生物破乳剂产生菌突变株的破乳性能及环境适应性的强化
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摘要
绿色生物制剂生物破乳剂以其低毒、无二次污染、可生物降解等独特的环境友好特性，逐渐成为该领域的研究热点。原油采出液因为使用水驱、蒸汽驱以及聚合物驱、碱驱、三元复合驱等强化驱油技术导致温度偏高，pH值变化范围大，使其破乳脱水增加了难度。运用生物破乳剂替代大量使用的化学破乳剂，对提高乳状液脱水率，降低环境污染风险具有深远意义。但随着生物破乳剂研究的不断深入，野生菌的产量或性能难以满足工业生产的要求、生物破乳剂的使用受环境条件制约以及生产成本高等问题，已成为阻碍其大规模生产和推广的关键。
本课题围绕以上问题开展相关研究，采用诱变方法改良生物破乳剂产生菌菌种的破乳能力，通过环境条件定向筛选的方法改良菌种极端环境适应能力；从蛋白质组学角度解析环境条件定向筛选对耐高温、耐碱和耐酸突变株的作用机制；初步开展破乳菌复配培养产复合型生物破乳剂的研究，为今后实际生产获得高效、稳定、广谱，廉价的生物破乳剂奠定理论基础。
为了提高野生菌株的破乳能力，本文对野生破乳菌XH1(Bacillus sp.)进行诱变育种。通过比较不同的诱变因子对破乳菌XH1的诱变效应，确定最佳诱变方式为紫外线+亚硝基胍（NTG）的复合诱变，筛选获得的最佳破乳突变株XN5，与出发菌株相比破乳能力提高15.12%。优化突变株XN5的培养条件，确定最佳培养条件为：培养温度35℃、摇床转数140min、培养基初始pH值7.0、培养时间21h。考察复合诱变对突变株XN5破乳性能的影响，发现与原始菌株XH1相比，诱变后所产生物破乳剂的投加量减少，破乳速率提高，但环境温度和pH值适应范围无显著变化。
通过诱变后进行环境条件定向筛选分别获得了耐高温、耐酸和耐碱的突变株XT3、XP3-3和XP11-2。突变株与原始菌株XH1相比在目的环境条件下的破乳性能大幅提高。利用SDS-PAGE、2-DE和nanoESI-Q-TOF MS/MS质谱结合的蛋白质组学研究手段，对原始菌株和突变株在相应条件下获得的差异表达蛋白质进行分离、鉴定和功能解析，从蛋白质组层面对环境条件定向筛选对与生物破乳剂产生菌的作用机制进行初步的分析。
采用生物表面活性剂检测方法及破乳试验相结合的筛选方法，从太平污水处理厂曝气池污泥泡沫中筛选出1株以液体石蜡为碳源的高效、稳定的破乳菌（24h排油率98.5%），实验室编号L1，经鉴定为Gordonia sihwensis。该菌全培养液的破乳能力不受升温处理，高压灭菌和反复冻融的影响(24h排油率>90%）。在无机盐培养基(MSM)中考察培养条件对破乳菌L1破乳效能的影响，获得最佳培养条件为：培养温度30℃、摇床转数140min、培养基初始pH值7.0、培养时间60h。
为了摸索复配培养产复合型生物破乳剂的最优培养条件，选择破乳菌XH1与L1在初始无葡萄糖的改进无机盐培养基(MMSM)中进行培养，利用响应面法(RSM)对复配培养条件进行优化，经模型的分析与验证，确定最佳培养条件为：种子液比例(L1:XH1)为3:2、葡萄糖投加时间为第4d，液体石蜡含量3.6%(v/v)，投加葡萄糖后再培养21h，获得复合型生物破乳剂排油率>95%(24h)。与单株菌培养相比，表现出投加量少、破乳接触时间短、对温度和pH的适应性高的优势。同时双株破乳菌复配培养有效的提高了培养中主要营养物质的利用率，生产每吨破乳粗产品与单独培养XH1相比可节约72.6%底物所需的成本。
Development and application of green biological agents in environmentalprotection fields has been taken more seriously. Bio-demulsifier has graduallybecome a hot research, because of its environmentally friendly property includinglow toxicity, non-secondary pollution and biodegradation. Bio-demulsifier can beused to treat the petroleum field emulsions and other industrial emulsionsin stead ofchemical demulsifier widely used, which has profound meaning for enhancing thedehydration rate of the emulsions and decreasing the environmental risks. However,with deep bio-demulsifier research, some significant problems have become theobstructions of large scale production and promotion. For example, bio-demulsifieryields and performance of wild strains cannot meet the requirements of industrialproduction, application of bio-demulsifier is restricted by environmental conditionsand incubation way is sole.
This paper conducts correlative studies according to above problems forfilling the gaps of this fields and improving application. That is mainly includingusing the mutation breeding approaches to improve the demulsifying ability andextreme environment adaptation of bio-demulsifier producing bacteria; based onproteomics analysis of environmental adaptation mutation mechanism ofthermotolerant, acid resistance and alkali resistance mutants; selection of highefficiency demulsifying new strains used to study complex culture and compoundbio-demulsifier development.
In order to enhance the demulsifying ability of wild strains, wild demulsifyingstrain XH1(Bacillus mojavensis) was proceed mutation breeding. Comparing themutation effect of the different mutation factors on demulsifying strain XH1, it isdetermined that the optimal mutation approach was UV+nitrosoguanidine(NTG) ofcompound mutation. The mutant with the highest demulsifying ability was strainXN5, which exhibited a15.12%increase of demulsifying ratios compared withoriginal strain XH1. Through study on the effect of cultivation condition on growthand demulsifying ability of mutant XN5, it obtained the optimal cultivationconditions were as follows:35℃cultivation temperature,140r/min shaking tablespeed,7.0initial pH-value,21h incubation time.
The correlative techniques of proteomics including SDS-PAGE,2-DE andnanoESI-Q-TOF MS/MS were used to analysis of environmental adaptationmutation mechanism of temperature-fast, acid-fast and alkali-fast mutants. Thedifferential expression protein was separated, identified and function analyzedwhich was from the original strain XH1grown on routine conditions, the mutant isolated by temperature grown on50℃incubation temperature, the mutant isolatedby acid environment grown on medium with pH3, the mutant isolated by alkalienvironment grown on medium with pH11, respectively. The results show thatsignificant variation of the numbers of the differential expression protein spots fromthermotolerant, acid resistance and alkali resistance mutants were14,8and9.Through identification and function analysis of differential expression protein ofdemulsifying strain XH1produced by mutation under the different environmentalconditions, the mutation mechanism of demulsifying bacteria was preliminaryanalyzed on the proteomics levels.Combination the detection methods ofbiosurfactants and demulsifying test was used to isolate the demulsifying bacteriafrom the activated sludge in aeration tank of Taiping waste water treatment plant.One strain with high and steady demulsifying characteristics (98.5%of24hdemulsifying ratios), named as L1, identified as Gordonia sihwensis. Thedemulsifying activity of bio-demulsifier produced by L1could not be influenced bythe temperature treatment, autoclaved sterilization and freeze thawing. Throughresearch on the effects of the cultivation conditions on demulsifying efficiency ofstrain L1grown on mineral salt medium(MSM), it obtained that the optimalcultivation conditions were as follows:30℃cultivation temperature,140minshaking table speed,7.0initial pH-value,60h incubation time.
In order to study on the optimal cultivation conditions of mixed cultureproducing compound bio-demulsifier, strain L1and XH1selected were incubated inmodified mineral salt medium(MMSM) without glucose. Then the response surfacemethodology (RSM) was used to optimize mixed culture conditions. According tothe results of the model analysis and verification, it was determined that the optimalconditions were follows:3:2inoculum proportion (strain L1:XH1),3.6%(v/v) liquidparaffin contents, the fourth day fed glucose, continue incubating21h after feedingglucose. Compared with single strain culture, the compound bio-demulsifierproduced by complex culture of L1and XH1exhibited a decrease of dose, anincrease of stability and a shortened demulsifying time. And the complex cultureincreased the utilization ratio of main nutrients in the medium, and the per tonbio-demulsifier cost decreased by72.6%.
