›› 2018, Vol. 61 ›› Issue (10): 1153-1159.doi: 10.16380/j.kcxb.2018.10.004

• RESEARCH PAPERS • Previous Articles     Next Articles

Purification and characterization of N-acetyl-β-D-glucosaminidase from the Italian honey bee, Apis mellifera ligustica (Hymenoptera: Apidae) (In English)

ZHANG Wei-Ni, HU Chong-Wei, ZHANG Meng-Xin, HAN Wei-Chao, HUANG Xiao-Hong*   

  1. (University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China)
  • Online:2018-10-20 Published:2018-10-20

Abstract: 【Aim】 N-acetyl-β-D-glucosaminidase (NAGase) is an important chitinase for degradation of chitin. It can cleave terminal N-acetyl-β-D-glucosamine residues from the nonreducing ends of N-acetyl-β-D-glucosides, participating in the ecdysis of exoskeletons in insects. Researching and characterizing this enzyme from honey bees may help to clarify its mechanism of action in the development of honey bees. 【Methods】 NAGase was purified from larvae of the Italian honey bee(Apis mellifera ligustica) by means of (NH4)2SO4 fractionation (40%-70% of saturation), DEAE-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration. The enzyme activity was determined by using p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) as the substrate. The purity of the enzyme was determined by native PAGE and SDS-PAGE. The isoelectric point (pI) of the enzyme was measured by IEF-PAGE, and its molecular weight was determined by Sephadex G-200 gel filtration. 【Results】The specific activity of purified NAGase was 803.09 U/mg and the molecular weight was 77.3 kD. The enzyme was composed of two subunits with the same molecular weight of 39 kD. Its pI was determined to be 4.8. Results of kinetic analysis indicated that the enzyme in the hydrolysis of pNP-NAG followed Michaelis -Menten kinetics with the Michaelis-Menten constant (Km) of 0.11 mmol/L and the maximum velocity (Vm) of 17.65 μmol/L·min, respectively. The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was pH 5.5 and 60℃, respectively. And the activation energy of the enzyme for the hydrolysis of pNP-NAG was determined to be 64.8 kJ/mol. Pb2+, Cu2+, Zn2+ and Al3+ inhibited the enzyme activity in different degrees. 【Conclusion】In this study the enzyme NAGase has been purified and characterized from A. mellifera ligustica, laying a foundation for further unveiling the structure and function of NAGase in bees.

Key words: Apis mellifera ligustica, N-Acetyl-β-D-glucosaminidase, purification, characterization, kinetics