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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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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Jian-zhong Xu

http://orcid.org/0000-0001-6555-6059

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

http://doi.org/10.1631/jzus.B1600127


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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Abstract: 
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.

带有二苯胺和结构类似物抗性的芽孢杆菌突变株以玉米水解液为底物合成甲萘醌-7的研究

目的:通过从自然界中筛选和传统诱变育种相结合的方法,获得一株以玉米水解液为底物且能高效合成甲萘醌-7(MK-7)的芽孢杆菌突变菌株。
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。

关键词:甲萘醌-7;解淀粉芽孢杆菌;类似物抗性;二苯胺抗性;玉米水解液

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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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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