Crab-Eating Monkey Acidic Chitinase (CHIA) Efficiently Degrades Chitin and Chitosan under Acidic and High-Temperature Conditions.

Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, anti-tumor, and anti-inflammatory activities. The enzymatic production of chitooligosaccharides may increase the interest in their potential biomedical or agricultural usability in terms of the safety and simplicity of the manufacturing process. Crab-eating monkey acidic chitinase (CHIA) is an enzyme with robust activity in various environments. Here, we report the efficient degradation of chitin and chitosan by monkey CHIA under acidic and high-temperature conditions. Monkey CHIA hydrolyzed α-chitin at 50 °C, producing N-acetyl-d-glucosamine (GlcNAc) dimers more efficiently than at 37 °C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (α-chitin, colloidal chitin, P-chitin, block-type, and random-type chitosan substrates) were exposed to monkey CHIS at pH 2.0 or pH 5.0 at 50 °C. P-chitin and random-type chitosan appeared to be the best sources of GlcNAc dimers and broad-scale chitooligosaccharides, respectively. In addition, the pattern of the products from the block-type chitosan was different between pH conditions (pH 2.0 and pH 5.0). Thus, monkey CHIA can degrade chitin and chitosan efficiently without inactivation under high-temperature or low pH conditions. Our results show that certain chitooligosaccharides are enriched by using different substrates under different conditions. Therefore, the reaction conditions can be adjusted to obtain desired oligomers. Crab-eating monkey CHIA can potentially become an efficient tool in producing chitooligosaccharide sets for agricultural and biomedical purposes.

Mouse Acidic Chitinase Effectively Degrades Random-Type Chitosan to Chitooligosaccharides of Variable Lengths under Stomach and Lung Tissue pH Conditions.

Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenesis reduction in mammals. The mechanism of the chitooligosaccharides' formation in vivo has been, however, poorly understood. Here we report that mouse acidic chitinase (Chia), which is widely expressed in mouse tissues, can produce chitooligosaccharides from deacetylated chitin (chitosan) at pH levels corresponding to stomach and lung tissues. Chia degraded chitin to produce N-acetyl-d-glucosamine (GlcNAc) dimers. The block-type chitosan (heterogenous deacetylation) is soluble at pH 2.0 (optimal condition for mouse Chia) and was degraded into chitooligosaccharides with various sizes ranging from di- to nonamers. The random-type chitosan (homogenous deacetylation) is soluble in water that enables us to examine its degradation at pH 2.0, 5.0, and 7.0. Incubation of these substrates with Chia resulted in the more efficient production of chitooligosaccharides with more variable sizes was from random-type chitosan than from the block-type form of the molecule. The data presented here indicate that Chia digests chitosan acquired by homogenous deacetylation of chitin in vitro and in vivo. The degradation products may then influence different physiological or pathological processes. Our results also suggest that bioactive chitooligosaccharides can be obtained conveniently using homogenously deacetylated chitosan and Chia for various biomedical applications.

Robust chitinolytic activity of crab-eating monkey (Macaca fascicularis) acidic chitinase under a broad pH and temperature range.

Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, including chitin-containing organisms such as crabs and insects. This omnivorous monkey has a high expression of acidic chitinase (CHIA) in the stomach and here, we report on its enzymatic properties under different conditions. When we compared with Mus musculus CHIA (Mm-CHIA), Macaca fascicularis CHIA (Mf-CHIA) exhibits higher chitinolytic activity at broad pH (1.0-7.0) and temperature (30-70 â„ƒ) range. Interestingly, at its optimum pH (5.0), Mf-CHIA showed the highest activity at 65 °C while maintaining it at robust levels between 50 and 70 °C. The degradation efficiency of Mf-CHIA was superior to Mm-CHIA toward both polymeric chitin as well as an artificial chromogenic substrate. Our results show that unique features of Mf-CHIA including its thermostability warrant the nomination of this enzyme for potential agricultural and biomedical applications.

High expression of acidic chitinase and chitin digestibility in the stomach of common marmoset (Callithrix jacchus), an insectivorous nonhuman primate.

Chitin is a polymer of N-acetyl-D-glucosamine (GlcNAc) and a main constituent of insects' exoskeleton. Insects are rich in protein with high energy conversion efficiency. Recently, we have reported that acidic chitinases (Chia) act as digestive enzymes in mouse, pig and chicken (omnivorous) but not in dog (carnivorous) and bovine (herbivorous), indicating that feeding behavior affects Chia expression levels, and determines chitin digestibility in the particular animals. Common marmoset (Callithrix jacchus) belongs to New World monkey family and provides a potential bridge between mouse models and human diseases. Common marmoset is an insectivorous nonhuman primate with unknown expression levels and enzymatic functions of the Chia homologue, CHIA. Here, we report that common marmoset highly expresses pepsin-, trypsin- and chymotrypsin-resistant CHIA in the stomach. We show that CHIA is most active at pH 2.0 and degrades chitin and mealworm shells into GlcNAc dimers under gastrointestinal conditions. Although common marmoset and crab-eating monkey (Old World monkey) have two CHIA genes in their genomes, they primarily express one gene in the stomach. Thus, this study is the first to investigate expression levels and enzymatic functions of CHIA in a New World primate, contributing to the understanding of dietary adaptation and digestion in this taxon.

Chitinase mRNA Levels Determined by QPCR in Crab-Eating Monkey (Macaca fascicularis) Tissues: Species-Specific Expression of Acidic Mammalian Chitinase and Chitotriosidase.

Mice and humans express two active chitinases: acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Both chitinases are thought to play important roles in specific pathophysiological conditions. The crab-eating monkey (Macaca fascicularis) is one of the most frequently used nonhuman primate models in basic and applied biomedical research. Here, we performed gene expression analysis of two chitinases in normal crab-eating monkey tissues by way of quantitative real-time polymerase chain reaction (qPCR) using a single standard DNA molecule. Levels of AMCase and CHIT1 messenger RNAs (mRNAs) were highest in the stomach and the lung, respectively, when compared to other tissues. Comparative gene expression analysis of mouse, monkey, and human using monkey⁻mouse⁻human hybrid standard DNA showed that the AMCase mRNA levels were exceptionally high in mouse and monkey stomachs while very low in the human stomach. As for the CHIT1 mRNA, we detected higher levels in the monkey lung when compared with those of mouse and human. The differences of mRNA expression between the species in the stomach tissues were basically reflecting the levels of the chitinolytic activities. These results indicate that gene expression of AMCase and CHIT1 differs between mammalian species and requiring special attention in handling data in chitinase-related studies in particular organisms.