摘要
β-胡萝卜素(β-carotene)是类胡萝卜素家族中的典型代表,不仅是体内维生素A的重要来源,而且其本身对人体也具有重要的生理功能,它可以预防、延缓和治疗某些疾病,尤其是癌症,同时也能提高机体的免疫功能。近年来,β-胡萝卜素的制备工艺,特别是发酵法生产,受到研究人员的重视。
杜氏盐藻(Dunaliella salina,简称盐藻)是商业生产β-胡萝卜素的主要微生物之一,能在强光照,高盐度,低营养的条件下积累大量的β-胡萝卜素,是公认的产β-胡萝卜素最好的天然资源。因此从分子生物学角度研究盐藻合成和累积β-胡萝卜素的机理,将从盐藻中分离得到的功能基因用于类胡萝卜素的基因工程和代谢工程研究具有重大意义。
盐藻番茄红素β-环化酶(LycB)的功能是催化番茄红素转化成β-胡萝卜素,盐藻高度累积β-胡萝卜素与该酶的基因调控及其酶催化机理直接相关。本研究根据盐藻LycB基因的开放阅读框(ORF)序列设计引物,采用RT-PCR的方法从盐藻中克隆得到LycB ORF,经测序确证后,将该段序列克隆入能合成LycB底物的原核表达载体pACCRT-EIB(携带欧文氏菌crtE,crtI和crtB基因,能合成番茄红素),构建得重组质粒pACCRT-EIB-LycB并转化大肠杆菌(Escherichia coli DH5α),经表达菌落的颜色筛选,获得了能稳定合成β-胡萝卜素的大肠杆菌基因工程菌株。
通过对该菌株的培养条件进行优化研究,确定了影响β-胡萝卜素积累的主要因素,接着通过设计四因素三水平的响应面优化实验,确定了积累β-胡萝卜素的最佳培养基配方。实验结果表明,最优培养基成分为:蛋白胨20.43g/L,酵母膏10g/L,NaCl 5g/L,甘油1.97mL/L,NH4Cl 1.09g/L,Na2HPO4?7H2O 12.84g/L。在此条件下培养该菌株,β-胡萝卜素平均产量为2.3896mg/L,比相同培养条件下含有欧文氏菌(Erwinia uredovora)环化酶基因crtY的pACCRT16ΔX参照菌株累积的β-胡萝卜素提高了39.26%。
As a typical representative of carotenoid family,β-carotene is not only an important source of vitamin A in human body, it also has some physiological functions to the health of a person. Recent research suggests thatβ-carotene is associated with reduced risk of certain disease, particularly in cacer. Simultaneouly, it can improve the immune ability of the body. In recent years, the producing technologies, especially fermentation, have been studied.
Dunaliella salina is one of the major microorganisms commercially employed to produceβ-carotene, it can accumulate much moreβ-carotene than any other organisms under the conditions of high light, high salinity and nutrient deprivation. Therefor, it is of great economical significance to study the mechanisms ofβ-carotene synthesis and accumulation from the perspective of molecular biology, which may explain the molecular regulation of key genes and enzymes of D. salina in the carotenogenic pathway.
Lycopeneβ-cyclase (LycB) is the key enzyme of catalyzing lycopene to formβ-carotene. The reason that D. salina highly accumulateβ-carotene is directly related to the catalytic mechanisms of LycB and the molecular regulation of its gene. In order to address the function of LycB, we cloned the open reading frame(ORF) of LycB by RT-PCR strategy. After confirmed by DNA sequencing, we constructed a recombinant plasmid pACCRT-EIB-LycB by cloning the ORF of LycB into the plasmid pACCRT-EIB which harbours the Erwinia uredovora genes necessary for lycopene formation. Than transformed the pACCRT-EIB-LycB into E.coli DH5α, so a genetically engineered bacteria strain which can accumulateβ-carotene stablely was gained.
In order to obtain the maximum yield ofβ-carotene, response surface analysis methodology (RSM) was applied to optimize the composition of fermentation medium. Based on single-factor experiments, four-factors-three-levels Box-behnken central compositon experiments design were applied. The results showed that the best fermentation medium was tryptone 20.43g/L, yeast extract 10g/L, NaCl 5g/L, glycerol 1.97g/L, NH4Cl 1.09g/L, Na2HPO4?7H2O 12.84g/L. Validation experiments were carried out at these optimum conditions and the average yield ofβ-carotene is 2.3896mg/L. The experiment data shows the production ofβ-carotene has increased 39.26% compared with the E.coli strain containing pACCRT16ΔX which contains crtY gene of Erwinia uredovora under the same culture conditions.
