Abstract
BackgroundSince chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and β-N-acetylglucosaminidases (GlcNAcases). We have previously reported the structure and function of chitinase A from Vibrio harveyi 650. This study t reports the identification of two GlcNAcases from the same organism and their detailed functional characterization.ResultsThe genes encoding two new members of family-20 GlcNAcases were isolated from the genome of V. harveyi 650, cloned and expressed at a high level in E. coli. VhNag1 has a molecular mass of 89 kDa and an optimum pH of 7.5, whereas VhNag2 has a molecular mass of 73 kDa and an optimum pH of 7.0. The recombinant GlcNAcases were found to hydrolyze all the natural substrates, VhNag2 being ten-fold more active than VhNag1. Product analysis by TLC and quantitative HPLC suggested that VhNag2 degraded chitooligosaccharides in a sequential manner, its highest activity being with chitotetraose. Kinetic modeling of the enzymic reaction revealed that binding at subsites (-2) and (+4) had unfavorable (positive) binding free energy changes and that the binding pocket of VhNag2 contains four GlcNAc binding subsites, designated (-1),(+1),(+2), and (+3).ConclusionsTwo novel GlcNAcases were identified as exolytic enzymes that degraded chitin oligosaccharides, releasing GlcNAc as the end product. In living cells, these intracellular enzymes may work after endolytic chitinases to complete chitin degradation. The availability of the two GlcNAcases, together with the previously-reported chitinase A from the same organism, suggests that a systematic development of the chitin-degrading enzymes may provide a valuable tool in commercial chitin bioconversion.
Highlights
Since chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and b-N-acetylglucosaminidases (GlcNAcases)
In the CAZy database, GlcNAcases are classified into glycosyl hydrolases family 3 (GH-3) or family 20 (GH-20), which differ in sequence and mode of enzyme action [10,11]
In an attempt to isolate the genes that encode GlcNAcases in a closely-related organism, three sets of oligonucleotides were designed based on the above-mentioned open reading frames (ORFs)
Summary
Since chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and b-N-acetylglucosaminidases (GlcNAcases). As well as functioning in chitin degradation by bacteria, GlcNAcases are known to be key enzymes in the catabolism of glycoconjugates containing N-acetylglucosamine residues [7,8] and mutations of the gene encoding a human GlcNAcase homologue (HexA) cause a fatal genetic lipid storage disorder, known as TaySachs disease [9]. GlcNAcases are thought to act by a standard retaining mechanism involving a covalent glycosyl-enzyme intermediate while family-20 enzymes employ a ‘substrateassisted’ mechanism involving the transient formation of an oxazolinium ion intermediate [12,13,14,15]. Only five bacterial GH-3 GlcNAcases have been characterized, including NagZ or ExoII from Vibrio furnissii [16], Nag3A from Clostridium paraputrificum M-2 [17], NagA from Streptomyces thermoviolaceus [18], and NagA and CbsA from Thermotoga maritima and T. neapolitana [19]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
