2.2 Den objektive gjerningsbeskrivelsen
2.2.6 De straffbare handlinger
A seqüência de 355 pb foi isolada da reação de PCR utilizando o plasmídeo pSTP4 como molde. O fragmento amplificado apresentou 100% de identidade com a região codificadora do domínio de afinidade da estreptavidina pela biotina (cStp). Com a finalidade de obter um sistema de expressão e secreção de proteínas em
Kluyveromyces lactis que permita isolar e/ou imobilizar proteínas recombinantes em
suportes biotinilados, a seqüência codificadora do domínio de afinidade da estreptavidina pela biotina (cStp) foi inserido no vetor de expressão e secreção pKLAC1 de K. lactis. A inserção do cStp nos sítios Xho I e Bgl II em fase com a seqüência do peptídeo líder do mating-α-factor.
O plasmídeo pKLAC1-cStp linearizado com a enzima Ahd I resultou um cassete cujas regiões 3’PLAC4 e 5’PLAC4, direcionaram o cassete para recombinação
homóloga na região promotora do gene da β-galactosidase (LAC4) presente no genoma das linhagens K. lactis MW 98.8C e CBS 2359 transformadas. O procedimento de transformação pela utilização de DMSO proposto por Hill el. al. (1991) resultou numa eficiência de transformação na ordem de 103 e 104 transformantes/µg de DNA nas linhagens CBS 2359 e MW 98.8C respectivamente. Os transformantes analisados foram positivos quanto a presença do cStp no genoma e demonstraram estabilidade mitótica.
Uma concentração de biomassa correspondente a A600 de 18 das linhagens
recombinantes MW 98.8C/cStp e CBS 2359/cStp foi obtida em Erlenmeyers de 250 mL contendo 50 mL de meio YPD. Posteriormente essa biomassa foi transferida para os meios YPL, YNBlac + ext lev e em soro de queijo, todos contendo lactose para a indução da
síntese da estreptavidina. As amostras foram coletadas em intervalos de 24 horas durante um período de 6 dias para análise de proteínas totais.
Para ambas as culturas recombinantes maiores concentrações de proteínas extracelulares totais foram obtidas durante as primeiras 72 horas de indução no meio YPL e após 96 horas no meio YNBlac + ext lev.
O fracionamento em condições desnaturantes das proteínas extracelulares totais do sobrenadantes das culturas recombinantes revelou uma banda de 15kDa ausente nas culturas controle. A atividade biológica da estreptavidina presente no sobrenadante das culturas recombinates foi analisada pela interação com biotina. Ambas as linhagens de K. lactis MW 98.8C/cStp e CBS 2359/cStp obtidas neste trabalho foram capazes de expressar e secretar o core da proteína estreptavidina funcionalmente ativo.
Mais estudos são necessários para determinar as condições fisiológicas que maximizam o rendimento de proteína nos meios de indução principalmente no soro de queijo.
A estabilidade mitótica das linhagens recombinantes favorece sua aplicação em larga escala, uma vez que células recombinantes mitoticamente estáveis reduzem as dificuldades com a manutenção da pressão seletiva no meio.
O plasmídeo pKLAC1 contendo o cStp está apto para a expressão e secreção de proteínas e de enzimas de interesse biotecnológico destinadas à adsorção biosseletiva em colunas biotiniladas. Sugere-se a fusão de genes de enzimas hidrolíticas tais como lipases, para modificação funcional de óleos e gorduras; pectinases, para clarificação de sucos e vinhos; α-glucosidases, para a obtenção de xarope de milho ou celulases úteis na obtenção de substratos fermentáveis de resíduos lignocelulolíticos para a indústria de biocombustível. Colunas biotiniladas contendo as enzimas imobilizadas podem ser incorporadas em uma linha de produção industrial.
6. REFERÊNCIAS
ARGARAÑA, C. E., KUNTZ, I. D. BIRKEN, S., AXEL, R., CANTOR, C. R. Molecular cloning and nucleotide sequence of the streptavidin gene. Nucleic Acids
Research, 14: 1871-1882, 1986.
BARRAL, P., BATANERO, E., VILLALBA, M., RODRÍGUEZ, R. Expression of the major olive pollen allergen Ole e 10 in the yeast Pichia pastoris: Evidence of post- translational modifications. Protein Expression and Purification, 44: 147–154, 2005.
BHAT, P.J., MURTHY, T.V. Transcriptional control of the GAL/MEL regulon of yeast
Saccharomyces cerevisiae: mechanism of galactose mediated signal transduction.
