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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 ZHOU1,2* 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 Received:
7 May 2018/Accepted: 20 March 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
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