ReviewRecent developments in chitosanase research and its biotechnological applications: A review
Introduction
Chitosan is a poly cationic natural polymer with a unique structure and functional properties. It is an unbranched copolymer consisting of β-(1→4)-2-acetamido-d-glucose (N-acetyl-d-glucosamine, GlcNAc) and β-(1→4)-2-amino-d-glucose (d-glucosamine, GlcN) units with the latter usually exceeding 80% and the GlcNAc and GlcN residues are randomly distributed and not blocked together (Zitouni et al., 2012) (Fig. 1). Chitosan is found in nature as a structural component mainly in the cell wall of Zygomycetes fungi and also found in the cell wall of Chlorophycean algae Chlorella sp. and in insect cuticle (Hsu, Chung, Chang, & Sung, 2012). Chitosan is the principal N-deacetylated derivative of chitin (a β-1,4 linked polymer of GlcNAc), although the N-deacetylation is almost never complete. Currently chitosan is produced commercially from crustacean’s (shrimp and crab) chitin by N-deacetylation, to different degree using hot concentrated alkali. Thus, there is no clear distinction between chitin and chitosan based on the degree of N-deacetylation; the term chitosan represents a collective name for a family of deacetylated chitins at different degrees (Fig. 1). The studies of chitosan with respect to their preparation, structure, functional properties and applications have been extensively reviewed (Zitouni et al., 2012).
Chitosan is usually susceptible to a number of enzymes; these include specific (chitosanases) and non-specific (carbohydrases, proteases, lipases etc.) chitosan hydrolysing enzymes. Chitosanases have been generally recognised as enzymes that specifically hydrolyse chitosan but not chitin (Kim & Rajapakse, 2005). In 2004, the Enzyme nomenclature committee had defined chitosanases (EC 3.2.1.132, Chitosan N-acetylglucosaminohydrolase) as the enzyme capable of performing endohydrolysis of β-1,4-linkages between GlcN residues in a partly acetylated chitosan, from the reducing end (Fig. 2a). Later, in 2008, the committee created a new (second) class of enzyme, exo-β-d-glucosaminidase (EC 3.2.1.165) that attack chitosan from its non reducing end (Fig. 2b). In addition, some other non specific enzymes such as common carbohydrases, proteases and lipases have also shown their hydrolytic ability on chitosan (Kim & Rajapakse, 2005). As on date a large number of specific chitosanolytic enzymes have been reported from different microorganisms including bacteria, fungi and cyanobacteria and plants.
Recent researches on chitosanases have received much attention due to their wide range of applications in various fields. Practical applications of chitosanase include the preparation of bioactive COSs (Kim and Rajapakse, 2005, Ming et al., 2006), preparation of fungal protoplasts particularly for Zygomycetes, a biocontrol agent to increase the resistance of plants against pathogenic fungi (Hsu et al., 2012), chitosan mediated gene delivery and the bioconversion of marine crustacean chitinous bio waste (Wang et al., 2009, Wang et al., 2011). Chitosanase mediated hydrolysis has advantages over the chemical/physical mediated hydrolytic production of COS, in which chitosanases can catalyse the hydrolysis under mild reaction conditions and do not produce monosaccharides (Kim and Rajapakse, 2005, Ming et al., 2006). Reviews on chitosanses with respect to its production and application (Somashekar & Joseph, 1996) have been reported earlier. The present review will focus not only on the current knowledge of chitosanases, their occurrence and classification but also on microbial production, biochemical properties, genetic improvement and applications based on research specifically carried out in the last ten years.
