title: Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy.
abstract: L-asparaginase (EC 3.5.1.1, 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 (28.91 U/mL). Second, Hpa II promoter was replaced by a strong promoter, P43 promoter, resulting in 38.1 % enhanced ASN activity. By two rounds of error-prone 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 51.13 U/mL. Third, after deletion of the N-terminal 25-residues, ASN activity reached 102.41 U/mL, which was 100 % higher than that of the intact ASN. At last, the extracellular ASN of the B. subtilis arrived at 407.6 U/mL (2.5 g/L of ASN protein) in a 3-L bioreactor by using a fed-batch strategy. The purified ASN showed maximal activity at 65 °C and its half-life at 65 °C was 61 min. The Kmand kcatof the ASN were 5.29 mM and 54.4 s-1, respectively. To the best of our knowledge, we obtained the highest yield of ASN in a food-grade host ever reported, which may benefit the industrial production and application of ASN.
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors: Yue
authors: Y
authors: Feng
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. liusong@jiangnan.edu.cn.|School of Biotechnology, Jiangnan University, Wuxi, 214122, China. liusong@jiangnan.edu.cn.
authors: Song
authors: S
authors: Liu
authors: School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors: Yun
authors: Y
authors: Jiao
authors: School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors: Hui
authors: H
authors: Gao
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
authors: Miao
authors: M
authors: Wang
authors: School of Biotechnology, Jiangnan University, Wuxi, 214122, China.|The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
authors: Guocheng
authors: G
authors: Du
authors: School of Biotechnology, Jiangnan University, Wuxi, 214122, China. jchen@jiangnan.edu.cn.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. jchen@jiangnan.edu.cn.
authors: Jian
authors: J
authors: Chen
mesh_terms: D001215:Asparaginase / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001215:Asparaginase
keywords: Bacillus subtilis; L-Asparaginase; N-terminal deletion; Promoter; Secretory expression; Signal peptide