引文
[1] Laurent D., Microbial and Microalgal Carotenoids as Colourants and Supplements [A]. Britton G.., Carotenoids[C]. Birkhauser, Basel., 2009: 83-98
[2] Kull D., Pfander H. Isolation and Analysis[A]. Britton G., Liaaen-Jensen S., Pfander H., Eds. Carotenoids[C]. Birkhauser, Basel., 1995: 295-317
[3] Gooin T. W., Britton G.. Distribution and analysis of carotenoids[A]. Gooin, T. W. , Ed. Plant Pigments[C] New York: Academic Press,1988: 133-182
[4]胡英考,李雅轩,蔡民华,等.提高植物维生素含量的基因工程[J].中国生物工程杂志, 2004, 24(5): 20-23
[5] Bartley G E., Scolnik P A. Plant carotenoid: pigment for photoprotection, visual attraction and human health[J]. Plant Cell, 1995, 7: 1027~1038
[6]朱秀灵,车振明,徐伟,等.β-胡萝卜素生理功能及提取技术的研究进展[J],西华大学学报, 2005, 24(1): 71-76
[7]黄健,刘建国.类胡萝卜素的分析测定[J].青岛化工学院学报(自然科学版), 2001, 22: 223-227
[8]王永华,梁世中.类胡萝卜素的结构和生理功能研究[J],广州食品工业科技, 1999, 16:1-4
[9]刘春朝,钱新民,高登文等.光合细菌饲料添加剂的生产及其对肉鸡生长性能的影响[J],中国饲料, 1995, 9: 14一15
[10]庞金钊.光合细菌及在水产养殖中的应用[J],齐鲁渔业, 1995, 12(5):11-14
[11] Yamauchi R., Miyake N., Inoue H., et al. Products formed by peroxyl radical oxidation ofβ-carotene. J Agr Food Chem, 1993, 41: 708-713
[12]陶俊,张上隆,徐昌杰,等.类胡萝卜素合成的相关基因及其基因工程[J].生物工程学报, 2002, 18(3): 276-281
[13] Ye X., Al Babili S., Kloti A., et al. Engineering the provitamin A (β-carotene) biosynthesis pathway into( carotenoid-free) rice endosperm[J]. Science, 2000, 287: 303-305
[14] Matsuno T. Xanthophylls as precursors of retinoids[J]. Pure Appl Chem, 1991, 63: 81-88
[15]韩雅珊.类胡萝卜素的功能研究进展[J].中国农业大学学报, 1999, 4(1): 5-9
[16] Street DA., Comstock GW., Salkeld RM., et al. Serum antioxidants and myocardialinfarction. Are low levels of carotenoids and alpha-tocopherol risk factors for myocardial infarction? [J]. Circulation 90, 1994, 90: 1154-1161
[17] Kohlmeier L., Kark J D., Gomez-Gracia E., et al. Lycopene and Myocardial Infarction Risk in the EURAMIC Study [J]. Am. J. Epidemiol, 1997, 146(1): 618-626
[18] Kontush A., Spranger T., Reich A. Lipophilic antioxidants in blood plasma as markers of atherosclerosis: the role ofα-carotene andγ-tocopherol[J].Atheroslerosis 1999, 144(1): 117-122
[19] Kardinaal A.F.M., Kok F.J., Ringstad J., et al. Antioxidants in adipose tissue and risk of myocardial infarction: the EURAMIC study [J]. Elsevier Science Ltd , 1993, 342(8884): 1379-1384
[20] Hsueh Y.M., Wu W.L., Huang Y.L. Low serum carotene level and increased risk of ischemic heart disease related to long-term arsenic exposure[J]. Atherosclerosis, 1998, 141(2): 249-257
[21] Malvy D.J.M., Burtschy B., J. Arnaud, et al. Serum Beta-Carotene and Antioxidant Micronutrients in Children with Cancer[J]. Int. J. Epidemiol, 1993, 22(5): 761-771
[22] Torun A.M., Yardim S., Gonenc A., et al. Serumβ–carotene, vitamin E, vitamin C and malondialdehyde levels in several types of cancer [J]. Clin. Pharm Ther, 1995, 20(5): 259-263
[23] Nagata C., Shimuzu H., Yoshikawa H., et al. Serum carotenoids and vitamins and risk of cervical dysplasia from a case–control study in Japan[J]. Br J Cancer, December, 1999, 81(7): 1234-1237
[24] Hao Chang-ming, Luo Yi. Applications of carotenoids and their research advances[J]. Science Knowledge, 2002, 4: 42-43
[25] Govannucci E., Ascherio A., Rimm EB., et al. Intake of carotenoids and ratinol in relation to risk of prostate cancer[J]. J Natl Cancer Inst, 1995, 87: 1767-1776
[26] Rao A V, Agarwal S. Effect of diet and smoking on serum lycopene and lipid Peroxidation[J]. Nutrition Research, 1998, 18: 713-721
