Acidic chitinase (Chia) has been implicated in asthma, allergic inflammations, and food processing. We have purified Chia enzymes with striking acid stability and protease resistance from chicken and pig stomach tissues using a chitin column and 8 M urea (urea-Chia). Here, we report that acetic acid is a suitable agent for native Chia purification from the stomach tissues using a chitin column (acetic acid-Chia). Chia protein can be eluted from a chitin column using 0.1 M acetic acid (pH 2.8), but not by using Gly-HCl (pH 2.5) or sodium acetate (pH 4.0 or 5.5). The melting temperatures of Chia are not affected substantially in the elution buffers, as assessed by differential scanning fluorimetry. Interestingly, acetic acid appears to be more effective for Chia-chitin dissociation than do other organic acids with similar structures. We propose a novel concept of this dissociation based on competitive interaction between chitin and acetic acid rather than on acid denaturation. Acetic acid-Chia also showed similar chitinolytic activity to urea-Chia, indicating that Chia is extremely stable against acid, proteases, and denaturing agents. Both acetic acid- and urea-Chia seem to have good potential for supplementation or compensatory purposes in agriculture or even biomedicine.

• Klíčová slova
• GlcNAc, acetic acid, acidic chitinase, chitin, chitin column, competitive manner, natural enzyme, supplementation purposes, therapeutic agents, urea,
• MeSH
• chitin chemie   metabolismus   MeSH
• chitinasy chemie   metabolismus   MeSH
• kur domácí MeSH
• kyselina octová chemie   MeSH
• prasata MeSH
• vazba proteinů MeSH
• žaludek enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chitin MeSH
• chitinasy MeSH
• kyselina octová MeSH

Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), is a major structural component in chitin-containing organism including crustaceans, insects and fungi. Mammals express two chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Here, we report that pig AMCase is stable in the presence of other digestive proteases and functions as chitinolytic enzyme under the gastrointestinal conditions. Quantification of chitinases expression in pig tissues using quantitative real-time PCR showed that Chit1 mRNA was highly expressed in eyes, whereas the AMCase mRNA was predominantly expressed in stomach at even higher levels than the housekeeping genes. AMCase purified from pig stomach has highest activity at pH of around 2-4 and remains active at up to pH 7.0. It was resistant to robust proteolytic activities of pepsin at pH 2.0 and trypsin and chymotrypsin at pH 7.6. AMCase degraded polymeric chitin substrates including mealworm shells to GlcNAc dimers. Furthermore, we visualized chitin digestion of fly wings by endogenous AMCase and pepsin in stomach extract. Thus, pig AMCase can function as a protease resistant chitin digestive enzyme at broad pH range present in stomach as well as in the intestine. These results indicate that chitin-containing organisms may be a sustainable feed ingredient in pig diet.

• MeSH
• chitin metabolismus   MeSH
• chitinasy genetika   izolace a purifikace   metabolismus   MeSH
• chymotrypsin metabolismus   MeSH
• dieta * MeSH
• Drosophila chemie   MeSH
• endopeptidasy metabolismus   MeSH
• gastrointestinální trakt metabolismus   MeSH
• křídla zvířecí chemie   MeSH
• messenger RNA genetika   metabolismus   MeSH
• orgánová specificita MeSH
• pepsinogen A metabolismus   MeSH
• prasata genetika   MeSH
• rozpustnost MeSH
• substrátová specifita MeSH
• Tenebrio MeSH
• tkáňové extrakty MeSH
• trypsin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chitin MeSH
• chitinasy MeSH
• chymotrypsin MeSH
• endopeptidasy MeSH
• messenger RNA MeSH
• pepsinogen A MeSH
• tkáňové extrakty MeSH
• trypsin MeSH