本课题围绕以上问题开展相关研究，采用诱变方法改良生物破乳剂产生菌菌种的破乳能力，通过环境条件定向筛选的方法改良菌种极端环境适应能力；从蛋白质组学角度解析环境条件定向筛选对耐高温、耐碱和耐酸突变株的作用机制；初步开展破乳菌复配培养产复合型生物破乳剂的研究，为今后实际生产获得高效、稳定、广谱，廉价的生物破乳剂奠定理论基础。
为了提高野生菌株的破乳能力，本文对野生破乳菌XH1(Bacillus sp.)进行诱变育种。通过比较不同的诱变因子对破乳菌XH1的诱变效应，确定最佳诱变方式为紫外线+亚硝基胍（NTG）的复合诱变，筛选获得的最佳破乳突变株XN5，与出发菌株相比破乳能力提高15.12%。优化突变株XN5的培养条件，确定最佳培养条件为：培养温度35℃、摇床转数140min、培养基初始pH值7.0、培养时间21h。考察复合诱变对突变株XN5破乳性能的影响，发现与原始菌株XH1相比，诱变后所产生物破乳剂的投加量减少，破乳速率提高，但环境温度和pH值适应范围无显著变化。
通过诱变后进行环境条件定向筛选分别获得了耐高温、耐酸和耐碱的突变株XT3、XP3-3和XP11-2。突变株与原始菌株XH1相比在目的环境条件下的破乳性能大幅提高。利用SDS-PAGE、2-DE和nanoESI-Q-TOF MS/MS质谱结合的蛋白质组学研究手段，对原始菌株和突变株在相应条件下获得的差异表达蛋白质进行分离、鉴定和功能解析，从蛋白质组层面对环境条件定向筛选对与生物破乳剂产生菌的作用机制进行初步的分析。
采用生物表面活性剂检测方法及破乳试验相结合的筛选方法，从太平污水处理厂曝气池污泥泡沫中筛选出1株以液体石蜡为碳源的高效、稳定的破乳菌（24h排油率98.5%），实验室编号L1，经鉴定为Gordonia sihwensis。该菌全培养液的破乳能力不受升温处理，高压灭菌和反复冻融的影响(24h排油率>90%）。在无机盐培养基(MSM)中考察培养条件对破乳菌L1破乳效能的影响，获得最佳培养条件为：培养温度30℃、摇床转数140min、培养基初始pH值7.0、培养时间60h。
为了摸索复配培养产复合型生物破乳剂的最优培养条件，选择破乳菌XH1与L1在初始无葡萄糖的改进无机盐培养基(MMSM)中进行培养，利用响应面法(RSM)对复配培养条件进行优化，经模型的分析与验证，确定最佳培养条件为：种子液比例(L1:XH1)为3:2、葡萄糖投加时间为第4d，液体石蜡含量3.6%(v/v)，投加葡萄糖后再培养21h，获得复合型生物破乳剂排油率>95%(24h)。与单株菌培养相比，表现出投加量少、破乳接触时间短、对温度和pH的适应性高的优势。同时双株破乳菌复配培养有效的提高了培养中主要营养物质的利用率，生产每吨破乳粗产品与单独培养XH1相比可节约72.6%底物所需的成本。
Development and application of green biological agents in environmentalprotection fields has been taken more seriously. Bio-demulsifier has graduallybecome a hot research, because of its environmentally friendly property includinglow toxicity, non-secondary pollution and biodegradation. Bio-demulsifier can beused to treat the petroleum field emulsions and other industrial emulsionsin stead ofchemical demulsifier widely used, which has profound meaning for enhancing thedehydration rate of the emulsions and decreasing the environmental risks. However,with deep bio-demulsifier research, some significant problems have become theobstructions of large scale production and promotion. For example, bio-demulsifieryields and performance of wild strains cannot meet the requirements of industrialproduction, application of bio-demulsifier is restricted by environmental conditionsand incubation way is sole.
This paper conducts correlative studies according to above problems forfilling the gaps of this fields and improving application. That is mainly includingusing the mutation breeding approaches to improve the demulsifying ability andextreme environment adaptation of bio-demulsifier producing bacteria; based onproteomics analysis of environmental adaptation mutation mechanism ofthermotolerant, acid resistance and alkali resistance mutants; selection of highefficiency demulsifying new strains used to study complex culture and compoundbio-demulsifier development.
In order to enhance the demulsifying ability of wild strains, wild demulsifyingstrain XH1(Bacillus mojavensis) was proceed mutation breeding. Comparing themutation effect of the different mutation factors on demulsifying strain XH1, it isdetermined that the optimal mutation approach was UV+nitrosoguanidine(NTG) ofcompound mutation. The mutant with the highest demulsifying ability was strainXN5, which exhibited a15.12%increase of demulsifying ratios compared withoriginal strain XH1. Through study on the effect of cultivation condition on growthand demulsifying ability of mutant XN5, it obtained the optimal cultivationconditions were as follows:35℃cultivation temperature,140r/min shaking tablespeed,7.0initial pH-value,21h incubation time.
The correlative techniques of proteomics including SDS-PAGE,2-DE andnanoESI-Q-TOF MS/MS were used to analysis of environmental adaptationmutation mechanism of temperature-fast, acid-fast and alkali-fast mutants. Thedifferential expression protein was separated, identified and function analyzedwhich was from the original strain XH1grown on routine conditions, the mutant isolated by temperature grown on50℃incubation temperature, the mutant isolatedby acid environment grown on medium with pH3, the mutant isolated by alkalienvironment grown on medium with pH11, respectively. The results show thatsignificant variation of the numbers of the differential expression protein spots fromthermotolerant, acid resistance and alkali resistance mutants were14,8and9.Through identification and function analysis of differential expression protein ofdemulsifying strain XH1produced by mutation under the different environmentalconditions, the mutation mechanism of demulsifying bacteria was preliminaryanalyzed on the proteomics levels.Combination the detection methods ofbiosurfactants and demulsifying test was used to isolate the demulsifying bacteriafrom the activated sludge in aeration tank of Taiping waste water treatment plant.One strain with high and steady demulsifying characteristics (98.5%of24hdemulsifying ratios), named as L1, identified as Gordonia sihwensis. Thedemulsifying activity of bio-demulsifier produced by L1could not be influenced bythe temperature treatment, autoclaved sterilization and freeze thawing. Throughresearch on the effects of the cultivation conditions on demulsifying efficiency ofstrain L1grown on mineral salt medium(MSM), it obtained that the optimalcultivation conditions were as follows:30℃cultivation temperature,140minshaking table speed,7.0initial pH-value,60h incubation time.
In order to study on the optimal cultivation conditions of mixed cultureproducing compound bio-demulsifier, strain L1and XH1selected were incubated inmodified mineral salt medium(MMSM) without glucose. Then the response surfacemethodology (RSM) was used to optimize mixed culture conditions. According tothe results of the model analysis and verification, it was determined that the optimalconditions were follows:3:2inoculum proportion (strain L1:XH1),3.6%(v/v) liquidparaffin contents, the fourth day fed glucose, continue incubating21h after feedingglucose. Compared with single strain culture, the compound bio-demulsifierproduced by complex culture of L1and XH1exhibited a decrease of dose, anincrease of stability and a shortened demulsifying time. And the complex cultureincreased the utilization ratio of main nutrients in the medium, and the per tonbio-demulsifier cost decreased by72.6%.
引文
[1] Mason S L, May K, Hartland S. Drop size and concentration profiledetermination in petroleum emulsion separation [J]. Colloids. Surf.1995, A96:85-92.
[2]张贤明，吴峰平，陈彬，等；油包水型乳化液破乳方法研究现状及展望[J].石化技术与应用，2010,28(2)：159-163.
[3]方云,夏咏梅.生物表面活性剂[M].北京：中国轻工业出版社，1992：210-265.
[4]纳米复合破乳剂的研究与应用[D].北京：北京交通大学,2008:1-27.
[5] Al-Sabagh A M, Kandile N G, El-Din M R N. Functions of Demuslifiers inthe Petroleum Industry [J]. Separation Science and Technology,2011,46:1144-1163.
[6]王海峰，包木太，耿雪丽，等.油田含聚合物污水生化处理室内模拟研究[J].西南石油大学学报：自然科学版，2008（1）：109-111.
[7]杨小刚，谭蔚，谭晓飞.高含水原油的热化学破乳方法[J].化学工业与工程,2007,24(3):236-239.
[8] Banat I M, Makkar R S, Cameotra S S. Potential Commercial Applicationsof Microbial Surfactants [J]. Appl Microbiol Biotechnol,2000,53:495-508.
[9]王世荣，李祥高，刘东志，等.表面活性剂化学[M].北京：化学工业出版社，2010：6-32.
[10]陈锋，杨总.乳状液破乳方法综述[J].石油化工应用,2009,28(2):1-3.
[11] Park S H, Lee J H, Ko S H, et al. Demulsification of Oil-in-waterEmulsions by Aerial Spores of a Streptomyces sp.[J]. Biotechnol Lett,2000,22:1389-1395.
[12]陆丽君.破乳菌破乳有效成分鉴定及对油田采出液水处理系统影响研究[D].上海：同济大学，2007:1-16.
[13]纳米复合破乳剂的研究与应用[D].北京：北京交通大学,2008:1-27.
[14] Tan W, Yang X G, Tan X F. Study on demulsification of crude oil emulsionsby microwave chemical method [J]. Separation Science and Technology,2007,42(6):1367-1377.
[15]杨小刚,谭蔚,谭晓飞.高含水原油的热化学破乳方法[J].化学工业与工程,2007,24(3):236-239.
[16]杜玉梅.优秀原油破乳剂所具备的性能初探[J].高分子通报，2006，11:47-50.
