temperature of keratinase were compared by ultraviolet spectrophotometer. The specific activities of Trichophyton mentagrophytes keratinase induced by different substrate (purified keratin or hair) were also compared by ultraviolet spectrophotometer.2. Cloning, expression and purification of Trichophyton mentagrophytes keratinase (Tri-M-K)Trichophyton mentagrophytes were cultured in presence of purified human keratins. Culture medium without keratins was established as controls. Total RNA from Trichophyton mentagrophytes were isolated and cDNA was prepared using the First Strand Synthesis Kit. Keratinase ds-DNA was generated by PCR using the template cDNAand specific primers. The resulting PCR products were double digested by BamHK EcoRI, and ligated into a pGEX-4T-l plasmid. After gel recycles, the obtained segments were ligated to pGEX-4T-l plasmid and the infusion expression GST vectors were established. The constructed vectors were further identified by digestion of restriction enzyme and gene sequencing. Then the constructed pGEX-4T-l/ Tri-M-K plasmid was transformed into JM109. The positive colony with a gene insert in the plasmid was identified by restriction digestion of the plasmid, followed by culture upon IPTG induction. The expression protein was purified by GST labeled columns and identified by SDS-PAGE and Transilluminator M-26 gel analysis.3. Biological roles of purified Tri-M-KUsing purified keratin as substrate, the activity, optimum pH value and temperature of Tri-M-K were determined by ultraviolet spectrophotometer. Human or rabbit keratins hydrolyzed by recombinant Tri-M-K were also subjected to SDS-PAGE and analyzed by Gel-Protein analyzer to quantificate the digestion rate of keratinase. Besides, recombinant Tri-M-K was incubated with human hair...
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