Molecular Microbiology, 40:1059– 66, 2001.
BLONDEAU, K., BOUTUR, O., BOZE, H., JUNG, G., MOULIN, G., GALZY, P. Development of high-cell-density fermentation for heterologous interleukin 1 beta production in Kluyveromyces lactis controlled by the PHO5 promoter. Applied
Microbiology Biotechnology, 41(3), 324 – 329, 1994.
BLONDEAU, K., BOZE, H., JUNG, G., MOULIN, G., GALZY, P. Physiological approach to heterologous human serum albumin production by Kluyveromyces lactis in chemostat culture; Yeast, 10(10), 1297 – 303, 1994.
BOZE, H., MOULIN, G., GALZY, P. Uptake of galactose and lactose by
Kluyveromyces lactis: biochemical characteristics and attempted genetical analysis.
BRADFORD, M. M. A rapid sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Analytical Biochemistry, 72: 248-254, 1976.
BRANDÃO, S.C.C. Soro: um desafio para as fábricas de queijo. Leite & Derivados, v. 15, p. 13-18, 1994.
CEREGHINO, J.L., CREGG, J.M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 24: 45–66, 2000.
CHAIET, I., WOLF, F.J. The properties of streptavidin, biotin-binding protein produced by Streptomycetes. Archives Biochemistry and Biophysics, 106: 1-5, 1964.
CHEN, X.J. Low- and high-copy-number shuttle vectors for replication in the budding yeast Kluyveromyces lactis. Gene, 172: 131–136, 1996.
CHEN, X.J., GAO, B., SHI, W., LI, Y. Expression and secretion of human interferon alpha A in yeast Kluyveromyces lactis. Yi Chuan Xue Bao, 19: 284-288, 1992.
CHEN, X.J., SALIOLA, M., FALCONE, C., BIACHINI, M.M., FUKUHARA, H. Sequence organization of the circular plasmid pKD1 from the yeast Kluyveromyces
drosophilarum. Nucleic Acids Research, 14: 4471-4481, 1986.
CHOI, D.B., PARK, E.Y. Enhanced production of mouse α-amilase by feeding combined nitrogen and carbon sources in fed-bath culture of recombinant Pichia
pastoris. Process Biochemistry – In press 2005.
CLARE, D.A., VALENTINE, V.W., CATIGNANI, G.L., SWAISGOOD, H.E. Molecular design, expression, and affinity immobilization of a trypsin-streptavidin fusion protein. Enzyme and Microbial Technology, 28: 483-491, 2001.
COLUSSI. P.A., TARON, C.H. Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in yeast. Applied Environmental
COS, O., SERRANO, A., MONTESINOS, J.L., FERRER, CREEG, J.M., VALERO, F. Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in P. pastoris fed-bath culture. Journal of
Biotechnology, 116: 321-335, 2005.
CREGG, J.M., VEDVICK, T.S., RASCHKE, W.C. Recents advances in expression of foreing genes in Pichia pastoris- a rewiew- Biotechnology, 11: 905-910, 1993.
DAMASCENO, L.M. Produção de estreptavidina em Pichia pastoris e sua imobilização em sílica biotinilada. Dissertação. Viçosa, UFV, 2001.
DAS, S., HOLLENBERG, C.P. A high-frequency transformation system for the yeast
Kluyveromyces lactis. Current Genetics, 6: 123-128, 1982.
DRIEGEN, S., FERREIRA, R., VAN ZON, A., STROUBOULIS, J., JAEGLE, M., GROSVELD, F., PHILIPSEN, S., MEIJER, D. A generic tool for biotinylation of tagged proteins in transgenic mice. Transgenic Research. 14(4):477-82, 2005.
DUJON, B., SHERMAN, D., FISHER, G. Genome evolution in yeasts. Nature 430: 35-44, 2004.
EL-HAWARY, F.I., MEHANNA, A.S. Production of single cell protein from yeast grown in whey. Acta Alimentaria, v. 20, p. 205-213, 1991.
FALCONE, C., SALIOLA, M., CHEN, X.J., FRONTALI, L., FUKUHARA, H. Analysis of a 1.6-micron circular plasmid from the yeast Kluyveromyces
drosophilarum: structure and molecular dimorphism. Plasmid, 15: 248–252, 1986.