[17]康万利，孙春柳.油田乳状液破乳方法研究进展[J].管道技术与设备，2006,2：1-4.
[18] Van Hamme J D, Singh A, Ward O P. Recent Advances in PetroleumMicrobiology [J]. Microbiol Mol Biol Rev,2003,67:503-549.
[19]彭伟，周华，陈永立．绿色生物破乳技术原理及研究状况[J]．新疆石油科技，2007,17（2）：67–69.
[20] Amézcua-Vega C, Poggi-Varaldo H M, Esparza-García F, et al. Effect ofCulture Conditions on Fatty Acids Composition of a BiosurfactantProduced by Candida ingens and Changes of Surface Tension of CultureMedia[J]. Bioresour Technol,2007,98：237-240.
[21]肖稳发，刘锡建，张红，等.微生物技术在油气田开发中的研究与应用进展[J].化学与生物工程，2006，23(10):1-3.
[22]缪永霞，易绍金.环保型生物破乳剂的研究及应用[J].油气田环境保护，2007，17(4):44-45.
[23] Cooper D G, Zajic J E, Cairns W L, et al. De-emulsification and Microbes,Biochemicals Engineering Research Reports. Canada: University ofWeatern Ontario,1980, Vol.Ⅵ.
[24] Gray N C C, Stewart A L, Cairns W L, et al. Bacteria-inducedDeemulsification of Oil-in-water Petroleum Emulsions [J]. Biotechnol Lett,1984,6:419-424.
[25] Stewart A L, Gray N C C. Bacteria-induced De-emulsification ofWater-in-oil Petroleum Emulsions [J]. Biotechnol Lett,1983,5:725-730.
[26] Duvnjak Z, Kosaric N. De-emulsification of Petroleum Water in OilEmulsions by Selected Bacterial and Yeast Cells [J]. Biotechnol Lett,1987,9：39-42.
[27] Kosaric N, Duvnjak Z, Cairns W L. De-emulsification of ComplexPetroleum Emulsions by Use of Microbial Biomass[J]. Environ Prog,1987,6:33-38.
[28] Kosaric N, Cairns W L, Gray N C C，et al.Microbial Deemulsifiers inBiosurfactant and Biotechnology[M]. New York: Marcel Dekker,1987:247-320.
[29] Janiyani K L, Purohit H J, Shanker R, et al. De-emulsification ofOil-inwater Emulsions by Bacillus subtilis[J]. World J MicrobiolBiotechnol,1994,10:452-456.
[30] Nishimaki, Fukumi, Takahashi, et al. Microorganisms Demulsifiers andProcesses for Breaking an Emulsion.1999, US5989892.
[31] Das M. Characterization of De-demulsification Capabilities of aMicrococcus sp.[J]. Bioresour Technol,2001,79:15-22.
[32] Lee J C, Lee K Y. Emulsification Using Enviromental CompatibleEmuldifiers and De-emulsification Using D.C. Field and ImmobilizedNocardia amarae [J]. Biotechnol Lett,2000,22:1157-1163.
[33] Nadarajah N, Singh A,Ward O P. De-emulsification of petroleum oilemulsion by a mixed bacterial culture.Process Biochem,2002,37(10):1135.
[34] Andrew J P, Ralf C R. Treatment of Strongflow Wool Scouring Effluent byBiological Emulsion Destabilization [J]. Water research,2004,38:1419-1426.
[35]苗芳,李连志.生物破乳剂的性能研究[J].河北职工医学院，2001，18（3）：5-8.
[36]韦良霞，任丽，肖英玉，等. HRB-4型生物破乳剂在纯梁采油厂的应用[J].油田化学，2004：21（1）：42-44.
[37]周劲,赵晓祥,冯庆安.环境因子对破乳优势菌生长的影响[J].广州化工，2005，33：44-45.
[38]马挺，梁凤来，奚艳伟，等.红球菌PR-1菌株破乳性能研究[J].环境科学，2006，27(6)：1192-1196.
[39]黄翔峰,闻岳，杨葆华，等.破乳菌种TR-1的筛选与破乳性能的实验研究[J].油田化学，2006，23（2）:136-139.
[40]张天胜.生物表面活性剂及其应用[M].北京：化学工业出版社,2005：1-14，294-316.
[41] Van D, Jonathan H, Ajay S, et al. Recent Advances in PetroleumMicrobiology [J]. Microbiology and Molecular Biology Reviews,2003,67(4):503-549.
[42] Ghojavand H, Vahabzadeh F, Azizmohseni F. A Halotolerant,Thermotolerant, and Facultative Biosurfactant Producer: Identification andMolecular Characterization of a Bacterium and Evolution of EmulsifierStability of a Lipopeptide Biosurfactant [J]. Biotechnology and BioprocessEngineering,2011,16(1):72-80.
[43]冯志强，杨永军，朱成军，等.原油生物破乳剂的研究与应用[J].石油大学学报，2004，28(3)：93-99.
[44]吴涓，李请彪，邓旭，等.重金属生物吸附的研究进展[J].离子交换与吸附，1998,14(2):180-187.
[45] Rosen M J. Surfactants and Interfacial Phenomena (2ed)[M]. New York:Wiley,1989:234-246.
[46] Cairns W L, Cooper D G, Zajic J E, et al. Characteriation of Nocardiaamarae as a Potent Biological Coalescing Agent of Water in Oil Emulsions[J]. Appl Evioron Microbiol,1982,43:362-366.
[47]徐燕莉。表面活性剂的功能[M].北京:化学工业出版社.2000,98-102.
[48] D·波塞尔特, W·施密特, M·古茨曼，等.蛋白质作为破乳剂的用途[P].2008, CN101151081.
[49]苗芳,李连志.生物破乳剂的性能研究[J].河北职工医学院，2001，18（3）：5-8.
[50]杨志生,孙宇.生物优势菌破乳剂的制备及性能研究.化学工业与工程.2004,21(1):8-11.
[51] E. Ristau, F. Wanger. Formation of novel anionic trehalosetetraesters fromRhodococcus erythropolis under growth limiting conditions [J]. Biotechnol.Lett.1993,5:95-100.
[52] Parra J L, Guinea J, Manresa M A, et al. Chemical Characterization andPhysicochemical Behaviour of Biosurfactants[J]. J Am Oil Chem Soc,1989,37:141-145.
[53] Peypoux F, Bonmatin J M, Wallach J. Recent trends in the biochemistry ofSurfactin [J]. Appl Microbiol Biotechnol,1999,51(5):553-563.
[54] M. Robert, M. E. Mercade, M. P. Bosch. Effect of the carbon source onbiosurfactant production by Pseudomonas aeruginosa44T1[J]. Biotechnol.Lett.1989,11:871-874.
[55] Demain A L, Adrio J L. Contributions of microorganisms to industrialbiology [J]. Mol Biotechnol,2008,38(1):41-55.
[56] Shikha A S, Darmwal N S. Improved production of alkaline protease from amutant of alkalophilic Bacillus pan totheneticus using molasses as asubstrate [J]. Bioresour Technol,2007,4:881-885.
[57]孙晓君，朱婷婷，秦智，等.紫外诱变获得耐高浓度丙烯腈菌株的研究［J］.哈尔滨工业大学学报，2005，37（10）：1376－1379.
[58]肖怀秋，李玉珍，兰立新.复合诱变原生质体选育高产中性蛋白酶菌株[J].中国酿造，2008（15）：52-54.
[59]施巧琴.工业微生物育种学[M].北京：科学出版社,2003.
[60]罗大珍，林稚兰.现代微生物发酵及技术教程[M].北京:北京大学出版社,2006:18-24.
[61]陈必链.微生物工程[M].北京：科学出版社，2010:23-25.
[62]郑国香，任南琪，林海龙，等.诱变育种选育产氢细菌[J].太阳能学报.2007,6(28):632-637.
[63]刘录祥，郑企成．空间诱变与作物改良［M］．北京:原子能出版社，1997．
[64]袁存权，李云，管耀义，等．航天诱变育种机理研究进展［J］．河北林业科技.2009，B09:39-43．
[65]杜丽平，闻建平，朱智勇。复合诱变菌处理氨氮废水[J]，上海环境科学.2002,21(1)15-23.
[66]冯建成,张容鹄,罗素兰。高产透明质酸菌种FJ-23的诱变选育[J]生物技术.2006,16(3)28-30.
[67]侯红萍,杜文娟.微波-亚硝基胍复合诱变筛选纤维素酶高产菌株[J].中国酿造，2009,8:53-56.
[68]郑国香,任南琪,李永峰,等.一株高效产氢突变体RF-9的筛选与产氢特性[J].中国环境科学2007,27(2)：184~188.
[69]鲍秀珍.高压静电场离子束组合诱变木聚糖酶产生菌(黑曲霉菌)的技术研究[D].内蒙古大学，2007.
[70] Wasinger V C，Cordewell S J，Cerpa P A，et al. Progress with Gene-productmapping of the Mollicutes: Mycoplasma genitalium [J]. Electrophoresis，1995,16(7):1090-1094.
