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Secretory production of enzymatically active endo-β-1,4-mannanase from Bacillus subtilis by ABC exporter in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
34146 45005 2016 7 PDF Available
Title
Secretory production of enzymatically active endo-β-1,4-mannanase from Bacillus subtilis by ABC exporter in Escherichia coli
Abstract

•ABC exporter and secretion signals of P. fluorescens were reconstituted in E. coli.•The enzymatically active mannanase with optimized signal sequence was secreted by ABC exporter.•The uncleaved signal sequences did not alter the enzymatic properties of mannanase.

To secrete the endo-β-1,4-mannanase of Bacillus subtilis WL7 in Escherichia coli, the ABC exporter (TliDEF) and the C-terminal signal sequences of lipase (TliA) and metalloprotease (PrtA) were used. Six C-terminal fragments of TliA (TliAC1, TliAC2 and TliAC3) and PrtA (PrtAC1, PrtAC2 and PrtAC3) were amplified by PCR, respectively, and were fused to the mature mannanase gene lacking its original N-terminal signal sequence. When coexpressed with TliDEF exporter gene in E. coli, all the fusion mannanases were successfully secreted to the culture supernatant as an enzymatically active form by TliDEF exporter in E. coli. However, the unspecific cell leakage occurred in the fusion mannanases containing TliA-derived signal sequences. Among the fusion mannanases containing the signal sequences of PrtA, the mannanase with the shortest signal sequence fragment (Man-PrtAC3) exhibited the highest secretion level (4.65 mg/L). Moreover, the specific activity, the effect of temperature and pH on activity and stability were almost identical to the wild-type mannanase, indicating that the fused signal sequence of PrtA did not affect the enzymatic properties of mannanase. It was concluded that the efficient secretion of enzymatically active mannanase from Bacillus subtilis WL-7 was achieved by using PrtAC3 as the most adequate signal sequence fragment.

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Keywords
Type I secretion system; Pseudomonas fluorescens ABC exporter; Bacillus subtilis endo-β-1; 4-mannanase; Secretory production in Escherichia coli; C-terminal signal sequence
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Secretory production of enzymatically active endo-β-1,4-mannanase from Bacillus subtilis by ABC exporter in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 51, Issue 8, August 2016, Pages 999–1005
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us