[27] Sakaki Keiji. Solubility ofβ-carotene in dense CO2 from 308 to 323 K and from 9.6 to 30 MPa[J]. Chem Eng. Data, 1992, 37(2): 249-251
[28] Siong Tee. Carotenoids and the immune response[J]. Pro Nutr Soc, 1991, 50: 263-274
[29] Patricia Botella-Pavía,óscar Besumbes, Michael A. Phillips, et al. Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors[J]. Plant J , 2004, 40(2): 188 - 199
[30] Van Dien S J. , Strovas T., Mary E. Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry[J]. Appl Environ Microbiol, 2003, 84 (1): 45-55
[31] Claudia Schmidt-Dannert. Engineering novel carotenoids in microorganisms[J]. Curr Opin Biotech, 2000, 11:255-261
[32] Armstrong GA., Alberiti M., Leach F., et al. Nucleotide sequence, organization and nature of the protein products of the caroteniod biosynthesis gene cluster of Rhodobacter capsulatus[J]. Mol Gen Genet, 1989, 216(3): 254-268
[33] Misawa N., Nakagawa M., Kobayashi K., et al. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli, 1990, J Bacterial, 172(12): 6704-6712
[34] Hodgson D.A., Murillo F.J. Genetics of regulation and pathway of synthesis of carotenoids[A]. orkin, M., KaiserD., Eds., Myxobacteria[C]. Washington D.C: ASM Press, 1993: 157-181
[35] Hirschberg J., Chamovitz D. Carotenoids in cyanobacteria[A]. Bryant, D., Kluwer D., Eds., Advances in Photosynthesis: The Molecular Biology of Cyanobacteria[C]. 1994: 559-579
[36] Armstrong G A. Genetic analysis and regulation of carotenoid biosynthesis: structure and function of the crt genes and gene products[A]. Blankenship R E., Madigan M T., Bauer C E., Eds. Anoxygenic Photosynthetic Bacteria[C]. The Netherlands: Kluwer Academic Publishers, 2004:1135-1157
[37] Breitenbach J., Misawa N., Kajiwara S., et al. Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus plucialis[J]. FEMS Microbiol Lett, 1996, 140: 241-246
[38] Harker M., Hirschberg J. Biosynthesis of ketocarotenoids in transgenic cyanobacteria expressing the algal gene for L-C-4-oxygenase, crtO[J]. FEBS Letters, 1997, 404: 129-134
[39] Bohne F., Linden H. Regulation of carotenoid biosynthesis genes in response to light in Chlamydomonas reinhardtii. Bba-Biomembranes, 2002, 1579: 26– 34
[40] Chien, Y H., Shiau, W C. The effects of dietary supplementation of algae and synthetic astaxanthin on body astaxanthin, survival, growth, and low dissolved oxygen stress resistance of kuruma prawn, Marsupenaeus japonicus Bate[J]. J Expmar Biol Ecol, 2005, 318: 201-211
[41] Barkovich R., Liao J C. Metabolic Engineering of Isoprenoids[J]. Metab Eng, 2001,3: 27-39
[42] Yan Y., Zhu Y H., Jiang J G.., et al. Cloning and sequence analysis of the phytoene synthase gene from a unicellular Chlorophyte, Dunaliella salina[J]. J Agr Food Chem, 2005, 53: 1466-1469
[43] Zhu Y H., Jiang J G., Yan Y., et al. Isolation and characterization of phytoene desaturase cDNA involved in theβ-carotene biosynthetic pathway in Dunaliella salina[J]. J Agr Food Chem, 2005, 53: 5593-5597
[44]严媛,姜建国,黄洋.类胡萝卜素及其基因工程的研究现状[J].食品科学. 2003, 4:118-121
[45] Schmimidt-Dannert C. Engineering novel carotenoids in microorganisms[J]. Curr Opin Biotech. 2000, 11: 255-261
[46] Al-Babili S., Hugueney P. Identification of a novel gene coding for neoxanthin synthase from Solanumtu berosum[J]. FEBS Lett, 2000, 485:168-172