[71]赵洪岩.采用非标记定量技术对变形链球菌耐氟菌株的差异蛋白质组学研究[D].吉林:吉林大学,2011:1-20.
[72] Cardoza J D, Parikh J R, Ficarro S B, et al. Mass Spectrometry-basedProteomics: Qualitative Identification to Activity-based Protein Profiling[J].Wiley Interdisciplinary Reviews. System Biology and Medicine,2012,4(2):141-162.
[73] Patel P S，Telang S D，Rawal R M，et al. A review of Proteomics in CancerResearch [J]. Asian Pac J Cancer Prev，2005，6(2):113-117.
[74] Chichester C，Nikitin F，Ravarini J C, et al. Consistency Checks forCharactering Protein Forms [J].Comput Biol Chem,2003,27(l):29-35.
[75]李丽莉,温博海,王恒樑.蛋白质组技术进展[J].生物技术通讯,2006,17(4):662-664.
[76] Farrell P H. High Resolution Two Dimensional Electorphoresis of Protein[J]. J Biol Chem,1975,250:4007-4021.
[77] Klose J. Protein Mapping by Combined Isoelectrophoresis of MouseTissues:A Novel Approach to Testing for Induced Point Mutations in Mammals [J].Humangenetik,1975,26:231-243.
[78]威尔金斯，阿尔佩，威廉斯，等.蛋白质组学研究[M].北京：科学出版社，2010:15-50.
[79]武霞.野大麦盐胁迫的差异蛋白质组学研究[D].长春：吉林大学,2007：24-27.
[80] Tonge R, Shaw J, Middleton B, et al. Validation and Different Gel ofFluorescence Electrophoresis Proteomics Technology [J]. Proteomics,2001,1:377-396.
[81] Zhou G, Li H M, DeCamp D, et al.2D-Differential in-Gel Electrophoresisfor the Identification Esophageal Scans Cancer Specific Protein Markers [J].Mol Cell Proteomics,2002,1:117-124.
[82] Lilley K S, Friedman D B. All about DIGE: Quantification Technology forDifferential Display2D-gel Proteomics [J]. Expert Rev Proteomics,2004,1(4):401-409.
[83] Fenn J B，Mann M，Meng C K，et al. Electrospray Ionization for MassSpectrometry of Large Biomolecules [J]. Science,1989,246(4926):64-71.
[84] Lopachin R M，Jones R C，Patterson T A，et al. Application of Proteomicsto the Study of Molecular Mechanisms in Neurotoxicology [J]. R M N euroToxicology,2003,24:761-765.
[85] Wang X N, Xu L N,Peng J Y, et al. Application of Recent Biological MassSpectrometry in Biomacromolecules Analysis and Reserch[J]. Chin JMAP,2008,25(2):105-109.
[86]吴祖建，高芳銮，沈建国.生物信息学分析实践[M].北京：科学出版社,2010:105-140.
[87] Mann M. Quantitative Proteomics [J]. Nat Bioltechnol,1999,17:954-955.
[88] Rotilio D, Della Corte A, D'lmperio M, et al. Proteomics: Bases for ProteinComplexity Understanding [J]. Thrombosis Reserarch,2012,129(3):257-262.
[89]李旭.生物破乳剂的开发及破乳效能研究[D].哈尔滨:哈尔滨工业大学.2008:18-30,45-96.
[90] Banat I M. The Isolation of a Thermoohilic Biosurfactant-Producingbacillus species [J]. Biotechnol Lett,1993,15(6):591-594.
[91]李衍达,孙之荣.生物信息学基因和蛋白质分析的实用指南[M].北京:清华大学出版社,2003:16-43,175-211.
[92]马放,任南琪,杨基先.污染控制微生物学实验[M].哈尔滨:哈尔滨工业大学出版社,2002:53-57.
[93]咸洪泉，郭立忠.微生物学实验教程[M].北京：高等教育出社,2010:21-25.
[94]于冰.产絮菌产絮过程差异表达蛋白质及其功能调控研究[D].哈尔滨:哈尔滨工业大学,2010:27-36.
[95]范国昌.微生物混合培养的研究与应用.生物学杂志，1996，4:28~29.
[96]侯宁.生物破乳剂产生菌的特性及破乳效能研究[D].哈尔滨:哈尔滨工业大学,2009:63-71.
[97]张向阳,刘登如,堵国成,等.辅酶Q10产生菌的抗性筛选及发酵条件优化[J].工业微生物，2007,37(3)：5-9.
[98] Hollywood, Katherine A.; Shadi, Iqbal T.; Goodacre, Royston.Monitoringthe succinate dehydrogenase activity isolated from mitochondria by surfaceenhanced Raman scattering [J]. Journal of Physical Chemistry C,2010,4:7308-7313.
[99] Khunderyakova N V， Zakharchenko M V， Zakharchenko A V.Hyperactivation of succinate dehydrogenase in lymphocytes of newbornrats [J]. Biochemistry (Moscow),2008,3:337-341.
[100] Cimen H，Han M J，Yang Y J. Regulation of succinate dehydrogenaseactivity by SIRT3in mammalian mitochondria [J]. Biochemistry,2010,1:304-311.
[101] Elisa P S，Sandra V V，Susana R N. The Cth2ARE-binding protein recruitsthe Dhh1helicase to promote the decay of succinate dehydrogenase SDH4mRNA in response to iron deficiency [J]. Journal of Biological Chemistry,2008,10:28527-28535.
[102]徐暘.一株破乳菌破乳有效成分分析及其强化培养条件优化[D].哈尔滨：哈尔滨工业大学，2010：72-87.
[103] Adriano N N，Fernando C，Yves G. Deficiency of mitochondrial fumaraseactivity in tomato plants impairs photosynthesis via an effect on stomatalfunction [J]. Plant Journal,2007,1:1093-1106.
[104] Koivunen P，Hirsil M，Remes A M. Inhibition of hypoxia-inducible factor(HIF) hydroxylases by citric acid cycle intermediates: Possible linksbetween cell metabolism and stabilization of HIF [J].2007,2:4524-4532.
[105] Guebel D V，Cánovas M，Torres N V. Analysis of the escherichia coliresponse to glycerol pulse in continuous, high-cell density culture using amultivariate approach [J]. Biotechnol. Bioeng.,2009,2:910-922.
[106] Vivoli M，Angelucci F，Ilari A. Role of a conserved active site cation-πinteraction in Escherichia coli serine hydroxymethyltransferase [J].Biochemistry,2009,12:12034-12046.
[107] Zuo Z Y，Zheng Z L，Liu Z G. Cloning, DNA shuffling and expressionof serine hydroxymethyltransferase gene from Escherichia coli strainAB90054[J]. Enzyme Microb. Technol.,2007,3:569-577.
[108] Jennifer T F，William K S， Marie A C. A UV-responsive internal ribosomeentry site enhances serine hydroxymethyltransferase1expression for DNAdamage repair [J]. Journal of Biological Chemistry,2009,11:31097-31108.
[109] Akiduki S，Ikemoto M J. Modulation of the neural glutamate transporterEAAC1by the addicsin-interacting protein Arl6ip1[J]. Journal ofBiological Chemistry,2008,11:31323-31332.
[110] Sanghamitra D，Rodney L B，Gregory A G. Structural analysis of substrateand effector binding in Mycobacterium tuberculosis D-3-phosphoglyceratedehydrogenase [J].Biochemistry,2008,8:8271-8282.
[111] Rodney L B，Chen S W，Xiao L X. Role of the anion-binding site incatalysis and regulation of Mycobacterium tuberculosisD-3-phosphoglycerate dehydrogenase [J]. Biochemistry,2009,6:4808-4815.
[112] Sanghamitra D，Hu Z Q，Xiao L X. The effect of hinge mutations oneffector binding and domain rotation in Escherichia coliD-3-phosphoglycerate dehydrogenase [J]. Journal of Biological Chemistry,2007,6:18418-18426.
[113] Alasdair F. B， Christopher F S， Zhang X J. Evidence from Ramanspectroscopy that InhA, the mycobacterial enoyl reductase, modulates theconformation of the NADH cofactor to promote catalysis [J]. Journal of theAmerican Chemical Society,2007,5:6425-6431.
[114] Xu H，Todd J S，Sekiguchi J I. Mechanism and inhibition of saFabI, theenoyl reductase from Staphylococcus aureus [J]. Biochemistry,2008,4:4228-4236.
[115] Sara M V，Patrick M S. The two-component system bacillus respiratoryresponse A and B (BrrA-BrrB) is a virulence factor regulator in Bacillusanthracis [J]. Biochemistry,2007,6:7343-7352.
[116] Yan L Y，Yang Q Q，Jiang, J H. Involvement of a putative responseregulator Brrg-1in the regulation of sporulation, sensitivity to fungicides,and osmotic stress in Botrytis cinerea [J]. Appl. Microbiol. Biotechnol.,2011,4:215-226.
