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Title of Journal: Appl Microbiol Biotechnol

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Abbravation: Applied Microbiology and Biotechnology

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Springer Berlin Heidelberg

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DOI

10.1007/bf01687862

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1432-0614

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Enhanced extracellular production of Lasparaginas

Authors: Yue Feng Song Liu Yun Jiao Hui Gao Miao Wang Guocheng Du Jian Chen
Publish Date: 2016/10/28
Volume: 101, Issue: 4, Pages: 1509-1520
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Abstract

Lasparaginase EC 3511 ASN exhibits great commercial value due to its uses in the food and medicine industry In this study we reported the enhanced expression of type II ASN from Bacillus subtilis 168 in B subtilis WB600 through a combined strategy First eight signal peptides the signal peptide of the ASN ywbN yvgO amyE oppA vpr lipA and wapA were used for ASN secretion in B subtilis by using Hpa II promoter respectively The signal peptide wapA achieved the highest extracellular ASN activity 2891 U/mL Second Hpa II promoter was replaced by a strong promoter P43 promoter resulting in 381  enhanced ASN activity By two rounds of errorprone PCR mutation the P43 promoter variants with remarkably enhanced strength D7 E2 H6 B2 and F3 were identified B2 −28 A → G −13 A → G achieved ASN activity up to 5113 U/mL Third after deletion of the Nterminal 25residues ASN activity reached 10241 U/mL which was 100  higher than that of the intact ASN At last the extracellular ASN of the B subtilis arrived at 4076 U/mL 25 g/L of ASN protein in a 3L bioreactor by using a fedbatch strategy The purified ASN showed maximal activity at 65 °C and its halflife at 65 °C was 61 min The K m and k cat of the ASN were 529 mM and 544 s−1 respectively To the best of our knowledge we obtained the highest yield of ASN in a foodgrade host ever reported which may benefit the industrial production and application of ASNThis work was supported by the National Natural Science Foundation of China No 31401638 the Natural Science Foundation of Jiangsu Province BK20130132 and the Program for Changjiang Scholars and Innovative Research Team in University No IRT 15R26

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