International Journal of Applied Microbiology and Biotechnology Research
ISSN: 2053-1818
Vol. 9(6), pp. 110-121, December, 2021
doi.org/10.33500/ijambr.2021.09.010



Maximization of extracellular mannanase KMAN production from the recombinant Escherichia coli KMAN-3 by auto-induction system

Piyanat Damrongteerapap1, Nawapan Pongsapipatana1, Oraya Sobprasong1, Wilawan Sintuprapa1, Achara Chaiongkarn2, Kittipong Rattanaporn1 and Sunee Nitisinprasert1*

1Specialized Research Unit: Prebiotics and Probiotics for Health, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, 50 Paholyothin Rd., Chatuchak, Bangkok 10900, Thailand.
2Thailand Institute of Scientific and Technological Research (TISTR), 35 Mu 3 Tambon Khlong Ha, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.

*To whom correspondence should be addressed. E-mail: fagisnn@ku.ac.th.

Received 02 October, 2021; Received in revised form 15 November, 2021; Accepted 18 November, 2021.

Abstract


Keywords:
Mannan degrading enzyme, Recombinant strain, Auto-induction, Lactose inducer.

Most of the recombinant proteins expressed in Escherichia coli were typically employed to isolate proteins from either cytoplasm or periplasm. Optimization of mannanase from the recombinant Escherichia coli KMAN-3 by induction system to obtain extracellular enzyme was presented. Three media as Luria-Bertani (LB), mM9 and mM9NG were studied with enzyme production from mM9 40% cheaper than the others were obtained. Further study on three induction systems of IPTG (IP), lactose (L) and lactose auto-induction (LA) resulted in maximum total mannanase activities of 77.90±1.23, 33.44±0.48 and 61.64±0.01 U/ml, respectively at 18-24 h, correlating with results of transcriptional analysis of the kman-3 gene, except for the LA treatment that presented disparities of its transcription and translation. The lactose auto-induction system showed higher extracellular relative activities of 69.39% while the other two displayed high intracellular activities of 54.16-83.69%. Supplementation of 0.05% (w/v) glucose in mM9 medium gave the highest mannanase production rate of 3.94±0.31 U/ml∙h, while induction temperatures of 26 and 30°C showed higher production rates than 20 and 37°C at 1.26 and 3.96 times, respectively. Results suggested the potential of auto-induction using lactose as a cheap inducer to facilitate upscaled major extracellular mannanase production to generate a value-added product such as manno-oligosaccharides in the future.

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