FENG, Y.M., ZHANG, B.Y., ZHANG, Y.S., HIROSHI, F. Secretory expression of porcine insulin precursor in Kluyveromyces lactis and its conversion into human insulin.
FLEER, R., CHEN, X.J., AMELLAL, N., YEH, P., FOURNIER, A., GUINET, F., GAULT, N., FAUCHER, D., FOLLIARD, F., FUKUHARA, H., MAYAYAUX, J.F. High-level secretion of correctly processed recombinant human interleukin-1 in
Kluyveromyces lactis. Gene, 107:285–295, 1991a.
FLEER, R., YEH, P., AMELLAL, N., MAURY, I., FOURNIER, A., BACCHETTA, F., BADUEL, P., JUNG, G., L’HOTE, H., BECQUART, J., FUKUHARA, H., MAYAYAUX, J.F. Stable multicopy vectors for high-level secretion of recombinant human serum albumin by Kluyveromyces lactis yeasts. Bio/Technology, 9: 968–975, 1991b.
FLORES, M.V., CUELLAS, A., VOGET, C.E. The proteolytic system of the yeast
Kluyveromyces lactis. Yeast, 15: 1437-1448, 1999.
FONSECA, M. C. Produção de estreptavidina recombinante pela levedura Pichia
pastoris. Dissertação, Viçosa, UFV, 2006.
GALLIZIA, A., LALLA, C. D.E., NARDONE, E., SANTAMBROGIO, P.,
BRANDAZZA, A., SIDOLI, A., AROSIO, P. Production of a soluble and functional recombinant streptavidin in Escherichia coli. Protein Expression and Purification, 14: 192–196, 1998.
GHALY, A.E., KAMAL, M., CORREIA, L.R. Kinetic modeling of continuous submerged fermentation of cheese whey for single cell protein production. Bioresource
Technology, 96: 1143-1152, 2005.
GELLISSEN, G. Heterologous protein production in methylotrophic yeasts. Applied
Microbiology and Biotechnology, 54: 741–750, 2000.
GITLIN, G., BAYER, E.A., WILCHEK, M. Studies of the biotin-binding site of avidin. Lysine residues involved in the active site. The Biochemical Journal, 242: 923-926, 1987.
HILL J., IAN, K.A., DONALD, G., GRIFFITHIS, D.E. DMSO-enhanced whole cell yeast transformation. Nucleic Acids Research, Vol. 19 (20) 5791, 1991.
HOFMANN, K., WOOD, S.W., BRINTON, C.C., MONTIBELLER, J.A., FINN, F.M. Iminobiotin affinity columns and their applications to retrieval o streptavidin.
Proceedings of the National Academy of Sciences of the United States of America,
77 (8) 4666-4668, 1980.
HSIEH H.P., DA SILVA, N.A. Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis. Biotechnology and Bioengineering, 67:408– 16, 2000.
HSIEH, H.P., DA SILVA, N.A. Partial-pKD1 plasmids provide enhanced structure stability for heterologous protein production in Kluyveromyces lactis. Applied
Microbiology and Biotechnology, 49:411–416, 1998.
HUA, Z., LIANG, X., ZHU, D. Expression and purification of a truncated macrophage colony stimulating factor in Kluyveromyces lactis; Biochemistry and Molecular
Biology International, 34: 419 – 27, 1994.
INOUE, H., NOJIMA, H., OKAYAMA, H. High efficiency transformation of
Escherichia coli with Plasmids. Gene, 96: 23 – 28, 1990.
IWATA, T., TANAKA, R., SUETSUGU, M., ISHIBASHI, M., TOKUNAGA, H., KIKUCHI, M., TOKUNAGA, M. Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis. Biotechnology Letters 26: 1803–1808, 2004.
JANOLINO, V.G., SWISGGOOD, H.E. Analysis and optimization of methods using water-soluble carbodiimide for immobilization of biochemicals to porous glass.
Biotechnology and Bioengineering, 24: 1069-1080, 1982.
JARVIK, J.W., TELMER, C.A., Epitope tagging. Annual Review of Genetics, 32: 601– 618. 1998.
JESKE, S., TIGGEMANN, M., MEINHARDT, F. Yeast autonomous linear plasmid pGKL2: ORF9 is an actively transcribed essential gene with multiple transcription start points. FEMS Microbiology Letters. 255(2):321-7, 2006.
JURETZEK, T., LE DALL, M.T., MAUERSBERGER, R., GAILLARDIN, C., BARTH, G., NICAUD, J.M. Vectors of gene expressions and amplification in the yeast
Yarrowia lipolytica. Yeast, 18: 97–113, 2001.