[117] Shobhana S G，Brendan N B，Timothy L C. Structural analysis of theDNA-binding domain of the Helicobacter pylori response regulator ArsR[J]. Journal of Biological Chemistry,2009,3:6536-6545.
[118] Helen R J，Kanchana R R，Lizbeth H. The Cys319Loop modulates thetransition between dehydrogenase and hydrolase conformations in inosine5-monophosphate dehydrogenase [J]. Biochemistry,2010,12:10674-10681.
[119] Isabelle L，Patrick C， Félix C. Liquid chromatography-coupled tandemmass spectrometry based assay to evaluate inosine-5-monophosphatedehydrogenase activity in peripheral blood mononuclear cells from stemcell transplant recipients [J]. Analytical Chemistry,2012,1:216-223.
[120] Arunkumar, K， Narayanan M. Molecular evolution of theN-acetylglucosamine-6-phosphate deacetylase gene [J]. InternationalSymposium on Biocomputing, ISB2010,2010.
[121] Jurgen R H， Mhairi S， Van-Duc L. Control and regulation of geneexpression: Quantitative analysis of the expression of phosphoglyceratekinase in bloodstream form Trypanosoma brucei [J]. Journal of BiologicalChemistry,2008,2:2495-2507.
[122] Hurth C，Tassius C，Talbot J C. Enzymatic activity of immobilized yeastphosphoglycerate kinase [J]. Biosensors and Bioelectronics,2007,5:2449-2455.
[123] Monica B，Luis G A，Sebastiao J F. The role of reaction energy andhydrogen bonding in the reaction path of enzymatic proton transfers [J].Journal of Physical Organic Chemistry,2009,3:254-263.
[124] Krahulec J，Szemes T，Krahulcová J. Bioinformatics characterization ofpotential new beta-glucuronidase from streptococcus equi subsp.Zooepidemicus [J]. Molecular Biotechnology,2010,3:232-241.
[125] Miguel S J，Casimir B，Jurgen S. Charge stabilization and entropy reductionof central lysine residues in fructose-bisphosphate aldolase [J].Biochemistry,2009,6:4528-4537.
[126] Chiu K H，Huang H W，Mok, H K. Differential proteome analysis ofhagfish dental and somatic skeletal muscles [J]. Marine Biotechnology,2007,12:689-700.
[127] Hallows W C, Yu W, Denu J M. Regulation of Glycolytic EnzymePhosphoglycerate Mutase-1by Sirt1Protein-mediated Deacetylation [J].Journal of Biological Chemistry,2012,287(6):3850-3858
[128] Papini M, Nookaew I, Scalcinati G, et al. Phosphoglycerate mutaseknock-out mutant Saccharomyces cerevisiae: Physiological investigationand transcriptome analysis [J]. Biotechnology journal,2012,5(10):1016-1027.
[129] Yamada S, Sugimoto H, Kobayashi M, et al. Structure of PAS-LinkedHistidine Kinase and the Response Regulator Complex [J]. Structure,2012,17(10):1333-1344.
[130] Song Y H, Wan L L, Wang Y, et al. Electron transfer and electrocatalyticsof cytochrome c and horseradish peroxidase on DNA modified electrode [J].Bioelectrochemistry,2012,85:29-35.
[131] Huynh H， Servant N, Chalifour L E, et al. Ubiquinol-cytochrome-creductase7.2kDa protein of mitochondrial complex III issteroid-responsive and increases in cardiac hypertrophy andhypertension[J]. Canadian journal of physiology and pharmacology,2012,85(10):986-996.
[132] Gutierrezcirlos E B, Antaramian A, Vazquezacevedo M, et al. Ahighly-active ubiquinol-cytochrome-creductase (BC (1) complex) from thecolorless ALGA polytomella SPP, a close relative ofchlamydomonas-characterization of the heme-binding site of cytochrome-c(1)[J]. Journal of biological chemistry,1994,269(12):9147-915.
[133] Hallows W C, Yu W, Denu J M. Regulation of Glycolytic EnzymePhosphoglycerate Mutase-1by Sirt1Protein-mediated Deacetylation [J].Journal of biological chemistry,2012,287(6):3850-3858.
[134] Niture S K, Jaiswal A K. Inhibitor of Nrf2(INrf2or Keap1) ProteinDegrades Bcl-xL via Phosphoglycerate mutase5and Controls CellularApoptosis [J]. Journal of biological chemistry,2011,286(52):44542-44556.
[135] Papini M, Nookaew I, Scalcinati G, et al. Phosphoglycerate mutaseknock-out mutant Saccharomyces cerevisiae: Physiological investigationand transcriptome analysis [J]. Biochemical journal,2010,5(10):1016-1027.
[136] Wang L, Chen Y, Jiang R R. Nanoparticle-supported consecutive reactionscatalyzed by alkyl hydroperoxide reductase [J]. Journal of MolecularCatalysisb-enzymatic,2012,76:9-14.
[137] Chien V, Aitken J F, Zhang S, et al. The chaperone proteins HSP70,HSP40/DnaJ and GRP78/BiP suppress misfolding and formation ofbeta-sheet-containing aggregates by human amylin: a potential role fordefective chaperone biology in Type2diabetes [J]. Biochemical journal,2012,432:113-121.
[138] Mohammadian T, Amini H, Panejad M. Electroelution of the stained alkylhydroperoxide reductase of Helicobacter pylori from preparative sodiumdodecyl sulfate polyacrylamide gels [J]. Clinical biochemistry,2012,44(13): S97-S97.
[139] Appleby T C, Anderson R, Fedorova O, et al. Visualizing ATP-DependentRNA Translocation by the NS3Helicase from HCV [J]. Journal ofmolecular biology,2011,405(5):1139-1153.
[140] Kikuta K, Kubota D, Saito T, et al. Clinical proteomics identifiedATP-dependent RNA helicase DDX39as a novel biomarker to predict poorprognosis of patients with gastrointestinal stromal tumor [J]. Journal ofpoteomics,2012,75(4):1089-1098.
[141] Lee J H, Kang G B, Lim H H, et al. Crystal structure of the GluR0ligand-binding core from Nostoc punctiforme in complex with L-glutamate:Structural dissection of the ligand interaction and subunit interface [J].Journal of molecular biology,2012,376(2):308-316.
[142] Lee J H, Park S J, Rho S H, et al. Crystallization and preliminary X-raycrystallographic analysis of the GluR0ligand-binding core from Nostocpunctiforme[J]. Acta Crystallographica Section F: Structural Biology andCrystallization Communications,2012,61:1020-1022.
[143] Friesen D E, Barakat K H, Semenchenko V, et al. Discovery of SmallMolecule Inhibitors that Interact with gamma-Tubulin [J]. ChemicalBiology&Drug Design,2012,79(5):639-652.
[144] Paul A, Lal L, Ahuja P S, et al. Alpha-tubulin (CsTUA) up-regulated duringwinter dormancy is a low temperature inducible gene in tea [Camelliasinensis (L.) O. Kuntze][J]. Molecular Biology Reports,2012,39(4):3485-3490.
[145] Rohani M M, Zydney A L. Protein transport through zwitterionicultrafiltration membranes [J]. Journal of Membrane Science Science,2012,397:1-8.
[146] Moinova H, Leidner R S, Ravi L. Aberrant Vimentin Methylation IsCharacteristic of Upper Gastrointestinal Pathologies [J]. CancerEpidemiology, Biomarkers&Prevention,2012,21(4):594-600.
[147] Triolo D, Dina G, Taveggia C, et al. Vimentin regulates peripheral nervemyelination [J]. Development,2012,139(7):1359-1367.
[148] Kim S J, Ise H, Goto M, et al. Interactions of vimentin-ordesmin-expressing liver cells with N-acetylglucosamine-bearingpolymers[J]. Biomaterials,2012,33(7):2154-2164.
[2]张贤明，吴峰平，陈彬，等；油包水型乳化液破乳方法研究现状及展望[J].石化技术与应用，2010,28(2)：159-163.
[3]方云,夏咏梅.生物表面活性剂[M].北京：中国轻工业出版社，1992：210-265.
[4]纳米复合破乳剂的研究与应用[D].北京：北京交通大学,2008:1-27.
[5] Al-Sabagh A M, Kandile N G, El-Din M R N. Functions of Demuslifiers inthe Petroleum Industry [J]. Separation Science and Technology,2011,46:1144-1163.
[6]王海峰，包木太，耿雪丽，等.油田含聚合物污水生化处理室内模拟研究[J].西南石油大学学报：自然科学版，2008（1）：109-111.
[7]杨小刚，谭蔚，谭晓飞.高含水原油的热化学破乳方法[J].化学工业与工程,2007,24(3):236-239.
[8] Banat I M, Makkar R S, Cameotra S S. Potential Commercial Applicationsof Microbial Surfactants [J]. Appl Microbiol Biotechnol,2000,53:495-508.
