Abstract: 【Objective】β-N-acetylglucosaminidase (NAG) is a key enzyme involved in degradation of chitin. The objective of this study is to obtain the high purified enzyme, and to analyze the enzymatic characteristics. It will be helpful for the biological function study of LmNAG1 during locust development, and will provide a theoretical and practical basis for developing molecular target of pest control. 【Method】The primers with BamH I, Hind III restriction sites and 6×His tags were designed according to the complete cDNA sequence deposited in GenBank (No. JX888720.1). The target sequence consisting of ORF was amplified by PCR, and then ligated to pFastBacTM-Dual vector after double enzyme digestion. The recombinant plasmid was transformed into E. coli DH10Bac competent cells, and the target gene was transposed to baculovirus genome through Tn7 transposon. The white clone was selected by blue-white selection combined with antibiotics screening and then the recombinant baculovirus plasmid (Bacmid) was verified by bacteria liquid PCR with the pUC/M13 primers. The recombinant Bacmid was transfected into Spodoptera frugiperda ovary cell line Sf9 using transfection reagent. The morphology of cell was observed within 72 h continuously. Once the infected phenomenon occurred, the P1 generation recombinant virions were obtained from the supernatant after cell collection and centrifugation, and then used to infect Sf9 cells again. The proteins obtained after lysing were performed western blot to examine whether the target protein was expressed or not. After that, large amounts of Sf9 cells were infected to extract proteins. The recombinant proteins were purified using Ni-NTA agarose beads and Q ion-exchange chromatography, and the protein concentration was determined according to the method of Bradford. The kinetic parameters of purified enzyme, the optimal pH and temperature were determined using the substrate, 4MU-GlcNAc. 【Result】 The recombinant plasmid pFastBac-LmNAG1 consisting of a full-length cDNA of LmNAG1 (1 845 bp) was verified by double enzyme digestion. Furthermore, it was transformed into DH10Bac competent cells, then combined into the baculovirus genome. The correct recombinant Bacmid selected by PCR was used for Sf9 cell transfection. The signs of transfection including cell enlargement and border irregularity were observed under the microscope after 72 h. The recombinant virions were collected after centrifugation, and used to infect Sf9 cells again. The infected Sf9 cells were collected for protein extraction. From western blot, an obvious band around 67 kD, which was in accordance with the molecular mass of LmNAG1. The fusion protein with 6×His tags was obtained successfully. Large amounts of protein were obtained from the infected cells and purified using Ni-NTA agarose beads followed by Q ion-exchange chromatography after dialysis to collect high purified target proteins. The top protein concentration of E3 fraction was 0.057 µg·µL^-1 determined using Bradford method. The in vitro activity detection showed that LmNAG1 exhibited the maximum activity at pH 8.0, and possessed higher stability when pH at 6.0-8.0. The optimum temperature for LmNAG1 was 40℃, the thermostability was high between 30-40℃. However, the enzyme activity was decreased rapidly when the temperature was higher than 45℃. The Km=(0.28±0.02) mmol·L^-1, Kcat= (902.88±38.15) s^-1, and the results showed that LmNAG1 could efficiently hydrolyze β-1,4 linked chitin oligosaccharide.【Conclusion】 The purified LmNAG1 was obtained in this study, and it has been shown that LmNAG1 is able to degrade β-1,4 linked chitin oligosaccharide. LmNAG1 is involved in chitin degradation, which exhibited the similar biological functions with NAG1 of other insects.

Key words: Locusta migratoria, β-N-acetylglucosaminidase (NAG), chitin degradation, protein expression, enzymatic characteristics