[47] Yokoyama A., Shizuria Y.,Misawa N. Production of new carotenoids, astaxanthin glucosides by Escherichia coli transformants carrying carotenoid biosynthesis genes[J]. Tetrahedron Lett, 1998, 39: 3709-3712
[48] Johnson E A., Schroeder W A. Microbial carotenoids[J]. Adv Biochem Eng Biot, 1995, 53: 119-178
[49] Misawa N., Schimada H. Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts[J]. J Biotechnol. 1998, 59: 169-181
[50] Wang CW, Oh MK, Liao JC. Engineered isoprenoid pathway enchances astaxanthin production in Escherichia coli. Biotechnol Bioeng[J]. 1999, 62: 235-241
[51] Albrecht M., Takaichi S., Steiger S., et al. Novel hydroxyl carotenoids with improved antioxidative properties produced by gene combination in Escherichia coli[J]. Nat Biotechnol. 2000,18: 843-846
[52] Umeno D., Arnold F H. Evolution of a pathway to novel long-chain carotenoids [J]. J Bacteriol, 2004, 186(5): 1531-1536
[53] Boze H., Moulin G., Galzy P. Production of food and fodder yeasts [J]. Crit Rev Biotechnol, 1992, 12:65-68
[54] Ishii T., Takehara S., Konno H., et al. Development of a new commercial-scale airlift fermentor for rapid growth of yeast [J]. J Ferment Bioeng, 1993, 75: 375-379
[55] Miura Y., Kondo K., Schimada H., et al. Production of lycopene by the food yeast Candida utilis that does not naturally synthesize carotenoid. Biotechnol Bioeng, 1998,58(2): 306-308
[56] Shimada H., Kondo K., Fraser P D, et al. Increased Carotenoid Production by the Food Yeast Candida utilis through Metabolic Engineering of the Isoprenoid Pathway [J]. Appl Environ Microbiol, 1998, 64: 2676-2680
[57] Shewmaker C K., Sheehy J A., Daley M., et al. Seed specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects[J]. Plant J, 1999, 20(4): 401~412
[58] Ye X., Al-Babili S., kloti A., etal. Engineering the provitamin A(β-carotene)biosynthetic pathway into(carotenoid-free)rice endosperm[J]. Science, 2000, 287: 303-305
[59] Romer S., Fraser P D., Kiano JW., et al. Elevation of the provitamin A content of transgenic tomato plants[J].Nat Biotechnol, 2000, 18(6): 666-669
[60] Schmidt - Dannert C., Umeno D., Arnold F H. Molecular breeding of carotenoid biosynthetic pathways[J]. Nat Biotechnol, 2000, 18: 750-753.
[61] Wang C., Liao J C. Alteration of product specificity of Rhodobacter sphaeroides phytoene desaturase by directed evolution[J]. Biol Chem, 2001 , 276(44): 41161-41164
[62] Rick W Y., Kristen J S., Henry Y., et al. Mutational and functional analysis of the-Carotene Ketolase involvedin the production of canthaxanthin and astaxanthin[J]. Appl Environ Microbiol, 2006, 72(9): 5829-5837
[63] Ettl H. Taxono mische Bemerkungen zuden phytononadina[J].Nova Hedvigia, 1983, 35: 731-736
[64] Michael A B., Lesley J B., David K.Effects of salinity increase on carotenoid accumulation in the green alga Dunaliella salina[J]. J Appl Phycol, 1990, 2(2): 111-119
[65] Loeblich L A. Photosynthesis and pigments influenced by light intensity and salinity in the halophile Dunaliella salina (Chlorophyta)[J]. J. Mar. Biol. Ass. U.K, 1982, 62: 493-508
[66] Jensen A.Chlorphylls and carotenoids[A]. Hellebust J A, Ed. Handbook of Phycological Methods[C]. Cambridge University Press,1978: 59-70
[67]周德庆.微生物学教程[M].北京:高等教育出版社, 1993, 99-106
[68] Borowitzka L J., Borowitzka M A., Moulton T. Mass culture of dunaliella: From laboratory to pilot plant [J]. Hydrobiologia, 1984, 116-117: 115-121.
