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Expression of aspergillus niger N5-5 in E. coli and purification and identification of products
المؤلفون المشاركون
Zhang, Shuai
Cao, Yong
Cheng, Hao
المصدر
Saudi Journal of Biological Sciences
العدد
المجلد 24، العدد 8 (31 ديسمبر/كانون الأول 2017)، ص ص. 1842-1848، 7ص.
الناشر
تاريخ النشر
2017-12-31
دولة النشر
السعودية
عدد الصفحات
7
التخصصات الرئيسية
العلوم الطبيعية والحياتية (متداخلة التخصصات)
الملخص EN
Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields.
However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a largescale.
Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper Aspergillus niger N5-5 was expressed using E.
coli system.
Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.
: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification.
Then, tannase gene tan was connected to expression vector by NdeI/HindIII enzyme cutting.
In this way, recombinant expression vector tan-pET43.1a was formed.
Then, the expression vector pET43.1a by NdeI / HindIII enzyme cutting was transformed into E.
coli BL21 (DE3) to induce expression of Aspergillus niger N5-5.
When the induced fungi were disrupted by the ultrasonic wave, the crude enzyme was extracted and purified by using the IMAC, and then the activity of the crude enzyme and pure enzyme was determined.
According to the results of determination of the tannase activity, the tannase activity of the crude enzyme was greatly improved after the crude enzyme was purified, and the specific activity of the pure enzyme was about 8 times of that of the crude enzyme.
The results of SDS-PAGE of the pure enzyme showed that the molecular mass of the pure enzyme was about 65 kDa / 64–65 kDa, which was consistent with the expected result (64.2 kDa), It can be concluded that the crude enzyme solution was purified successfully.
The results of pure enzyme’s protein identification by Western Blotting showed that clear protein bands pro-3 were observed.
Molecular mass of clear protein bands pro-3 was about 65 kDa, which was in line with the expected results (64.2 kDa).
It can be seen that the aforementioned expression protein could be specifically combined with His tag.
It proved expression protein to be a recombinant fusion protein with 6 His tag.
نمط استشهاد جمعية علماء النفس الأمريكية (APA)
Zhang, Shuai& Cao, Yong& Cheng, Hao. 2017. Expression of aspergillus niger N5-5 in E. coli and purification and identification of products. Saudi Journal of Biological Sciences،Vol. 24, no. 8, pp.1842-1848.
https://search.emarefa.net/detail/BIM-780610
نمط استشهاد الجمعية الأمريكية للغات الحديثة (MLA)
Zhang, Shuai…[et al.]. Expression of aspergillus niger N5-5 in E. coli and purification and identification of products. Saudi Journal of Biological Sciences Vol. 24, no. 8 (Dec. 2017), pp.1842-1848.
https://search.emarefa.net/detail/BIM-780610
نمط استشهاد الجمعية الطبية الأمريكية (AMA)
Zhang, Shuai& Cao, Yong& Cheng, Hao. Expression of aspergillus niger N5-5 in E. coli and purification and identification of products. Saudi Journal of Biological Sciences. 2017. Vol. 24, no. 8, pp.1842-1848.
https://search.emarefa.net/detail/BIM-780610
نوع البيانات
مقالات
لغة النص
الإنجليزية
الملاحظات
Includes bibliographical references : p. 1848
رقم السجل
BIM-780610
قاعدة معامل التأثير والاستشهادات المرجعية العربي "ارسيف Arcif"
أضخم قاعدة بيانات عربية للاستشهادات المرجعية للمجلات العلمية المحكمة الصادرة في العالم العربي
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