Dubey A K, Suresh C, Kavitha R, Karanth N G, Umesh-Kumar S
Department of Food Microbiology, Central Food Technological Research Institute, Mysore, India.
FEBS Lett. 2000 Apr 14;471(2-3):251-5. doi: 10.1016/s0014-5793(00)01410-1.
A 125-kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673-675] was also found to possess activity towards raw starch. Segregation of these activities in the 71-kDa glucoamylase and a 53-kDa alpha-amylase-like enzyme supported by antibody cross-reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125-kDa starch hydrolysing enzyme as their precursor. N-terminal sequence analysis further revealed that the 71-kDa glucoamylase was the N-terminal product of the precursor enzyme. Immunological cross reactivity of the 53-kDa amylase with antibodies raised against the precursor enzyme but not with the 71- and 61-kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C-terminal fragment of the precursor. The N-terminal sequence of the 53-kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross-reactivity to a 10-kDa non-enzymic peptide and a 61-kDa glucoamylase described these proteins as products of the 71-kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger.
黑曲霉的一种125 kDa淀粉水解酶，其特征在于能够将淀粉糊精化和糖化[苏雷什等人（1999年），《应用微生物学与生物技术》，第51卷，第673 - 675页]，还发现它对生淀粉具有活性。通过抗体交叉反应研究以及基于特定酶形式分泌的测定筛选分离突变体，将这些活性在71 kDa葡萄糖淀粉酶和一种53 kDaα -淀粉酶样酶中进行分离，结果表明125 kDa淀粉水解酶是它们的前体。N端序列分析进一步表明，71 kDa葡萄糖淀粉酶是前体酶的N端产物。53 kDa淀粉酶与针对前体酶产生的抗体具有免疫交叉反应，但与71 kDa和61 kDa葡萄糖淀粉酶抗体没有交叉反应，这表明这种酶活性由前体的C端片段代表。53 kDa蛋白的N端序列与报道的米曲霉Taka淀粉酶相似。对一种10 kDa非酶肽和一种61 kDa葡萄糖淀粉酶的抗体交叉反应表明这些蛋白是71 kDa葡萄糖淀粉酶的产物。在真菌原生质体的蛋白质提取物中仅鉴定出前体淀粉水解酶，这表明细胞周质空间中的蛋白水解加工是黑曲霉分泌多种形式淀粉酶的原因。
