TmChi is not acquired from food and concentrates in the anterior part of midgut contents. The enzyme is unstable, due to attack by the proteolytic enzymes of the insect, and is stabilized in the presence of E64 and PMSF in homogenizing and chromatographic buffers. TmChi is purified by sequential chromatographies in HiTrap Phenyl, Resource Phenyl and Resource Q columns in FPLC system, with only one major activity being eluted in all steps. After second Resource Q chromatography, a protein with 44 kDa is recovered, with an yield of 11% and 135 times enrichment. TmChi has low activity against colloidal chitin, and is strongly inhibited by high concentrations of the substrates 4-methyl-umbelliferyl-b-N',N'-diacetyl-chitobioside (MUC2) and 4-methyl-umbelliferyl-b-N',N',N'-triacetylchitotrioside (MUC3). Based on kinetic parameters, the best synthetic and natural substrates of this enzyme are MUC3 and chitopentaose, respectively. TmChi shows an endolytic oligosaccharide cleavage pattern. It has two ionizable groups involved in catalysis, with pK_E1 = 5.78 ± 0.08 and pK_E2 = 7.35 ± 0.07. Substrate binding shifts ionization constants of these groups to pK_ES1 = 5.12 ± 0.01 and pK_ES2 = 7.62 ± 0.02. Chemical modification experiments showed that TmChi has at least one carboxylate group and one Trp residue at its active site. TmChi activity against 4-methyl-umbelliferyl-b-N-acetylglucosamine is not affected by phenylglyoxal, diethylpyrocarbonate, tetranitromethane or EDTA, ruling out the presence of Arg, His and Tyr residues and metal cations at the enzyme active site.