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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",
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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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T1 - Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance
A1 - Jian-zhong Xu
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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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[1]Armougom, F., Bittar, F., Stremler, N., et al., 2009. Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing. Eur. J. Clin. Microbiol. Infect. Dis., 28(9):1151-1154.

[2]Berenjian, A., Mahanama, R., Talbot, A., et al., 2011. Efficient media for high menaquinone-7 production: response surface methodology approach. New Biotechnol., 28(6): 665-672.

[3]Chen, J.N., Yang, W.S., Dick, K., et al., 2008. Tip-enhanced Raman scattering of p-thiocresol molecules on individual gold nanoparticles. Appl. Phys. Lett., 92:093110.

[4]Chen, Z.M., Li, Q., Liu, H.M., et al., 2010. Greater enhancement of Bacillus subtilis spore yields in submerged cultures by optimization of medium composition through statistical experimental designs. Appl. Microbiol. Biotechnol., 85(5):1353-1360.

[5]Fernandez, F., Collins, M.D., 1987. Vitamin K composition of anaerobic gut bacteria. FEMS Microbiol. Lett., 41(2): 175-180.

[6]Fujii, H., Sagami, H., Koyama, T., et al., 1980. Variable product specificity of solanesyl pyrophosphate synthetase. Biochem. Biophys. Res. Commun., 96(4):1648-1653.

[7]AQSIQ (General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China), SAC (Standardization Administration of the People’s Republic of China), 2007. GB/T 20885-2007: Glucose Syrup.

[8]Howard, L.M., Payne, A.C., 2006. Health Benefits of Vitamin K2: A Revolutionary Natural Treatment for Heart Disease and Bone Loss. Basic Health Publications, California.

[9]Kim, Y.K., Kim, S.M., Kim, J.Y., et al., 2011. The culture filtrates from Bacillus subtilis natto lowers blood pressure via renin-angiotensin system in spontaneously hypertensive rats fed with a high-cholesterol diet. J. Korean Soc. Appl. Biol. Chem., 54(6):959-965.

[10]Li, H.G., Ofosu, F.K., Li, K.T., et al., 2014. Acetone, butanol, and ethanol production from gelatinized cassava flour by a new isolates with high butanol tolerance. Bioresour. Technol., 172:276-282.

[11]Li, H.P., Wang, L.Y., Li, G., et al., 2011. Manipulation of lipase activity by the helium radio-frequency, atmospheric-pressure glow discharge plasma jet. Plasma Proc. Polym., 8(3):224-229.

[12]Liu, Y., Zhang, Z.M., Qiu, H.W., et al., 2014. Surfactant supplementation to enhance the production of vitamin K2 metabolites in shake flask cultures using Escherichia sp. mutant FM3-1709. Food Technol. Biotechnol., 52(3): 269-275.

[13]Lorenzi, V., Muselli, A., Bernardini, A.F., et al., 2009. Geraniol restores antibiotic activities against multidrug-resistant isolates from Gram-negative species. Antimicrob. Agents Chemother., 53(5):2209-2211.

[14]Patil, S.R., Dayanand, A., 2006. Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-state conditions. Bioresour. Technol., 97(18):2340-2344.

[15]Rosa-Putra, S., Hemmerlin, A., Epperson, J., et al., 2001. Zeaxanthin and menaquinone-7 biosynthesis in Sphingobacterium multivorum via the methylerythritol phosphate pathway. FEMS Microbiol. Lett., 204(2):347-353.

[16]Sagami, H., Ogura, K., Seto, S., 1977. Solanesyl pyrophosphate synthetase from Micrococcus lysodeikticus. Biochemistry, 16(21):4616-4622.

[17]Saito, Y., Ogura, K., 1981. Biosynthesis of menaquinones. Enzymatic prenylation of 1,4-dihydroxy-2-naphthoate by Micrococcus luteus membrane fractions. J. Biochem., 89(5):1445-1452.

[18]Sato, T., Yamada, Y., Ohtani, Y., et al., 2001a. Efficient production of menaquinone (vitamin K2) by a menadione-resistant mutant of Bacillus subtilis. J. Ind. Microbiol. Biotech., 26(3):115-120.

[19]Sato, T., Yamada, Y., Ohtani, Y., et al., 2001b. Production of menaquinone (vitamin K2)-7 by Bacillus subtilis. J. Biosci. Bioeng., 91(1):16-20.

[20]Song, J.Y., Liu, H.X., Wang, L., et al., 2014. Enhanced production of vitamin K2 from Bacillus subtilis (natto) by mutation and optimization of the fermentation medium. Braz. Arch. Biol. Technol., 57(4):606-612.

[21]Takahashi, I., Ogura, K., Seto, S., 1980. Heptaprenyl pyrophosphate synthetase from Bacillus subtilis. J. Biol. Chem., 255:4539-4543.

[22]Tani, Y., Asahi, S., Yamada, H., 1985. Production of menaquinone (vitamin K2)-5 by a hydroxynaphthoate-resistant mutant derived from Flavobacterium meningosepticum, a menaquinone-6 producer. Agric. Biol. Chem., 49(1): 111-115.

[23]Tsukamoto, Y., Kasai, M., Kakuda, H., 2001. Construction of a Bacillus subtilis (natto) with high productivity of vitamin K2 (menaquinone-7) by analog resistance. Biosci. Biotechnol. Biochem., 65(9):2007-2015.

[24]Unnanuntana, A., Bonsignore, L., Shirtliff, M.E., et al., 2009. The effects of farnesol on Staphylococcus aureus biofilms and osteoblasts. An in vitro study. J. Bone Joint. Surg. Am., 91(11):2683-2692.

[25]Walther, B., Karl, J.P., Booth, S.L., et al., 2013. Menaquinones, bacteria, and the food supply: the relevance of dairy and fermented food products to vitamin K requirements. Adv. Nutr., 4:463-473.

[26]Wee, Y.J., Reddy, L.V.A., Ryu, W.H., 2008. Fermentative production of L(+)-lactic acid from starch hydrolyzate and corn steep liquor as inexpensive nutrients by batch culture of Enterococcus faecalis RKY1. J. Chem. Technol. Biotechnol., 83(10):1387-1393.

[27]Wu, W.J., Ahn, B.Y., 2011. Isolation and identification of Bacillus amyloliquefaciens BY01 with high productivity of menaquinone for cheonggukjang production. J. Korean Soc. Appl. Biol. Chem., 54(5):783-789.

[28]Xu, J.Z., Han, M., Zhang, J.L., et al., 2014. Metabolic engineering Corynebacterium glutamicum for the L-lysine production by increasing the flux into L-lysine biosynthetic pathway. Amino Acids, 46(9):2165-2175.

[29]Yanagisawa, Y., Sumi, H., 2005. Natto bacillus contains a large amount of water-soluble vitamin K (menaquinone-7). J. Food Biochem., 29(3):267-277.

[30]Zhang, X., Zhang, X.F., Li, H.P., et al., 2014. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl. Microbiol. Biotechnol., 98(12): 5387-5396.

[31]Zhang, X., Zhang, C., Zhou, Q.Q., et al., 2015. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis. Appl. Microbiol. Biotechnol., 99(13): 5639-5646.

[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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