[9]王世荣，李祥高，刘东志，等.表面活性剂化学[M].北京：化学工业出版社，2010：6-32.
[10]陈锋，杨总.乳状液破乳方法综述[J].石油化工应用,2009,28(2):1-3.
[11] Park S H, Lee J H, Ko S H, et al. Demulsification of Oil-in-waterEmulsions by Aerial Spores of a Streptomyces sp.[J]. Biotechnol Lett,2000,22:1389-1395.
[12]陆丽君.破乳菌破乳有效成分鉴定及对油田采出液水处理系统影响研究[D].上海：同济大学，2007:1-16.
[13]纳米复合破乳剂的研究与应用[D].北京：北京交通大学,2008:1-27.
[14] Tan W, Yang X G, Tan X F. Study on demulsification of crude oil emulsionsby microwave chemical method [J]. Separation Science and Technology,2007,42(6):1367-1377.
[15]杨小刚,谭蔚,谭晓飞.高含水原油的热化学破乳方法[J].化学工业与工程,2007,24(3):236-239.
[16]杜玉梅.优秀原油破乳剂所具备的性能初探[J].高分子通报，2006，11:47-50.
[17]康万利，孙春柳.油田乳状液破乳方法研究进展[J].管道技术与设备，2006,2：1-4.
[18] Van Hamme J D, Singh A, Ward O P. Recent Advances in PetroleumMicrobiology [J]. Microbiol Mol Biol Rev,2003,67:503-549.
[19]彭伟，周华，陈永立．绿色生物破乳技术原理及研究状况[J]．新疆石油科技，2007,17（2）：67–69.
[20] Amézcua-Vega C, Poggi-Varaldo H M, Esparza-García F, et al. Effect ofCulture Conditions on Fatty Acids Composition of a BiosurfactantProduced by Candida ingens and Changes of Surface Tension of CultureMedia[J]. Bioresour Technol,2007,98：237-240.
[21]肖稳发，刘锡建，张红，等.微生物技术在油气田开发中的研究与应用进展[J].化学与生物工程，2006，23(10):1-3.
[22]缪永霞，易绍金.环保型生物破乳剂的研究及应用[J].油气田环境保护，2007，17(4):44-45.
[23] Cooper D G, Zajic J E, Cairns W L, et al. De-emulsification and Microbes,Biochemicals Engineering Research Reports. Canada: University ofWeatern Ontario,1980, Vol.Ⅵ.
[24] Gray N C C, Stewart A L, Cairns W L, et al. Bacteria-inducedDeemulsification of Oil-in-water Petroleum Emulsions [J]. Biotechnol Lett,1984,6:419-424.
[25] Stewart A L, Gray N C C. Bacteria-induced De-emulsification ofWater-in-oil Petroleum Emulsions [J]. Biotechnol Lett,1983,5:725-730.
[26] Duvnjak Z, Kosaric N. De-emulsification of Petroleum Water in OilEmulsions by Selected Bacterial and Yeast Cells [J]. Biotechnol Lett,1987,9：39-42.
[27] Kosaric N, Duvnjak Z, Cairns W L. De-emulsification of ComplexPetroleum Emulsions by Use of Microbial Biomass[J]. Environ Prog,1987,6:33-38.
[28] Kosaric N, Cairns W L, Gray N C C，et al.Microbial Deemulsifiers inBiosurfactant and Biotechnology[M]. New York: Marcel Dekker,1987:247-320.
[29] Janiyani K L, Purohit H J, Shanker R, et al. De-emulsification ofOil-inwater Emulsions by Bacillus subtilis[J]. World J MicrobiolBiotechnol,1994,10:452-456.
[30] Nishimaki, Fukumi, Takahashi, et al. Microorganisms Demulsifiers andProcesses for Breaking an Emulsion.1999, US5989892.
[31] Das M. Characterization of De-demulsification Capabilities of aMicrococcus sp.[J]. Bioresour Technol,2001,79:15-22.
[32] Lee J C, Lee K Y. Emulsification Using Enviromental CompatibleEmuldifiers and De-emulsification Using D.C. Field and ImmobilizedNocardia amarae [J]. Biotechnol Lett,2000,22:1157-1163.
[33] Nadarajah N, Singh A,Ward O P. De-emulsification of petroleum oilemulsion by a mixed bacterial culture.Process Biochem,2002,37(10):1135.
[34] Andrew J P, Ralf C R. Treatment of Strongflow Wool Scouring Effluent byBiological Emulsion Destabilization [J]. Water research,2004,38:1419-1426.
[35]苗芳,李连志.生物破乳剂的性能研究[J].河北职工医学院，2001，18（3）：5-8.
[36]韦良霞，任丽，肖英玉，等. HRB-4型生物破乳剂在纯梁采油厂的应用[J].油田化学，2004：21（1）：42-44.
[37]周劲,赵晓祥,冯庆安.环境因子对破乳优势菌生长的影响[J].广州化工，2005，33：44-45.
[38]马挺，梁凤来，奚艳伟，等.红球菌PR-1菌株破乳性能研究[J].环境科学，2006，27(6)：1192-1196.
[39]黄翔峰,闻岳，杨葆华，等.破乳菌种TR-1的筛选与破乳性能的实验研究[J].油田化学，2006，23（2）:136-139.
[40]张天胜.生物表面活性剂及其应用[M].北京：化学工业出版社,2005：1-14，294-316.
[41] Van D, Jonathan H, Ajay S, et al. Recent Advances in PetroleumMicrobiology [J]. Microbiology and Molecular Biology Reviews,2003,67(4):503-549.
[42] Ghojavand H, Vahabzadeh F, Azizmohseni F. A Halotolerant,Thermotolerant, and Facultative Biosurfactant Producer: Identification andMolecular Characterization of a Bacterium and Evolution of EmulsifierStability of a Lipopeptide Biosurfactant [J]. Biotechnology and BioprocessEngineering,2011,16(1):72-80.
[43]冯志强，杨永军，朱成军，等.原油生物破乳剂的研究与应用[J].石油大学学报，2004，28(3)：93-99.
[44]吴涓，李请彪，邓旭，等.重金属生物吸附的研究进展[J].离子交换与吸附，1998,14(2):180-187.
[45] Rosen M J. Surfactants and Interfacial Phenomena (2ed)[M]. New York:Wiley,1989:234-246.
[46] Cairns W L, Cooper D G, Zajic J E, et al. Characteriation of Nocardiaamarae as a Potent Biological Coalescing Agent of Water in Oil Emulsions[J]. Appl Evioron Microbiol,1982,43:362-366.
[47]徐燕莉。表面活性剂的功能[M].北京:化学工业出版社.2000,98-102.
[48] D·波塞尔特, W·施密特, M·古茨曼，等.蛋白质作为破乳剂的用途[P].2008, CN101151081.
[49]苗芳,李连志.生物破乳剂的性能研究[J].河北职工医学院，2001，18（3）：5-8.
[50]杨志生,孙宇.生物优势菌破乳剂的制备及性能研究.化学工业与工程.2004,21(1):8-11.
[51] E. Ristau, F. Wanger. Formation of novel anionic trehalosetetraesters fromRhodococcus erythropolis under growth limiting conditions [J]. Biotechnol.Lett.1993,5:95-100.
[52] Parra J L, Guinea J, Manresa M A, et al. Chemical Characterization andPhysicochemical Behaviour of Biosurfactants[J]. J Am Oil Chem Soc,1989,37:141-145.
[53] Peypoux F, Bonmatin J M, Wallach J. Recent trends in the biochemistry ofSurfactin [J]. Appl Microbiol Biotechnol,1999,51(5):553-563.
[54] M. Robert, M. E. Mercade, M. P. Bosch. Effect of the carbon source onbiosurfactant production by Pseudomonas aeruginosa44T1[J]. Biotechnol.Lett.1989,11:871-874.
[55] Demain A L, Adrio J L. Contributions of microorganisms to industrialbiology [J]. Mol Biotechnol,2008,38(1):41-55.
[56] Shikha A S, Darmwal N S. Improved production of alkaline protease from amutant of alkalophilic Bacillus pan totheneticus using molasses as asubstrate [J]. Bioresour Technol,2007,4:881-885.
[57]孙晓君，朱婷婷，秦智，等.紫外诱变获得耐高浓度丙烯腈菌株的研究［J］.哈尔滨工业大学学报，2005，37（10）：1376－1379.
[58]肖怀秋，李玉珍，兰立新.复合诱变原生质体选育高产中性蛋白酶菌株[J].中国酿造，2008（15）：52-54.
[59]施巧琴.工业微生物育种学[M].北京：科学出版社,2003.
[60]罗大珍，林稚兰.现代微生物发酵及技术教程[M].北京:北京大学出版社,2006:18-24.
[61]陈必链.微生物工程[M].北京：科学出版社，2010:23-25.
[62]郑国香，任南琪，林海龙，等.诱变育种选育产氢细菌[J].太阳能学报.2007,6(28):632-637.
