title: Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria.
abstract: Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development.
authors.affiliation: Research Institute of Applied Biology, Shanxi University, Taiyuan, China.|College of Life Science, Shanxi University, Taiyuan, China.
authors.forename: Ying-Long
authors.initials: YL
authors.lastname: Li
authors.affiliation: Research Institute of Applied Biology, Shanxi University, Taiyuan, China.|College of Life Science, Shanxi University, Taiyuan, China.
authors.forename: Hui-Fang
authors.initials: HF
authors.lastname: Song
authors.affiliation: Research Institute of Applied Biology, Shanxi University, Taiyuan, China.
authors.forename: Xue-Yao
authors.initials: XY
authors.lastname: Zhang
authors.affiliation: Research Institute of Applied Biology, Shanxi University, Taiyuan, China.
authors.forename: Da-Qi
authors.initials: DQ
authors.lastname: Li
authors.affiliation: Research Institute of Applied Biology, Shanxi University, Taiyuan, China.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Zhang
authors.affiliation: Research Institute of Applied Biology, Shanxi University, Taiyuan, China.
authors.forename: En-Bo
authors.initials: EB
authors.lastname: Ma
authors.affiliation: Research Institute of Applied Biology, Shanxi University, Taiyuan, China.
authors.forename: Jian-Zhen
authors.initials: JZ
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D048188:Locusta migratoria / Q000201:enzymology*; D011485:Protein Binding; D061987:Sf9 Cells
chemical_list: D019476:Insect Proteins; D002686:Chitin; D002688:Chitinases
keywords: Locusta migratoria; chitinase 5; enzyme kinetics; heterologous expression