Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 68, Issue 3, Pages 818-825Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.9b06246
Keywords
beta-mannanase; Thermoanaerobacterium aotearoense; plate assay; thermostability improvement
Funding
- National Key R&D Program of China [2018YFA0901504]
- National Natural Science Foundation of China [31800063, 21878104, U1701243]
- Science & Technology Planning Project of Guangdong Province, China [2017A010105019]
- Natural Science Foundation of Guangdong Province, China [2019A1515011702]
- Project on the Integration of Industry, Education, and Research of Guangzhou, China [201704020183, 201903010086]
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beta-Mannanase was expressed in Thermoanaerobacterium aotearoense SCUT27 induced by locust bean gum (LBG). The open reading frame encoding a GH26 beta-mannanase was identified and encoded a preprotein of 515 amino acids with a putative signal peptide. The enzyme without a signal sequence (Man25) was overexpressed in Escherichia coli with a specific activity of 1286.2 U/mg. Moreover, a facile method for beta-mannanase activity screening was established based on agar plates. The optimum temperature for the purified Man25 using LBG as a substrate was 55 degrees C. The catalytic activity and thermostability of Man25 displayed a strong dependence on calcium ions. Through saturation mutagenesis at the putative Ca2+ binding sites in Man25, the best mutant ManM3-3 (D143A) presented improvements in thermostability with 3.6-fold extended half-life at 55 degrees C compared with that of the wild-type. The results suggest that mutagenesis at metal binding sites could be an efficient approach to increase enzyme thermostability.
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