Section snippets
Occurrence and distribution of chitosanases
Chitosanase is produced by microbes and plants, where they play an important role in nutrition and defence. Chitosanase was first described in 1973 from different soil microorganisms. Over the past 40 years, several research papers have been published on the occurrence, production, purification and characterization of chitosanase from different microorganisms including bacteria, fungi and cyanobacteria; and plants. Detailed literature is available on various microbial sources of chitosanase (
Definition and classification of chitosanases
Chitosanases have been generally recognised as enzymes that specifically attack chitosan but not chitin. In addition, some non specific enzymes such as common carbohydrases, proteases and lipases also have shown their hydrolytic ability on chitosan (Gupta et al., 2012, Kim and Rajapakse, 2005). The definition of chitosanase (EC 3.2.1.132, Chitosan N-acetyl glucosaminohydrolase) created in 1990 by the Enzyme Nomenclature Commission and has been amended in 2004 (
Fermentative production of chitosanase
Table 1 presents data available in the literature regarding the microbial production of extracellular chitosanses, using both submerged fermentation (SmF) and solid-state fermentation (SSF) systems. Most of the chitosanases reported so far have been inducible. The use of colloidal chitosan as a supplement for culture media in production of chitosanses is a common strategy and in the majority of cases, its inductor effect has been established. Other substrates such as chitosan powder, squid pen
Measurement of chitosanase activity
Most of the chitosanases cannot act on solid/crystalline chitosan, so colloidal chitosan or glycol chitosan are generally used as substrates for the chitosanase assays (Somashekar & Joseph, 1996). Chitosanase activity is influenced by various factors such as the degree of deacetylation of chitosan, viscosity and concentration of chitosan solution, kind of acid used for dissolving the chitosan, the amount of enzyme and other reaction conditions such as pH, temperature, and agitation. Chitosanase
Purification of microbial chitosanases
Industrial enzymes usually prepared in crude form for their commercial applications. The use of chitosanases in COS preparation usually does not require purification of the enzyme, but chitosanses in their purified form is required to study the biochemical properties, structure–function relationships and their biotechnological applications. The purification of chitosanases from microbial sources in most cases has involved classical enzyme purification methods. These methods involve removal of
Basic biochemical properties of chitosanases
The basic biochemical properties of chitosanases vary depending on the source of enzymes. The biochemical properties of chitosanases from different microorganisms have been thoroughly studied and reported. Table 2 represents the data available in the literature regarding the basic properties of chitosanses from various microorganisms.
Immobilised chitosanase
Immobilisation of enzymes is one of the method for protecting and stabilizing the enzymes, thereby enhancing their properties and their repetitive utilisation either in batch, or continuous mode. Immobilisation of enzymes prevents their deactivation by various physical and chemical denaturing agents and thereby enhancing their operational stability. Furthermore, utilisation of immobilised enzymes permits control of the progress of the hydrolysis reaction (Ming et al., 2006). Particularly in the
Cloning and genetic improvement
Cloning of genes has been carried out extensively for the molecular study of proteins, hyper production and protein engineering. Cloning of chitosanase genes has been carried for studying their sequence, characteristics, hyper production and expression, change in enzyme induction pattern and for enzyme engineering. Chitosanase genes from different bacteria and fungi have been cloned and expressed mainly in Escherichia coli or in heterologous host Pichia pistoris. Many chitosanase genes have
Biological roles of chitosanase
Microbial chitosanases with different biological roles have been found in nature. Chitosan degrading microorganisms are widely distributed in nature and microorganisms secrete chitosanase extracellularly to degrade chitosan for their nutritional purpose (Somashekar & Joseph, 1996). Chitosanase together with chitinase, chitin deacetylase and glucosaminidase, involve in the decomposition and recycling of enormous quantity of crustaceans shell produced in nature. Since chitosan is the major
Potential applications
Chitosanases have industrial, as well as biotechnological applications that require different types of formulations. One of the most important applications of chitosanase is the preparation of COS from chitosan (Ming et al., 2006, Zhang et al., 2012). Enzymatic hydrolysis of chitosan has some advantages for the production of COS, the chitosanases can catalyse the hydrolysis under mild conditions and do not produce monosaccharides (Liu et al., 2009). COS produced by enzymatic hydrolysis of
Futuristic considerations
Bioactive COS has significant applications especially in the food and biomedical industries. Chitosanase is the key enzyme required for the preparation of biologically active COS from chitosan. The use of chitosanase for the biocontrol of phyto pathogens and for developing transgenic plants is one of the major areas of research. The success in using chitosanase for diverse applications depends on the production of highly active enzyme at a reasonable cost. Research is also focused on developing
Acknowledgement
NT expresses his gratitude to the University Grant Commission (UGC), New Delhi, India for the award of Research Fellowship. The authors would like to thank anonymous reviewers for the valuable comments provided to improve the manuscript.
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