[69]Borowitzka L J, Borowitzka M A, Moulton T P. The mass culture of Dunaliella salina for fine chemicals: from laboratory to pilot plant[J]. Hydrobiologia, 1984, 116/117:115-121
[70] Gooin T W. Metabolism, Nutrition, and Function of Carotenoids[J]. Annu Rev Nurt, 1986, 6: 273-297
[71] Semenenko V E., Abdulaev A A. Parametric control ofβ-carotene biosynthesis in Dunaliella salina cells under conditions of intensive cultivation[J]. Fiziologya Rastenii, 1980, 27:31-34
[72] Lers A., Biener Y., Zamir A. Photoinduction of massiveβ-carotene accumulation by the Dunaliella bardawil[J]. Plant Physiol. 1990, 93:389-395
[73] Pace F., Ferrara R., Carratore G. D. Effect of sub-lethal doses of copper sulphate and lead nitrate on growth and pigment composition of Dunaliella salina Teod[J]. B Environ Contam Tox, 1977, 17(6): 679-685
[74] Ko J H, Kim JY, Lee S H. Molecular cloning and characterization of cDNA encoding caltractin from Dunaliella salina [J]. Plant Cell Physiol, 1999, 40(4):457-461
[75]李淑清,索全伶,杨伟.吉兰泰杜氏盐藻中营养成分分析[J ].内蒙古工业大学学报, 2000, 19(1): 22– 24
[76]姜建国,姚汝华.五种盐藻生化组成及β-胡萝卜素异构体分析[J ].华南理工大学学报, 1997, 25(10): 38– 41
[77]李建宏,翁永萍.不同氮源对盐生杜氏藻生长和β-胡萝卜素积累的影响[J ].南京师大学报(自然科学版), 1999, 22(3): 73– 76
[78] Avron M., Ben-Amotz A. Dunaliella: physiology, biochemistry and biotechnology[J]. J Exp Mar Biol Ecol, 1993, 171(1): 142-144
[79] Walker T L., Purton S., Becker D K., et al. Mocroalgae as bioreactors[J]. Plant Cell Rep, 2005, 24(11): 629-641
[80]柴玉荣,王天云,薛乐勋.新型生物反应器——杜氏盐藻研究进展[J].中国生物工程杂志, 2004, 24(2): 30-33
[81]白林含,乔代蓉,郑鸣.改进简并PCR法克隆盐生杜氏藻烯醇酶基因[J].北京林业大学学报, 2004, 26(4): 25-29
[82]刘敏,白林含,郑鸣,等.盐生杜氏藻核糖体蛋白L38部分cDNA的克隆及分析[J].四川大学学报(自然科学版), 2003, 40 (2): 346-348
[83]李音,乔代蓉,易弋,等.盐生杜氏藻Ugd基因的cDNA克隆及序列分析[J].四川大学学报(自然科学版), 2004, 41( 2): 409-412
[84]郑鸣,白林含,马梵辛,等.盐生杜氏藻cbr基因的克隆[J].四川大学学报(自然科学版), 2003, 40 (3): 590-593
[85]秦松,王希华,曾呈奎.植物遗传转化手册[M].北京:中国科学技术出版社, 1994
[86]潘卫东,袁保梅,谢华,等.杜氏盐藻叶绿体转化载体pDS16S-CAT的构建[J].郑州大学学报(医学版), 2004, 39(1): 25-28
[87]耿德贵,韩燕,王义琴,等.杜氏盐藻德耐盐机制研究进展和基因工程研究的展望[J].植物学通报, 2002, 19(3): 290-295
[88]柴玉荣,侯卫红,谢华,等.杜氏盐藻荧光素酶基因表达载体的构建[J].郑州大学学报(医学版), 2004, 39(1): 29-31
[89]李杰,任宏伟,黄洁虹,等.外源报告基因EGFP在盐藻中实现瞬时表达[J].中国生物化学和分子生物学报, 2003, 19(3): 338-342
[90] Ben-Amotz A, Avron M. The biotechnology of cultivating the halotolerant alga Dunaliella[J]. Trends Biotechno1, 2002, 18(6): 257-263
[91]周世水,姜建国,林炜铁,等.盐藻生长及其β-胡萝卜素累积的作用因子和最适条件探讨[J].食品与发酵工业, 2002, 28(11): 1-3
[92]周世水,姜建国,姚汝华.二步法培养盐藻生产天然β-胡萝卜素的研究[J].食品科学, 1999, 5: 11-14
