ABSTRACT
Chitin, a β-1,4 polymer of N-acetyl-d-glucosamine (GlcNAc), is the second abundant biopolymer found in nature after cellulose (Muzzarelli 1999). This natural resource is relatively easily accessible, for example, as crab and shrimp shell waste. N-acetyl-chito-oligosaccharides and chito-oligosaccharides have varied biological functions and many potential applications in a wide range of elds (Tokoro et al. 1988, Hirano and Nagao 1989). To obtain enzymes that can be applied
27.1 Introduction .......................................................................................................................... 371 27.2 Purication and Characterization of Chitinolytic Enzymes from the Moderately
Thermophilic Bacterium Ralstonia sp. A-471 ...................................................................... 372 27.2.1 Enzyme Assay .......................................................................................................... 372 27.2.2 Purication and Characterization of Chitinases A and B ........................................ 372
27.2.2.1 Cultivation and Purication of Chitinases (Chitinases A and B) .............. 372 27.2.2.2 Molecular Mass and N-Terminal Amino Acid Sequence .......................... 372 27.2.2.3 Effects of pH and Temperature .................................................................. 373 27.2.2.4 Substrate Specicity .................................................................................. 373 27.2.2.5 HPLC Analysis of Hydrolysis Products from (GlcNAc)2-6
and N-Acetylated Chitosans ....................................................................... 373 27.3 Cloning, Sequencing, and Expression of a Novel Goose-Type Lysozyme Gene
with Chitinase (Ra-ChiC) Activity from the Moderately Thermophilic Bacterium Ralstonia sp. A-471 .............................................................................................................. 374 27.3.1 Cloning and Sequencing of a Novel G-Type Lysozyme Gene .................................. 374 27.3.2 Expression of a Novel G-Type Lysozyme Gene and Purication
of the Recombinant Protein ...................................................................................... 374 27.3.3 Characterization of a Recombinant Protein ............................................................. 374
27.3.3.1 Optimum pH and Temperature .................................................................. 374 27.3.3.2 Substrate Specicity .................................................................................. 375 27.3.3.3 HPLC Analysis of Hydrolysis Products from (GlcNAc)2-6 ..................... 375
References ...................................................................................................................................... 375
to develop environment-friendly techniques in chitin-oligosaccharide production, we have isolated a thermophilic strain belonging to the genus Ralstonia (Sutrisno et al. 2004). Chitinase A was excreted into the culture medium and was constantly produced until the colloidal chitin was wholly degraded. The other chitinase, chitinase B, showed a trace amount of protein in the culture medium, and had weaker activity than Ralstonia chitinase A. We have isolated and puried chitinases A (Ra-ChiA) and B (Ra-ChiB) from Ralstonia sp. A-471. The enzymatic properties of these two enzymes were similar to each other (Ueda et al. 2005). To understand the genetic basis for the production of chitinases in Ralstonia sp. A-471, we attempted to clone and sequence the chitinase genes. In the cloning of the chitinase genes from Ralstonia sp. A-471, we got several positive clones and one of them indicated a homology to amino acid sequences of G-type lysozyme (Pooart et al. 2005) and Clostridium lysozyme-like enzyme (NCBI accession no. YP_001309935). This transformant had activities against several chitins and chitosans but not against the cell wall of Micrococcus lysodeikticus (Ueda et al. 2009). In this chapter, we review (1) the purication and characterization of chitinolytic enzymes from moderately thermophilic bacterium Ralstonia sp. A-471 and (2) cloning, sequencing, and the expression of G-type lysozyme gene with chitinase (Ra-ChiC) activity from the bacterium.
