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CLC number: Q56

On-line Access: 2017-06-05

Received: 2016-03-27

Revision Accepted: 2016-07-21

Crosschecked: 2017-05-08

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Jian-zhong Xu


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Journal of Zhejiang University SCIENCE B 2017 Vol.18 No.6 P.462-473


Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance

Author(s):  Jian-zhong Xu, Wei-guo Zhang

Affiliation(s):  The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China

Corresponding email(s):   xujz126@126.com, zwgjnedu@sina.cn

Key Words:  Menaquinone-7, Bacillus amyloliquefaciens, Analog resistance, Diphenylamine resistance, Maize meal hydrolysate

Jian-zhong Xu, Wei-guo Zhang. Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance[J]. Journal of Zhejiang University Science B, 2017, 18(6): 462-473.

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author="Jian-zhong Xu, Wei-guo Zhang",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

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%T Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance
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%A Wei-guo Zhang
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%DOI 10.1631/jzus.B1600127

T1 - Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance
A1 - Jian-zhong Xu
A1 - Wei-guo Zhang
J0 - Journal of Zhejiang University Science B
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PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B1600127

A menaquinone-7 (MK-7) high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK-7 high-producing strains via screening and mutagenesis by an atmospheric and room temperature plasma (ARTP) mutation breeding system. We isolated an MK-7-producing strain Y-2 and identified it as Bacillus amyloliquefaciens, which produced (7.1±0.5) mg/L of MK-7 with maize meal hydrolysate as carbon source. Then, an MK-7 high-producing strain B. amyloliquefaciens H.β.D.R.-5 with resistance to 1-hydroxy-2-naphthoic acid, β-2-thienylalanine, and diphenylamine was obtained from the mutation of the strain Y-2 using an ARTP mutation breeding system. Using strain H.β.D.R.-5, efficient production of MK-7 was achieved ((30.2±2.7) mg/L). In addition, the effects of nitrogen sources, prenyl alcohols, and MgSO4 on MK-7 production were investigated, suggesting that soymeal extract combined with yeast extract, isopentenol, and MgSO4 was beneficial. Under the optimized condition, the MK-7 production and biomass-specific yield reached (61.3±5.2) mg/L and 2.59 mg/L per OD600 unit respectively in a 7-L fermenter. These results demonstrated that strain H.β.D.R.-5 has the capacity to produce MK-7 from maize meal hydrolysate, which could reduce the substrate cost.


创新点:首次在中国的发酵豆制品--豆豉中分离得到一株能以玉米水解液为底物合成MK-7的解淀粉芽孢杆菌Y-2(Bacillus amyloliquefaciens Y-2),并通过传统诱变育种获得一株带有二苯胺和结构类似物抗性的、以玉米水解液为底物的、高产MK-7的菌株Bamyloliquefaciens H.β.D.R.-5。
方法:以来自中国不同省市地区的豆豉为分离样品,筛选高产纳豆激酶的菌株,再从中挑选出高产MK-7的菌株,并通过16S rDNA分析对其种属进行鉴定。采用常压室温等离子体(ARTP)系统,对分离到的高产MK-7菌株进行诱变处理,获得解除3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶(即结构类似物抗性)和聚丙烯焦磷酸合成酶(即二苯胺抗性)反馈调节的菌株。最后,考察不同氮源、乙戊烯醇和镁离子(Mg2+)对突变菌株合成MK-7的影响,并分析在7 L发酵罐中合成MK-7的区别。
结论:从中国豆豉中分离到了一株以玉米水解液为底物合成MK-7的菌株,经16S rDNA分析比对,鉴定为Bacillus amyloliquefaciens(图1)。通过比较MK-7产量,发现利用ARTP可以有效获得解除反馈调节作用的且高产MK-7的突变菌株H.β.D.R.-5(表1)。以大豆水解液和酵母水解液为氮源,异戊醇和MgSO4有利于突变菌株H.β.D.R.-5合成MK-7(图2、表2和表3)。综上所述,利用ARTP处理从中国豆豉中分离到的以玉米水解液为底物的合成MK-7的菌株,可获得高产的MK-7菌株,该方法对选育工业化合成MK-7的菌株有重要参考价值。


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[32]List of electronic supplementary materials

[33]Fig. S1 Biosynthetic pathway of MK-7 and regulation mechanism by inhibition of aromatic amino acids and diphenylamine (Armougom et al., 2009)

[34]Fig. S2 Mutation rate and lethality rate of B. amyloliquefaciens Y-2 by ARTP

[35]Fig. S3 Cell growth, MK-7 production, and sugar utilization of the mutant B. amyloliquefaciens H.β.D.R.-5 after several generations

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