Aneutral trehalase gene (NTH1) was isolated from a kEMBL3 genomic library of the insect pathogenic fungus Metarhizium anisopliae. Sequencing of the gene revealed extensive homology with other fungal neutral trehalases. The NTH1 gene exists as a single copy in the genome. Two STREs exist in the 50UTR of NTH1, which may mediate transcriptional activation of the NTH1 gene in response to various stresses. The NTH1 gene encodes a protein of 737 amino acids with a calculated Mr of 83.1kDa. Acyclic adenosine 30,50-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site were found in the amino-terminal domain of NTH1, consistent with a regulatory enzyme. Expression of the trehalase cDNAwas achieved in Saccharomyces cerevisiae. Southern blot analysis of RT-PCR products indicated that the neutral trehalase gene is transcribed in vitro in cell-free haemolymph of the tobacco hornworm Manduca sexta and in vivo in the early stage of infection.

以蝉蜕为底物诱导绿僵菌产生分解昆虫外壳蛋白酶。发酵液经超滤、Ultrogel AcA54凝胶层析、制备IEF电泳，纯化了一种蛋白酶MAP-21，SDS-PAGE电泳后经银染色呈单带。该酶的Mr 为27kD左右，pI为716。它的特异识别氨基酸为Arg，其活性可被PMSF和TLCK抑制，表明其活性中心有Ser和His残基。它还可被胰蛋白酶的典型抑制剂Leu-peptin、Antipain及STI等所抑制，而胰凝乳蛋白酶抑制剂TPCK和胰凝乳弹性蛋白酶抑制剂TEI对其活性无影响。专一底物和抑制剂特性试验结果表明MAP-21是类胰蛋白酶。此外，该酶还可被EDTA所抑制，表明金属离子为其活性所必需。另外还研究了MAP-21的最适作用温度和pH，以及温度耐受性等特性。

Trehalose is the main haemolymph sugar in most insects including the tobacco hornworm, Manduca sexta, and is potentially a prime target for an invading pathogenic fungus. There was considerably more trehalose-hydrolysing activity in the haemolymph of caterpillars infected with Metarhizium anisopliae than in controls. This appeared to be due primarily to additional isoforms; one of which could also hydrolyse maltose and was designated an a-glucosidase. A comparable isoform was identified in in vitro culture of the fungus, supporting a fungal origin for the in vivo enzyme. The in vitro fungal enzyme, a-glucosidase-1 (a-gluc-1), was purified to homogeneity and partially characterised. A study with the trehalase inhibitor trehazolin and C14 trehalose suggested that extracellular hydrolysis is important for fungal mobilisation of trehalose. Haemolymph glucose increases significantly during mycosis of tobacco hornworm larvae by M. anisopliae, consistent with the hydrolysis of trehalose by extracellular fungal enzymes. The implications for the host insect are discussed.

以几丁质为底物，加入基本盐培养基中，诱导球孢白僵菌（Beauveria bassiana）产生分解昆虫表皮的蛋白酶。诱导物中，蝉蜕诱导的蛋白酶总活性、比活较高，经超滤、离子交换层析、亲和层析、制备性IEF电洗脱纯化了一种有凝乳弹性蛋白酶（Prl）活性的蛋白酶BbPrl。经SDS-PAGE电泳银染后呈单带，HILC凝胶过滤显示单峰。BbPrl为单体酶，分子量为33.6kD左右，pI为7.4。底物专一性测定显示，BbPrl能水解Phe或ku形成的酰胺键和肽键。BbPrl可被PMSF抑制，表明其活性中心有Ser残基；BbPrl还可被胰凝乳蛋白酶抑制剂TPCK和凝乳弹性蛋白酶抑制剂TEI等所抑制；胰蛋白酶抑制剂LeupepUin和Epianstatin，及胃蛋白酶抑制剂Pepstain对BbPrl活性无影响。还研究了BbPrl的最适作用pH和pH稳定性。