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

作者：Sukprasirt Pannawich; Wititsuwannakul Rapepun^*

来源：Phytochemistry, 2014, 104: 5-11.

DOI：10.1016/j.phytochem.2014.04.001

• Hevea brasiliensis
• Euphorbiaceae
• Rubber latex serum
• Chitinase
• Endochitinase
• beta-N-Acetylglucosaminidases
• N-Acetylglucosamine

An endochitinase and beta-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, K-m and V-max, for the endochitinase using 4-MU-beta-(NAG)(3) as a substrate were 99.73 mu M and 29.49 pkat mg(-1), respectively. For NAGase, using 4-MU-beta-NAG as a substrate, the corresponding K-m and V-max values were 20.4 mu M and 25.82 pkat mg(-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 beta-chitin and 20% from alpha-chitin. These yields were obtained after 4 days of hydrolysis of equal amounts of beta-chitin and alpha-chitin in the mixture. Thus, beta-chitin was the preferred substrate compared to alpha-chitin by a ratio of nearly five to one. Mass spectroscopic analysis, using electrospray ionisation mass spectrometry (ESI-MS), of the product obtained from beta-chitin after digestion (for 24 h) 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.

• 出版日期2014-8