[63]刘录祥，郑企成．空间诱变与作物改良［M］．北京:原子能出版社，1997．
[64]袁存权，李云，管耀义，等．航天诱变育种机理研究进展［J］．河北林业科技.2009，B09:39-43．
[65]杜丽平，闻建平，朱智勇。复合诱变菌处理氨氮废水[J]，上海环境科学.2002,21(1)15-23.
[66]冯建成,张容鹄,罗素兰。高产透明质酸菌种FJ-23的诱变选育[J]生物技术.2006,16(3)28-30.
[67]侯红萍,杜文娟.微波-亚硝基胍复合诱变筛选纤维素酶高产菌株[J].中国酿造，2009,8:53-56.
[68]郑国香,任南琪,李永峰,等.一株高效产氢突变体RF-9的筛选与产氢特性[J].中国环境科学2007,27(2)：184~188.
[69]鲍秀珍.高压静电场离子束组合诱变木聚糖酶产生菌(黑曲霉菌)的技术研究[D].内蒙古大学，2007.
[70] Wasinger V C，Cordewell S J，Cerpa P A，et al. Progress with Gene-productmapping of the Mollicutes: Mycoplasma genitalium [J]. Electrophoresis，1995,16(7):1090-1094.
[71]赵洪岩.采用非标记定量技术对变形链球菌耐氟菌株的差异蛋白质组学研究[D].吉林:吉林大学,2011:1-20.
[72] Cardoza J D, Parikh J R, Ficarro S B, et al. Mass Spectrometry-basedProteomics: Qualitative Identification to Activity-based Protein Profiling[J].Wiley Interdisciplinary Reviews. System Biology and Medicine,2012,4(2):141-162.
[73] Patel P S，Telang S D，Rawal R M，et al. A review of Proteomics in CancerResearch [J]. Asian Pac J Cancer Prev，2005，6(2):113-117.
[74] Chichester C，Nikitin F，Ravarini J C, et al. Consistency Checks forCharactering Protein Forms [J].Comput Biol Chem,2003,27(l):29-35.
[75]李丽莉,温博海,王恒樑.蛋白质组技术进展[J].生物技术通讯,2006,17(4):662-664.
[76] Farrell P H. High Resolution Two Dimensional Electorphoresis of Protein[J]. J Biol Chem,1975,250:4007-4021.
[77] Klose J. Protein Mapping by Combined Isoelectrophoresis of MouseTissues:A Novel Approach to Testing for Induced Point Mutations in Mammals [J].Humangenetik,1975,26:231-243.
[78]威尔金斯，阿尔佩，威廉斯，等.蛋白质组学研究[M].北京：科学出版社，2010:15-50.
[79]武霞.野大麦盐胁迫的差异蛋白质组学研究[D].长春：吉林大学,2007：24-27.
[80] Tonge R, Shaw J, Middleton B, et al. Validation and Different Gel ofFluorescence Electrophoresis Proteomics Technology [J]. Proteomics,2001,1:377-396.
[81] Zhou G, Li H M, DeCamp D, et al.2D-Differential in-Gel Electrophoresisfor the Identification Esophageal Scans Cancer Specific Protein Markers [J].Mol Cell Proteomics,2002,1:117-124.
[82] Lilley K S, Friedman D B. All about DIGE: Quantification Technology forDifferential Display2D-gel Proteomics [J]. Expert Rev Proteomics,2004,1(4):401-409.
[83] Fenn J B，Mann M，Meng C K，et al. Electrospray Ionization for MassSpectrometry of Large Biomolecules [J]. Science,1989,246(4926):64-71.
[84] Lopachin R M，Jones R C，Patterson T A，et al. Application of Proteomicsto the Study of Molecular Mechanisms in Neurotoxicology [J]. R M N euroToxicology,2003,24:761-765.
[85] Wang X N, Xu L N,Peng J Y, et al. Application of Recent Biological MassSpectrometry in Biomacromolecules Analysis and Reserch[J]. Chin JMAP,2008,25(2):105-109.
[86]吴祖建，高芳銮，沈建国.生物信息学分析实践[M].北京：科学出版社,2010:105-140.
[87] Mann M. Quantitative Proteomics [J]. Nat Bioltechnol,1999,17:954-955.
[88] Rotilio D, Della Corte A, D'lmperio M, et al. Proteomics: Bases for ProteinComplexity Understanding [J]. Thrombosis Reserarch,2012,129(3):257-262.
[89]李旭.生物破乳剂的开发及破乳效能研究[D].哈尔滨:哈尔滨工业大学.2008:18-30,45-96.
[90] Banat I M. The Isolation of a Thermoohilic Biosurfactant-Producingbacillus species [J]. Biotechnol Lett,1993,15(6):591-594.
[91]李衍达,孙之荣.生物信息学基因和蛋白质分析的实用指南[M].北京:清华大学出版社,2003:16-43,175-211.
[92]马放,任南琪,杨基先.污染控制微生物学实验[M].哈尔滨:哈尔滨工业大学出版社,2002:53-57.
[93]咸洪泉，郭立忠.微生物学实验教程[M].北京：高等教育出社,2010:21-25.
[94]于冰.产絮菌产絮过程差异表达蛋白质及其功能调控研究[D].哈尔滨:哈尔滨工业大学,2010:27-36.
[95]范国昌.微生物混合培养的研究与应用.生物学杂志，1996，4:28~29.
[96]侯宁.生物破乳剂产生菌的特性及破乳效能研究[D].哈尔滨:哈尔滨工业大学,2009:63-71.
[97]张向阳,刘登如,堵国成,等.辅酶Q10产生菌的抗性筛选及发酵条件优化[J].工业微生物，2007,37(3)：5-9.
[98] Hollywood, Katherine A.; Shadi, Iqbal T.; Goodacre, Royston.Monitoringthe succinate dehydrogenase activity isolated from mitochondria by surfaceenhanced Raman scattering [J]. Journal of Physical Chemistry C,2010,4:7308-7313.
[99] Khunderyakova N V， Zakharchenko M V， Zakharchenko A V.Hyperactivation of succinate dehydrogenase in lymphocytes of newbornrats [J]. Biochemistry (Moscow),2008,3:337-341.
[100] Cimen H，Han M J，Yang Y J. Regulation of succinate dehydrogenaseactivity by SIRT3in mammalian mitochondria [J]. Biochemistry,2010,1:304-311.
[101] Elisa P S，Sandra V V，Susana R N. The Cth2ARE-binding protein recruitsthe Dhh1helicase to promote the decay of succinate dehydrogenase SDH4mRNA in response to iron deficiency [J]. Journal of Biological Chemistry,2008,10:28527-28535.
[102]徐暘.一株破乳菌破乳有效成分分析及其强化培养条件优化[D].哈尔滨：哈尔滨工业大学，2010：72-87.
[103] Adriano N N，Fernando C，Yves G. Deficiency of mitochondrial fumaraseactivity in tomato plants impairs photosynthesis via an effect on stomatalfunction [J]. Plant Journal,2007,1:1093-1106.
[104] Koivunen P，Hirsil M，Remes A M. Inhibition of hypoxia-inducible factor(HIF) hydroxylases by citric acid cycle intermediates: Possible linksbetween cell metabolism and stabilization of HIF [J].2007,2:4524-4532.
[105] Guebel D V，Cánovas M，Torres N V. Analysis of the escherichia coliresponse to glycerol pulse in continuous, high-cell density culture using amultivariate approach [J]. Biotechnol. Bioeng.,2009,2:910-922.
[106] Vivoli M，Angelucci F，Ilari A. Role of a conserved active site cation-πinteraction in Escherichia coli serine hydroxymethyltransferase [J].Biochemistry,2009,12:12034-12046.
[107] Zuo Z Y，Zheng Z L，Liu Z G. Cloning, DNA shuffling and expressionof serine hydroxymethyltransferase gene from Escherichia coli strainAB90054[J]. Enzyme Microb. Technol.,2007,3:569-577.
[108] Jennifer T F，William K S， Marie A C. A UV-responsive internal ribosomeentry site enhances serine hydroxymethyltransferase1expression for DNAdamage repair [J]. Journal of Biological Chemistry,2009,11:31097-31108.
[109] Akiduki S，Ikemoto M J. Modulation of the neural glutamate transporterEAAC1by the addicsin-interacting protein Arl6ip1[J]. Journal ofBiological Chemistry,2008,11:31323-31332.
[110] Sanghamitra D，Rodney L B，Gregory A G. Structural analysis of substrateand effector binding in Mycobacterium tuberculosis D-3-phosphoglyceratedehydrogenase [J].Biochemistry,2008,8:8271-8282.
[111] Rodney L B，Chen S W，Xiao L X. Role of the anion-binding site incatalysis and regulation of Mycobacterium tuberculosisD-3-phosphoglycerate dehydrogenase [J]. Biochemistry,2009,6:4808-4815.
