Tolerance mechanism of Aspergilus niger C2J6 to cyclohexanone

doi:10.11949/j.issn.0438-1157.20170277

Abstract

Abstract:

The mycelium growth and enzyme activity of Aspergillus niger C2J6 in organic solvent are related to cell tolerance in the solvent. In order to reveal the regulation and mechanism of organic solvent tolerance, fifteen kinds of organic solvents were used to investigate the organic solvent tolerance of Aspergillus niger C2J6. Then the tolerance mechanism for cyclohexanone, which is a less hydrophobic solvent, was studied in the cellular level. The results indicate that Aspergillus niger C2J6 can grow in a variety of organic solvents for enzyme production. Organic solvents whose lgP are between -0.24 and 3.0 will inhibit the cell growth and enzyme production to different degrees, whereas organic solvents whose lgP are greater than 3.0 will promote the growth and production. Under the stress of cyclohexanone, the cell membrane is damaged. The intracellular organelles shrunk. The hydrophobicity of cell surface decreased. Extracellular nucleotide concentration increased. The content of unsaturated fatty acid decreased while the content of saturated fatty acid increased. These phenomena illustrate that cellular tolerance to cyclohexanone is influenced in many aspects, and there are variety of tolerance mechanisms working together which help to reduce the toxicity of cyclohexanone on the cells, so as to improve the tolerance of the microbes.

Key words: Aspergillus niger, lipase, organic solvent, cyclohexanone, cell tolerent mechanism

摘要：

鉴于黑曲霉C2J6在有机溶剂中的菌体生长和酶活性与其细胞耐受性有关，为揭示菌体对有机溶剂的耐受规律和耐受性机制，考察了菌体对15种有机溶剂的耐受性，并从细胞水平上探究了菌体在疏水性较低的环己酮中的耐受性机制。结果表明：黑曲霉C2J6可以在多种有机溶剂中生长产酶，lgP为-0.24~3.0的有机溶剂对菌体生长及产酶有不同程度的抑制作用，而lgP>3.0的有机溶剂则起到相应的促进作用。环己酮胁迫下，细胞膜表面出现破损，胞内细胞器有皱缩现象，细胞表面疏水性下降，胞外核苷酸浓度上升，细胞膜不饱和脂肪酸含量降低，饱和脂肪酸含量增加。说明菌体对环己酮的耐受性涉及多个方面，多种耐受机制同时存在，使环己酮对细胞的毒害大大降低，从而提高了微生物的耐受能力。

关键词: 黑曲霉, 脂肪酶, 有机溶剂, 环己酮, 细胞耐受性机制

CLC Number:

SU Xuelin, ZHANG Zihan, LIU Ya. Tolerance mechanism of Aspergilus niger C2J6 to cyclohexanone[J]. CIESC Journal, 2017, 68(8): 3218-3224.

苏雪林, 张梓涵, 刘娅. 黑曲霉C2J6对环己酮的细胞耐受性机制[J]. 化工学报, 2017, 68(8): 3218-3224.

