A chitinolytic endochitinase and β-N-acetylglucosaminidase-based system from Hevea latex in generating N-acetylglucosamine from chitin

Abstract

An endochitinase and β-N-acetylglucosaminidase (NAGase) were purified and characterised from fresh rubber latex serum. These enzymes were used in a total enzyme-based system to produce pure N-acetylglucosamine (NAG) from chitin. The N-terminal amino acid sequences of both purified endochitinase (KEESRRRRHR) and NAGase (AAVDSDTLEI) lacked homology with other known chitinases, including hevamine from rubber latex lutoids. The apparent kinetic parameters, Km and Vmax, for the endochitinase using 4-MU-β-(NAG)3 as a substrate were 99.73μM and 29.49pkatmg−1, respectively. For NAGase, using 4-MU-β-NAG as a substrate, the corresponding Km and Vmax values were 20.4μM and 25.82pkatmg−1. When an enzyme incubation mixture containing a 1:1 (pkat/pkat) activity mixed ratio of endochitinase: NAGase was employed, the maximum yield of N-acetylglucosamine (NAG) obtained was 98% from β-chitin and 20% from α-chitin. These yields were obtained after 4days of hydrolysis of equal amounts of β-chitin and α-chitin in the mixture. Thus, β-chitin was the preferred substrate compared to α-chitin by a ratio of nearly five to one. Mass spectroscopic analysis, using electrospray ionisation mass spectrometry (ESI-MS), of the product obtained from β-chitin after digestion (for 24h) depicted one distinct major molecular ion peak m/z 260.1, a small minor ion peak m/z 481.2, a potassium adduct of NAG and a potassium adduct of two NAG molecules. Furthermore, experiments to establish the commercial production of NAG using crude enzymes of Hevea latex serum are currently in progress.