KELLIS, M., BIRREN, B.W., LANDER, E.S. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature, 428:617– 24, 2004.
KIM, C.S., JI, E.S., OH, D.K. Expression and characterization of Kluyveromyces lactis beta-galactosidase in Escherichia coli. Biotechnology Letters, 25:1769– 74, 2003.
KUMURA, H., TANOUE, Y., TSUKAHARA, M., TANAKA, T., SHIMAZAKA, K. Screening of dairy yeasts strains for probiotic application. Journal of Dairy Science, 87:4050-4056, 2004.
LU, J., HUANG, B., ESBERG, A., MARCUS, J.O.J., BYSTRÖM, A.S. The
Kluyveromyces lactis g-toxin targets tRNA anticodons. RNA, 11:1648–1654, 2005.
MACÊDO, C.S. Expressão heteróloga de β-galactosidade de Kluyveromyces lactis: transformação estável de Pichia pastoris com o gene quimérico. Dissertação. Viçosa,
UFV, 2001.
MARTINEZ. E., MORALES, J., AGUIAR, J., PINEDA, Y., IZQUIERDO, M., FERBEYRE, G. Cloning and expression of hepatitis B surface antigen in the yeast
Kluyveromyces lactis. Biotechnology Letters, 14: 83–86, 1992.
MAULLU, C., LAMPIS, G., BASILE, T., INGIANNI, A., ROSSOLINI, G.M., POMPEI, R. Production of lysozyme-enriched biomass from cheese industry by- products. Journal of Applied Microbiology, 86 (2): 182-186, 1999.
MORESI, M. Cost/benefit analysis of yeast and yeast autolysate production from cheese whey. Italian Journal of Food Science, v. 6, p. 357-370, 1994.
MÜLLER, S., SANDAL, T., KAMP-HANSEN, P., DALBOGE, H. Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha,
Kluyveromyces lactis, Schizosacchromyces pombe and Yarrowia lipolytica. Cloning of
two novel promoters from Yarrowia lipolytica. Yeast, 14:1267– 1283, 1998.
NAGARAJAN, V., RAMALEY, R., ALBERTSON, H., CHEN, M. Secretion of streptavidin from Bacillus subtilis, Applied Environmental Microbiology, 59: 3894– 3898, 1993.
NEUHOFF, V., AROLD, N., TAUBE, D., EHRHARDT, W. Improved staining of proteins in polyacrylamide gels including isoeletric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.
Electrophoresis, 9: 255-262, 1988.
PANUWATSUK, W., DA SILVA, N.A. Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis. Applied Microbiology
and Biotechnology, 58:195–201, 2002.
PANUWATSUK, W., DA SILVA, N.A. Application of a gratuitous induction system in
Kluyveromyces lactis for the expression of intracellular and secreted proteins during
fed-batch culture. Biotechnology and Bioengineering, 81:712– 8, 2003.
PENG, G., HOPPER, J. E. Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein. Proceedings of the National Academy Sciences of the
United States of America, 99: 8548–8553, 2002.
PILAURI, V., BEWLEY, M., DIEP, C., HOPPER, J. Gal80 dimerization and the GAL genes switch. Genetics, 169 (4): 1903-1914, 2005.
PIONTEK, M., HAGEDORN, J., HOLLENBERG, C.P., GELLISSEN, G., STRASSER, A.W. Two novel gene expression systems based on the yeasts
Schwanniomyces occidentalis and Pichia stipitis. Applied Microbiology and
Biotechnology, 50: 331-338, 1998.
PLATT, A., ROSS, H.C., HANKIN, S., REECE, R.J. Insertion of two amino acids into a transcriptional inducer converts it into a galactokinase. Proceedings of the National
Academy Sciences of the United States of America, 97:3154– 9, 2000.
PORRO, D., MATTANOVICH, D. Recombinant protein production in yeasts.
Methods in Molecular Biology, 267: 241–258, 2004.
RIVERO-URGELL, M., SANTAMARIA-ORLEANS, A. Oligosaccharides: application in infant food. Early Human Development, 65 Suppl:S43– 52, 2001.
ROBIN, S., PETROV, K., DINTINGER, T., KUJUMDZIEVA, A., TELLIER, C., DION, M. Comparison of three microbial hosts for expression of an active catalytic scFv. Molecular Immunology, 39: 729-738, 2003.