[93]郑阳霞,枸杞类胡萝卜素合成酶基因(PSY、LycB)的克隆及其转化洋桔梗的研究,四川:四川农业大学, 2006.6
[94] Misawa N., Shimada H., Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts[J]. J Biotech, 1998,59: 169-181
[95] Misawa N.,Satomi Y., Kondo K. Structure and Functional Analysis of a Marine Bacterial Carotenoid Biosynthesis Gene Cluster and Astaxanthin Biosynthetic Pathway Proposed at the Gene Leve[J], American Society for Microbiology., 1995, 177(22), 6575-6584
[96]朱跃辉,房科腾,姜建国,杜氏藻类胡萝卜素的最佳提取条件的研究及比较[J],广州食品工业科技, 2003, 19(4), 18-19
[97] Misawa N., Nakagawa M., K. Kobayashi., et al. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli[J]. J. Bacteriol, 1990, 172: 6704-6712.
[98] Misawa N., Satomi Y., Kondo K., et al. Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level[J]. J. Bacteriol, 1995, 177:6575–6584.
[99] Matthews P D., Wurtzel E T. Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid pool with expression of deoxyxylulose phosphate synthase[J]. Appl Microbiol Biot, 2000, 53(4), 396-400
[100]黄健,刘建国.类胡萝卜素的分析测定,青岛化工学院学报, 2001, 22(3), 224-227
[101]欧阳琴,陈兴才,黄亚治.雨生红球藻虾青素提取工艺条件研究[J],福建轻纺,2003,12(12):9-13
[102]王海滨.类胡萝卜素的紫外可见光谱特性及其应用[J].武汉工业学院学报, 2004, 23(4), 10-13
[103]逯家辉,姜鑫,李昊龙等.应用响应面法优化超声波法提取甘草中总黄酮的工艺[J],吉林大学学报, 2008, 38(2): 293-297
[104] Sung-Woo Kim, Jae-Bum Kim, W.-H. Jung, Jung-Hoe Kim, Joon-Ki Jung, Over-production of b-carotene from metabolically engineered Escherichia coli, Biotechnol Lett [J], 2006, 28: 897-904.
[105] Hausmann A., Sandmann G. A Single Five-Step Desaturase Is Involved in the Carotenoid Biosynthesis Pathway to b-Carotene and Torulene in Neurospora crassa[J]. Fungal Genet Biol, 2000, 30:147–153
[106] Ruther A., Misawa N.,Boger,P., et al. Production of zeaxanthin in Escherichia coli transformed with different carotenogenic plasmids[J]. Appl Microbiol Biotechnol, 1997, 48: 162-167
[107]刘贺,朱丹实,徐学明,等.低酯桔皮果胶凝胶全质参数及持水性响应面分析[J],食品科学, 2009, 30(3): 81-87
[108]肖卫华,韩鲁佳,杨增玲,等.响应面法优化黄芪黄酮提取工艺的研究[J].中国农业大学学报, 2007, 12 (5): 52-56
[109] Sandmann G., Kuhn S., B?ger P. Evaluation of structurally different carotenoids in Escherichia coli transformants as protectants against UV-B radiation. Appl[J]. Environ Microbiol, 1998;64:1972-1974
[110]苏东林,单杨,李高阳,等.柑桔皮总黄酮提取工艺优化及其数学模型研究[J].食品科学, 2008, 29(5): 167-172
[111] Bezerraa M A., Santelli R E., Oliveiraa E P., et al. Response Surface Methodology(RSM) as a Tool for Optimization in analytical chemistry[J]. Talanta, 2008, 76(5): 965-977