Chen H M, Ford C, Reilly P J
Department of Chemical Engineering, Iowa State University, Ames 50011, USA.
Protein Eng. 1995 Jun;8(6):575-82. doi: 10.1093/protein/8.6.575.
Both native Aspergillus niger glucoamylase and wild-type Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae, which have identical primary structures, undergo hydrolysis at aspartyl bonds at low pH values and elevated temperatures. In native A.niger enzyme the Asp126-Gly127 bond was preferentially cleaved at pH 3.5, while at pH 4.5 cleavage of the Asp257-Pro258 and Asp293-Gly294 bonds was dominant. In wild-type A.awamori glucoamylase, cleavage of the latter was dominant at both pH 3.5 and 4.5. Site-directed mutations Asp126-->Glu and Gly127-->Ala in wild-type enzyme decreased specific activities by approximately 60 and 30%, respectively, and increased irreversible thermoinactivation rates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Glu and Asp293 with Glu or Gln decreased specific activities by approximately 20%, but greatly reduced cleavage of the Asp257-Pro258 and Asp293-Gly294 bonds. The Asp257-->Glu mutant was produced very slowly and was more thermostable than wild-type glucoamylase at pH 4.5 up to 70 degrees C. Replacement of Asp293 with either Glu or Gln significantly raised protein production and slightly increased thermostability at pH 3.5 and 4.5, but not at pH 5.5.
在酿酒酵母中表达的天然黑曲霉葡萄糖淀粉酶和野生型泡盛曲霉葡萄糖淀粉酶，它们具有相同的一级结构，在低pH值和高温下会在天冬氨酰键处发生水解。在天然黑曲霉酶中，Asp126-Gly127键在pH 3.5时优先被切割，而在pH 4.5时，Asp257-Pro258和Asp293-Gly294键的切割占主导。在野生型泡盛曲霉葡萄糖淀粉酶中，后者的切割在pH 3.5和4.5时均占主导。野生型酶中的定点突变Asp126→Glu和Gly127→Ala分别使比活性降低了约60%和30%，并在pH 4.5时使不可逆热失活速率提高了3至4倍。用Glu取代Asp257以及用Glu或Gln取代Asp293使比活性降低了约20%，但大大减少了Asp257-Pro258和Asp293-Gly294键的切割。Asp257→Glu突变体产生非常缓慢，并且在pH 4.5至70℃时比野生型葡萄糖淀粉酶更耐热。用Glu或Gln取代Asp293显著提高了蛋白质产量，并在pH 3.5和4.5时略微提高了热稳定性，但在pH 5.5时没有提高。
