Sains Malaysiana 48(5)(2019): 965–974

http://dx.doi.org/10.17576/jsm-2019-4805-04

 

Yield Enhancement of Recombinant α-Amylases in Bacillus amyloliquefaciens by ARTP Mutagenesis-Screening and Medium Optimization

(Hasil Peningkatan Rekombinan α-Amilase dalam Bacillus amyloliquefaciens ARTP Mutagenesis-Penyaringan dan Pengoptimuman Sederhana)

 

TING-LIANG XU¹, JING PENG¹, YU-LING ZHU¹, SU LI¹, KAI-YAN ZHOU¹, HAI-NA CHENG¹, SHI-ZHE TANG¹ & HONG-BO ZHOU^1,2*

 

¹School of Minerals Processing and Bioengineering, Central South University, 410083 No.932 South Lushan Road, Changsha, China

 

²Key Laboratory of Biometallurgy of Ministry of Education, Central South University, 410083 No.932 South Lushan Road, Changsha, China

 

Diserahkan: 7 Mei 2018/Diterima: 20 Mac 2019

 

ABSTRACT

α-Amylase is the most extensively applied enzyme in industry. There is an urgent need for improvement on the yield of α-amylases currently. Herein, a strategy which combined Atmospheric and Room Temperature Plasma (ARTP) mutagenesis tool for construction of mutant library of Bacillus amyloliquefaciens with a 24-well plates screening technique was adopted to improve the yield of recombinant Bacillus amyloliquefaciens α-amylases (BAA). A mutant strain named B. amyloliquefaciens ZN mut-7# was obtained, and the activity of BAA produced by this mutant strain was 86.92% higher than that of the original strain. B. amyloliquefaciens ZN mut-7# has an unchanged BAA gene and genetic stability. This successful application proved that ARTP can be applied to the genetically engineering strains that contain recombinant plasmid. Furthermore, response surface methodology offers an achievable and efficient strategy to optimize the composition of medium used to generate BAA in B. amyloliquefaciens ZN mut-7#. A 1.28-fold increase had been obtained compared to the production of non-optimized fermentation medium. This study demonstrates that ARTP mutagenesis and medium optimization are efficient and feasible methods for increasing recombinant enzyme production in the genetically engineering strains.

 

Keywords: ARTP mutagenesis; Bacillus amyloliquefaciens α-amylases; medium optimization; response surface methodology

 

ABSTRAK

α-Amilase adalah enzim yang diaplikasi secara meluas dalam industri. Terdapat keperluan segera untuk menambah baik hasil daripada α-amilase pada masa ini. Di sini, satu strategi yang menggabungkan alat mutagenesis atmosfera dan plasma suhu bilik (ARTP) untuk pembinaan perpustakaan mutan Bacillus amyloliquefaciens dengan teknik saringan plat 24-telaga yang telah diguna pakai untuk meningkatkan hasil recombinan Bacillus amyloliquefaciens α-amylases (BAA). Terikan mutan yang dipanggil B. amyloliquefaciens ZN # mut-7 telah diperoleh dan aktiviti BAA yang dihasilkan oleh terikan mutan ini adalah 86.92% lebih tinggi daripada terikan asal. B. amyloliquefaciens ZN mut-7 # mempunyai gen BAA tidak berubah dan kestabilan genetik. Aplikasi ini berjaya membuktikan bahawa ARTP boleh digunakan untuk kejuruteraan genetik terikan yang mengandungi plasmid recombinan. Selain itu, kaedah gerak balas permukaan menawarkan strategi yang boleh dicapai dan cekap untuk mengoptimumkan komposisi medium yang digunakan untuk menjana BAA dalam B. amyloliquefaciens ZN # mut-7. Peningkatan sebanyak 1.28-lipatan telah diperoleh berbanding pengeluaran sederhana penapaian tidak optimum. Kajian ini menunjukkan bahawa mutagenesis ARTP dan pengoptimuman sederhana adalah kaedah yang cekap dan boleh dilaksanakan untuk meningkatkan penghasilan enzim recombinan dalam kejuruteraan genetic terikan.

 

Kata kunci: Bacillus amyloliquefaciens α-amilase; mutagenesis ARTP; pengoptimuman sederhana; kaedah gerak balas permukaan

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*Pengarang untuk surat-menyurat; email: zhouhb@csu.edu.cn