References 30

[1]	MARIANO A P, QURESHI N, EZEJI T C. Bioproduction of butanol in bioreactors:new insights from simultaneous in situ butanol recovery to eliminate product toxicity[J]. Biotechnology and Bioengineering, 2011, 108(8):1757-1765.
[2]	STANCU M M. Physiological cellular responses and adaptations of Rhodococcus erythropolis IBB Po1 to toxic organic solvents[J]. Journal of Environmental Sciences, 2014, 26(10):2065-2075.
[3]	BAHARUM S N, RAHMAN R N Z R A, BASRI M, et al. Chaperone-dependent gene expression of organic solvent-tolerant lipase from Pseudomonas aeruginosa strain S5[J]. Process Biochemistry, 2010, 45(3):346-354.
[4]	SANDERS A, COLLIER R, TRETHEWY A, et al. AAP1 regulates import of amino acids into developing Arabidopsis embryos[J]. The Plant Journal, 2009, 59(4):540-552.
[5]	LIMA V M G, KRIEGER N, MITCHELL D A, et al. Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents[J]. Biochemical Engineering Journal, 2004, 18(1):65-71.
[6]	SⅡROLA E, GRISCHEK B, CLAY D, et al. Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents[J]. Biotechnology and Bioengineering, 2011, 108(12):2815-2822.
[7]	KABELITZ N, SANTOS P M, HEIPIEPER H J. Effect of aliphatic alcohols on growth and degree of saturation of membrane lipids in Acinetobacter calcoaceticus[J]. FEMS Microbiology Letters, 2003, 220(2):223-227.
[8]	GAUR R, KHARE S K. Solvent tolerant Pseudomonads as a source of novel lipases for applications in non-aqueous systems[J]. Biocatalysis and Biotransformation, 2011, 29(5):161-171.
[9]	DUNLOP M J. Engineering microbes for tolerance to next-generation biofuels[J]. Biotechnology for Biofuels, 2011, 4(1):2-9.
[10]	TAKATSUKA Y, CHEN C, NIKAIDO H. Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli[J]. Proceedings of the National Academy of Sciences, 2010, 107(15):6559-6565.
[11]	江欢, 舒正玉, 吴继光, 等. 耐有机溶剂极端微生物的耐受机制及应用[J]. 微生物学通报, 2009, 36(11):1744-1749. JIANG H, SHU Z Y, WU J G, et al. Mechanisms for solvent tolerance and application of extremophile with organic solvent tolerance[J]. Microbiology China, 2009, 36(11):1744-1749.
[12]	SHIMIZU K, HAYASHI S, KAKO T, et al. Discovery of glpC, an organic solvent tolerance-related gene in Escherichia coli, using gene expression profiles from DNA microarrays[J]. Applied and Environmental Microbiology, 2005, 71(2):1093-1096.
[13]	HEIPIEPER H J, NEUMANN G, CORNELISSEN S, et al. Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems[J]. Applied Microbiology and Biotechnology, 2007, 74(5):961-973.
[14]	OH H Y, LEE J O, KIM O B. Increase of organic solvent tolerance of Escherichia coli by the deletion of two regulator genes, fadR and marR[J]. Applied Microbiology and Biotechnology, 2012, 96(6):1619-1627.
[15]	HEIPIEPER H J, MEINHARDT F, SEGURA A. The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio:biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism[J]. FEMS Microbiology Letters, 2003, 229(1):1-7.
[16]	唐啸宇, 孙洪林, 何冰芳. 耐有机溶剂微生物及酶类在生物催化中的应用[J]. 化学进展, 2009, 21(12):2726-2733. TANG X Y, SUN H L, HE B F. Organic solvent tolerant bacteria and enzymes for application in biocatalysis[J]. Progress in Chemistry, 2009, 21(12):2726-2733.
[17]	LIU G, HU S, LI L, et al. Purification and characterization of a lipase with high thermostability and polar organic solvent-tolerance from Aspergillus niger AN0512[J]. Lipids, 2015, 50(11):1155-1163.
[18]	VAN HOLLE A, MACHADO M D, SOARES E V. Flocculation in ale brewing strains of Saccharomyces cerevisiae:re-evaluation of the role of cell surface charge and hydrophobicity[J]. Applied Microbiology and Biotechnology, 2012, 93(3):1221-1229.
[19]	SEDLÁ?KOVÁ P, ?E?OVSKÝ M, HORSÁKOVÁ I, et al. Cell surface characteristic of Asaia bogorensis-spoilage microorganism of bottled water[J]. Czech Journal of Food Sciences, 2011, 29(4):457-461.
[20]	BARRENTO S, MARQUES A, TEIXEIRA B, et al. Chemical composition, cholesterol, fatty acid and amino acid in two populations of brown crab Cancer pagurus:ecological and human health implications[J]. Journal of Food Composition and Analysis, 2010, 23(7):716-725.
[21]	LAANE C, BOEREN S, VOS K, et al. Rules for optimization of biocatalysis in organic solvents[J]. Biotechnology and Bioengineering, 1987, 30(1):81-87.
[22]	MANDER P, YOO H Y, KIM S W, et al. Transesterification of waste cooking oil by an organic solvent-tolerant alkaline lipase from Streptomyces sp. CS273[J]. Applied Biochemistry and Biotechnology, 2014, 172(3):1377-1389.
[23]	RAJAGOPAL A N. Growth of Gram-negative bacteria in the presence of organic solvents[J]. Enzyme and Microbial Technology, 1996, 19(8):606-613.
[24]	ZHANG H F, CHONG H Q, CHING C B, et al. Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance[J]. Applied Microbiology and Biotechnology, 2012, 94(4):1107-1117.
[25]	SARDESSAI Y, BHOSLE S. Tolerance of bacteria to organic solvents[J]. Research in Microbiology, 2002, 153(5):263-268.
[26]	王鑫昕, 王少华, 李维, 等. 细菌的有机溶剂耐受机制[J]. 生物工程学报, 2009, 25(5):641-649. WANG X X, WANG S H, LI W, et al. Tolerant mechanisms of bacteria to organic solvents[J]. Chinese Journal of Biotechnology, 2009, 25(5):641-649.
[27]	ISAR J, RANGASWAMY V. Improved n-butanol production by solvent tolerant Clostridium beijerinckii[J]. Biomass and Bioenergy, 2012, 37:9-15.
[28]	HEIPIEPER H J, MEULENBELD G, VAN OIRSCHOT Q, et al. Effect of environmental factors on the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida S12[J]. Applied and Environmental Microbiology, 1996, 62(8):2773-2777.
[29]	CARIO A, GROSSI V, SCHAEFFER P, et al. Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus[J]. Frontiers in Microbiology, 2015, 6:1-12.
[30]	OGER P M, CARIO A. Adaptation of the membrane in Archaea[J]. Biophysical Chemistry, 2013, 183:42-56.