ROMANOS, M.A., SCORER, C.A., CLARE, J.J. Foreign gene expression in yeast: a review. Yeast, 8: 423–488, 1992.
RÖSEL, H., KUNZE, G. Integrative transformation of the dimorphic yeast Arxula
adeninivorans LS3 based on hygromycin B resistance. Current Genetics, 33:157–163,
1998.
RUBIO-TEXEIRA, M.A. comparative analysis of the GAL genetic switch between not- so-distant cousins: Saccharomyces cerevisiae versus Kluyveromyces lactis. FEMS
Yeast Research, 5 (12) 1115-28, 2005.
SALIOLA, M., MAZZONI, C., SOLIMANDO, N., CRISA, A., FALCONE, C., JUNG, G., FLEER, R. Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumin in Kluyveromyces lactis. Applied Environmental
Microbiology, 65: 53-60, 1999.
SAMBROOK, J., FRITSCH, E.F., MANIATIS, T. Molecular Cloning: A laboratory manual, second edition. Cold Spring Harbor Press, Cold Spring, NY, 1989.
SANO, T., CANTOR, C.R. Expression of a cloned streptavidin gene in Escherichia
coli. Proceedings of the National Academy Sciences of the United States of
America, 87: 142–146, 1990.
SANO, T., PADORI, M.W., CHEN, X., SMITH, C. L., CANTOR, C. R. Recombinant core streptavidin. A minimum-sized core streptavidin has enhanced structural stability and higher accessibility to biotinylated macromolecules. The Journal of Biological
Chemistry, 270(47): 28204-28209, 1995.
SANO, T., VAJDA, S., CANTOR, C., R. Genetic engineering of streptavidin, a versatile affinity tag. Journal of Chromatography. B, Biomedical Sciences and
Applications, 715: 85-91, 1998.
SCHAFFRATH, R., BREUNIG, K.D. Genetics and molecular physiology of the yeast
Kluyveromyces lactis. Fungal Genetics and Biology, 30:173– 90, 2000.
SOLOMON, B., HOLLANSER, Z., KOPPEL, R., KATCHALSKI-KATZIR, E. Use of monoclonal antibodies for the preparation of highly active immobilized enzymes.
Methods in Enzymology, 135: 160-170,1987.
SREEKRISHNA, K., DICKSON, R.C. Construction of strains of Saccharomyces
cerevisiae that grow on lactose. Proceedings of the National Academy Sciences of the
United States of America, 82:7909– 13, 1985.
STAYTON, P.S., FREITAG, S., KLUMB, L.A., CHILKOTI, A., CHU, V., PENZOTTI, J.E., TO, R., HYRE, D., LE TRONG, I., LYBRAND, T.P., STENKAMP, R.E. Streptavidin-biotin binding energetic; Biomolecular Engineering, 16(1-4):39-44, 1999.
SWENNEN, D., PAUL, M.F., VERNIS, L., BECKRICH, J.M., FOURNIER, A., GAILLARDIN, C. Secretion of active anti-Ras single-chain Fv antibody by yeasts
SWINKELS, B.W., VAN OOYEN, A.J.J., BONEKAMP, F.J. The yeast Kluyveromyces
lactis as an efficient host for heterologous gene expression. Antonie Van
Leeuwenhoek, 64: 187–201, 1993.
SWAISGOOD, H.E., LEE, P. Cloning and expression of a streptavidin-lipase fusion gene in Escherichia coli and characterization of the immobilization fusion protein.
Enzyme and Microbial Technology, 22: 246-254, 1998.
SWAISGOOD, H.E, WALSH, M.K. An Escherichia coli plasmid vector system for production of streptavidin fusion proteins: Expression and biosselective adsorption of streptavidin- β-galactosidase. Biotechnology and Bioengineering, 44: 1348-1354, 1994.
TUNTASUVAN, D., MIMAPAN, S., SARATAPHAN, N., TRONGWONGSA, L., INTRARAKSA, R., LUCKINS, A.G. Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidin-biotin immunohistochemistry. Veterinary
Parasitology, 89: 337-338, 2000.
VAN DEN BERG, J., A., VAN DER LAKEN, K., J., VAN OOVEN, A., J., RENIERS, T., C., RIETVELD, K., SCHAAP, A., BRAKE, A., J., BISHOP, R., J., SCHULTZ, K., MOYER, D., RICHMAN, M., SHUSTER, J.R. Kluyveromyces lactis as a host for heterologous gene expression: expression and secretion of prochymosin.