[112] Sanghamitra D，Hu Z Q，Xiao L X. The effect of hinge mutations oneffector binding and domain rotation in Escherichia coliD-3-phosphoglycerate dehydrogenase [J]. Journal of Biological Chemistry,2007,6:18418-18426.
[113] Alasdair F. B， Christopher F S， Zhang X J. Evidence from Ramanspectroscopy that InhA, the mycobacterial enoyl reductase, modulates theconformation of the NADH cofactor to promote catalysis [J]. Journal of theAmerican Chemical Society,2007,5:6425-6431.
[114] Xu H，Todd J S，Sekiguchi J I. Mechanism and inhibition of saFabI, theenoyl reductase from Staphylococcus aureus [J]. Biochemistry,2008,4:4228-4236.
[115] Sara M V，Patrick M S. The two-component system bacillus respiratoryresponse A and B (BrrA-BrrB) is a virulence factor regulator in Bacillusanthracis [J]. Biochemistry,2007,6:7343-7352.
[116] Yan L Y，Yang Q Q，Jiang, J H. Involvement of a putative responseregulator Brrg-1in the regulation of sporulation, sensitivity to fungicides,and osmotic stress in Botrytis cinerea [J]. Appl. Microbiol. Biotechnol.,2011,4:215-226.
[117] Shobhana S G，Brendan N B，Timothy L C. Structural analysis of theDNA-binding domain of the Helicobacter pylori response regulator ArsR[J]. Journal of Biological Chemistry,2009,3:6536-6545.
[118] Helen R J，Kanchana R R，Lizbeth H. The Cys319Loop modulates thetransition between dehydrogenase and hydrolase conformations in inosine5-monophosphate dehydrogenase [J]. Biochemistry,2010,12:10674-10681.
[119] Isabelle L，Patrick C， Félix C. Liquid chromatography-coupled tandemmass spectrometry based assay to evaluate inosine-5-monophosphatedehydrogenase activity in peripheral blood mononuclear cells from stemcell transplant recipients [J]. Analytical Chemistry,2012,1:216-223.
[120] Arunkumar, K， Narayanan M. Molecular evolution of theN-acetylglucosamine-6-phosphate deacetylase gene [J]. InternationalSymposium on Biocomputing, ISB2010,2010.
[121] Jurgen R H， Mhairi S， Van-Duc L. Control and regulation of geneexpression: Quantitative analysis of the expression of phosphoglyceratekinase in bloodstream form Trypanosoma brucei [J]. Journal of BiologicalChemistry,2008,2:2495-2507.
[122] Hurth C，Tassius C，Talbot J C. Enzymatic activity of immobilized yeastphosphoglycerate kinase [J]. Biosensors and Bioelectronics,2007,5:2449-2455.
[123] Monica B，Luis G A，Sebastiao J F. The role of reaction energy andhydrogen bonding in the reaction path of enzymatic proton transfers [J].Journal of Physical Organic Chemistry,2009,3:254-263.
[124] Krahulec J，Szemes T，Krahulcová J. Bioinformatics characterization ofpotential new beta-glucuronidase from streptococcus equi subsp.Zooepidemicus [J]. Molecular Biotechnology,2010,3:232-241.
[125] Miguel S J，Casimir B，Jurgen S. Charge stabilization and entropy reductionof central lysine residues in fructose-bisphosphate aldolase [J].Biochemistry,2009,6:4528-4537.
[126] Chiu K H，Huang H W，Mok, H K. Differential proteome analysis ofhagfish dental and somatic skeletal muscles [J]. Marine Biotechnology,2007,12:689-700.
[127] Hallows W C, Yu W, Denu J M. Regulation of Glycolytic EnzymePhosphoglycerate Mutase-1by Sirt1Protein-mediated Deacetylation [J].Journal of Biological Chemistry,2012,287(6):3850-3858
[128] Papini M, Nookaew I, Scalcinati G, et al. Phosphoglycerate mutaseknock-out mutant Saccharomyces cerevisiae: Physiological investigationand transcriptome analysis [J]. Biotechnology journal,2012,5(10):1016-1027.
[129] Yamada S, Sugimoto H, Kobayashi M, et al. Structure of PAS-LinkedHistidine Kinase and the Response Regulator Complex [J]. Structure,2012,17(10):1333-1344.
[130] Song Y H, Wan L L, Wang Y, et al. Electron transfer and electrocatalyticsof cytochrome c and horseradish peroxidase on DNA modified electrode [J].Bioelectrochemistry,2012,85:29-35.
[131] Huynh H， Servant N, Chalifour L E, et al. Ubiquinol-cytochrome-creductase7.2kDa protein of mitochondrial complex III issteroid-responsive and increases in cardiac hypertrophy andhypertension[J]. Canadian journal of physiology and pharmacology,2012,85(10):986-996.
[132] Gutierrezcirlos E B, Antaramian A, Vazquezacevedo M, et al. Ahighly-active ubiquinol-cytochrome-creductase (BC (1) complex) from thecolorless ALGA polytomella SPP, a close relative ofchlamydomonas-characterization of the heme-binding site of cytochrome-c(1)[J]. Journal of biological chemistry,1994,269(12):9147-915.
[133] Hallows W C, Yu W, Denu J M. Regulation of Glycolytic EnzymePhosphoglycerate Mutase-1by Sirt1Protein-mediated Deacetylation [J].Journal of biological chemistry,2012,287(6):3850-3858.
[134] Niture S K, Jaiswal A K. Inhibitor of Nrf2(INrf2or Keap1) ProteinDegrades Bcl-xL via Phosphoglycerate mutase5and Controls CellularApoptosis [J]. Journal of biological chemistry,2011,286(52):44542-44556.
[135] Papini M, Nookaew I, Scalcinati G, et al. Phosphoglycerate mutaseknock-out mutant Saccharomyces cerevisiae: Physiological investigationand transcriptome analysis [J]. Biochemical journal,2010,5(10):1016-1027.
[136] Wang L, Chen Y, Jiang R R. Nanoparticle-supported consecutive reactionscatalyzed by alkyl hydroperoxide reductase [J]. Journal of MolecularCatalysisb-enzymatic,2012,76:9-14.
[137] Chien V, Aitken J F, Zhang S, et al. The chaperone proteins HSP70,HSP40/DnaJ and GRP78/BiP suppress misfolding and formation ofbeta-sheet-containing aggregates by human amylin: a potential role fordefective chaperone biology in Type2diabetes [J]. Biochemical journal,2012,432:113-121.
[138] Mohammadian T, Amini H, Panejad M. Electroelution of the stained alkylhydroperoxide reductase of Helicobacter pylori from preparative sodiumdodecyl sulfate polyacrylamide gels [J]. Clinical biochemistry,2012,44(13): S97-S97.
[139] Appleby T C, Anderson R, Fedorova O, et al. Visualizing ATP-DependentRNA Translocation by the NS3Helicase from HCV [J]. Journal ofmolecular biology,2011,405(5):1139-1153.
[140] Kikuta K, Kubota D, Saito T, et al. Clinical proteomics identifiedATP-dependent RNA helicase DDX39as a novel biomarker to predict poorprognosis of patients with gastrointestinal stromal tumor [J]. Journal ofpoteomics,2012,75(4):1089-1098.
[141] Lee J H, Kang G B, Lim H H, et al. Crystal structure of the GluR0ligand-binding core from Nostoc punctiforme in complex with L-glutamate:Structural dissection of the ligand interaction and subunit interface [J].Journal of molecular biology,2012,376(2):308-316.
[142] Lee J H, Park S J, Rho S H, et al. Crystallization and preliminary X-raycrystallographic analysis of the GluR0ligand-binding core from Nostocpunctiforme[J]. Acta Crystallographica Section F: Structural Biology andCrystallization Communications,2012,61:1020-1022.
[143] Friesen D E, Barakat K H, Semenchenko V, et al. Discovery of SmallMolecule Inhibitors that Interact with gamma-Tubulin [J]. ChemicalBiology&Drug Design,2012,79(5):639-652.
[144] Paul A, Lal L, Ahuja P S, et al. Alpha-tubulin (CsTUA) up-regulated duringwinter dormancy is a low temperature inducible gene in tea [Camelliasinensis (L.) O. Kuntze][J]. Molecular Biology Reports,2012,39(4):3485-3490.
[145] Rohani M M, Zydney A L. Protein transport through zwitterionicultrafiltration membranes [J]. Journal of Membrane Science Science,2012,397:1-8.
[146] Moinova H, Leidner R S, Ravi L. Aberrant Vimentin Methylation IsCharacteristic of Upper Gastrointestinal Pathologies [J]. CancerEpidemiology, Biomarkers&Prevention,2012,21(4):594-600.
[147] Triolo D, Dina G, Taveggia C, et al. Vimentin regulates peripheral nervemyelination [J]. Development,2012,139(7):1359-1367.
[148] Kim S J, Ise H, Goto M, et al. Interactions of vimentin-ordesmin-expressing liver cells with N-acetylglucosamine-bearingpolymers[J]. Biomaterials,2012,33(7):2154-2164.