Abstract
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.
Highlights
Chitin is a polymer of (β-1,4)-linked N-acetyl-D-glucosamine (GlcNAc), which is an integral component of the exoskeleton of crustaceans and insects, microfilarial sheaths of parasites, and cell walls in fungi [1,2,3]
We recently reported that Chia can be purified from chicken and pig stomach tissues using a chitin column and 8 M urea [16,17]
The eluted fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and SYPRO Ruby staining
Summary
Chitin is a polymer of (β-1,4)-linked N-acetyl-D-glucosamine (GlcNAc), which is an integral component of the exoskeleton of crustaceans and insects, microfilarial sheaths of parasites, and cell walls in fungi [1,2,3]. Herbivorous and carnivorous animals such as bovines and dogs have very low capability to digest chitin when compared with omnivorous animals [29] This knowledge could potentially lead to the development of Chia-related supplementation for chitin digestion enhancement or as promising therapeutic agents for the treatment of specific diseases resulting from low levels and/or activity of Chia. We recently reported that Chia can be purified from chicken and pig stomach tissues using a chitin column and 8 M urea [16,17]. We report that a Chia-chitin complex is dissociated in a competitive manner by 0.1 M acetic acid This acetic acid competition toward chitin can be applied to the purification of endogenous chicken and pig Chia, potentially having numerous applications for agricultural or biomedical Chia-related supplementation purposes
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