Biotechnology, 8: 138-139, 1990.
WANER, M. J., NAVROTSKAYA, I., BRAIN, A., OLDHAM, E.D., MASCOTTI, D.P. Thermal and sodium dodecylsulfate induced transitions of streptavidin.
Biophysical Journal, 87: 2701-2713, 2004.
WEBER, P.C., OHLENDORF, D.H., WENDOLOSKI, J.J., SALEMME, F.R. Strutural origins of high-affinity biotin binding to streptavidin. Science Direct, 243: 85-88, 1989.
WÉSOLOWSKI-LOUVEL, M., BREUNIG, K.D., FUKUHARA, H. Kluyveromyces
lactis. In: Wolf K (ed) Genetics, biochemistry, and molecular biology of non-
WILLIAMS, M.G., WILSHER, J., NUGENT, P., MILLS, A., DHANARAJ, V., FABRY M., SEDLACEK, J., UUSITALO, J.M., PENTTILA, M.E., PITTS J.E., BLUNDELL T.L. Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin, Protein Engineering, 10: 991–997, 1997.
WHITE, D. The physiology and biochemistry of prokaryotes. Oxford University Press, 2nd edition, 2000.
WU, J., CHIENG, L., HSU, T., LEE, C. Sequential expression of recombinant proteins and their separate recovery from a Pichia pastoris cultivation. Biochemical
Engineering Journal, 16: 9-16, 2003.
WU, S.C., QURESHI. M.H., WONG, S.J. Secretory production and purification of functional full-length streptavidin from Bacillus subtilis. Protein Expression and
Purification, 24: 348–356, 2002a.
WU, S.C., WONG, S.L. Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin, Applied
Environmental Microbiology, 68: 1102–1108, 2002b.
YANG, S.T., SILVA, E.M. Novel products and new technologies for use of a familiar carbohydrate, milk lactose. Journal of Dairy Science. 78: 2541–62, 1995
APÊNDICE A
Apêndice A: Curva padrão de Albumina Bovina Sérica (BSA) utilizada como
referência para o cálculo de proteínas totais pelo método de Bradford.
y = 0,0426x + 0,0089
R
2= 0,9959
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
0
2,5
5
7,5
10
12,5 15
17,5 20
22,5
[BSA] μg.mL
-1A
b
s 595 n
m
Apêndice B
Tempo (h) Soro de Queijo (μg.mL-1)
YNBlac +ext lev
(μg.mL-1) YPL (μg.mL-1) 0 0 0 0 24 23,33 ± 3,57 67,88 ± 10,48 101,00 ± 7,17 48 38,29 ± 11,5 76,49 ± 10,98 178,20 ± 8,97 72 44,93 ± 11,99 99,44 ± 17,59 140,38 ± 15,40 96 42,07 ± 15,05 150,81 ± 5,64 117,69 ± 15,42 120 75,89 ± 9,31 192,54 ± 10,81 173,76 ± 4,41 144 51,95 ± 17,26 195,93 ± 5,96 103,35 ± 2,06
Apêndice B: Quantificação de proteínas totais secretadas presente no sobrenadante da
cultura de K. lactis MW 98.8C cStp nos diferentes meios de cultivos em regime de batelada pelo método de Bradford.
APÊNDICE C
Tempo (h) Soro de queijo (μg.mL-1)
YNBlac +ext lev
(μg.mL-1) YPL (μg.mL-1) 0 0 0 0 24 40,25 ± 11,37 15,98 ± 7,85 131,78 ± 6,62 48 38,43 ± 5,01 54,32 ± 7,72 152,90 ± 10,39 72 58,20 ± 5,31 90,31 ± 12,14 178,72 ± 3,86 96 40,51 ± 5,11 179,24 ± 16,01 137,78 ± 4,96 120 56,12 ± 14,07 166,72 ± 6,67 96,31 ± 17,66 144 69,65 ± 9,88 127,08 ± 1,65 117,96 ± 15,22
Apêndice C: Quantificação de proteínas totais secretadas presente no sobrenadante da
cultura de K. lactis CBS 2359 cStp nos diferentes meios de cultivos em regime de batelada, pelo método de Bradford.
APÊNDICE D
Apendice D: Fracionamento das proteínas extracelulares totais em gel de poliacrilamida
15% (p/v) sob condições desnaturantes das amostras do sobrenadante da cultura recombinante de K. lactis CBS 2359/cStp em meio de soro de queijo.