Simultaneous production of alpha and beta amylase enzymes using separate gene bearing recombinant vectors in the same Escherichia coli cells

The present study describes the simultaneous expression of thermostable industrial alpha (alpha) and beta (beta) amylase enzymes that have been used widely in starch industry. Genomic DNA of Bacillus stearothermophilus DSM 22 strain for alpha amylase and, Thermoanaerobacterium (Clostridium) thermosulfurogenes DSM 2229 strain for beta amylase were used as gene sources. Both genes were ligated into pETDuet-1 expression vector separately and resulting recombinant vectors were transformed into Escherichia coli BL21 competent cells by electroporation. The cells were first transformed by pETDuet-1/ alpha Amy recombinant plasmid, then the competent cells carrying this plasmid were prepared for the transformation of pETDuet-1/beta Amy plasmid. Enzymatic activities of bacterial colonies were detected on LB agar staining with iodide. Both enzymes were more produced by IPTG induction in BL21 cells and were purified using Ni-NTA agarose column. SDS-PAGE and western blot analyses showed that the molecular weight of purified alpha and beta amylase to be approximately 60 kDa and 55kDa, respectively. The concentration of the purified alpha and beta amylase were calculated as 4.59 mu g/mL and 3.17 mu g/mL with IPTG as an inducer in LB medium. The present study proposes a novel and efficient method for the production of thermostable alpha and beta amylases at the same E coli cells containing separate engineered plasmid vectors.

Keywords:

Alpha amylase, beta amylase dual gene expression, purification of recombinant enzyme,

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Bertoldo C, 2002, CURR OPIN CHEM BIOL, V6, P151, DOI 10.1016/S1367-5931(02)00311-3
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
CASTRO GR, 1992, BIOTECHNOL LETT, V14, P49, DOI 10.1007/BF01030913
Cowan DA, 1991, BIOTECHNOLOGY SCI BU, P311
Ertugay Z, 2010, ATATURK U ZIRAAT FAK, P173
Gandhi S, 2015, BIOMED RES INT, V2015, DOI 10.1155/2015/529059
Gozukara F, 2009, THESIS .
Gupta R, 2003, PROCESS BIOCHEM, V38, P1599, DOI 10.1016/S0032-9592(03)00053-0
Haki GD, 2003, BIORESOURCE TECHNOL, V89, P17, DOI 10.1016/S0960-8524(03)00033-6
He Zhenggui, 2014, BMC Biotechnol, V14, P114, DOI 10.1186/s12896-014-0114-8
Kiran O., 2005, TURK J BIOL, V29, P99 .
LAEMMLI UK, 1970, NATURE, V227, P680, DOI 10.1038/227680a0
Li S, 2011, APPL BIOCHEM BIOTECH, V164, P581, DOI 10.1007/s12010-011-9159-5
Lin LL, 1997, LETT APPL MICROBIOL, V24, P365, DOI 10.1046/j.1472-765X.1997.00146.x
Liu HL, 2003, COLLOID SURFACE B, V28, P215, DOI 10.1016/S0927-7765(02)00142-X
Lo HF, 2001, CURR MICROBIOL, V43, P170, DOI 10.1007/s002840010282
Ozcan N, 2001, TURK J VET ANIM SCI, V25, P197
Radley JA, 1976, PRODUCTION MICROBIAL, P295
Rajagopalan G, 2008, BIORESOURCE TECHNOL, V99, P3044, DOI 10.1016/j.biortech.2007.06.001
Reddy N. S., 2003, African Journal of Biotechnology, V2, P645
REED G., 1966, ENZYMES FOOD PROCESS .
Sambrook J, 1989, MOL CLONING LAB MANU, P633
Shiina S, 2007, J ELECTROSTAT, V65, P30, DOI 10.1016/j.elstat.2005.03.093
SU P, 1993, BIOTECHNOL LETT, V15, P979, DOI 10.1007/BF00131768
Tatar S, 2007, THESIS .
Yamabhai M, 2008, J BIOTECHNOL, V133, P50, DOI 10.1016/j.jbiotec.2007.09.005
Zafar A, 2019, RSC ADV, V9, P984, DOI 10.1039/c8ra06554c