Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties

被引:7
作者
Trabelsi, Sahar [1 ]
Sahnoun, Mouna [1 ]
Elgharbi, Fatma [1 ]
Ameri, Rihab [1 ]
Ben Mabrouk, Sameh [1 ]
Mezghani, Monia [1 ]
Hmida-Sayari, Aida [1 ]
Bejar, Samir [1 ]
机构
[1] Univ Sfax, CBS, LMBEE, Sidi Mansour Rd Km 6,POB 1177, Sfax 3018, Tunisia
关键词
AmyA isoform amylase; Aspergillus oryzae; Pichia pastoris; Thermostability; Glycosylation; RECOMBINANT ALPHA-AMYLASE; BIOCHEMICAL-CHARACTERIZATION; BACILLUS-ACIDICOLA; GENE; OVEREXPRESSION; GLYCOSYLATION; PHYTASE; CLONING; STRAIN; CLEAVAGE;
D O I
10.1007/s11033-018-4548-2
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60 degrees C against 50 degrees C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60 degrees C, r-AmyA reached 45min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4mM Fe2+ and Mg2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.
引用
收藏
页码:921 / 932
页数:12
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