茂原链霉菌谷氨酰胺转氨酶合成与菌体形态分化的关系
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摘要
目的:研究茂原链霉菌(Streptomyces mobaraensis)合成谷氨酰胺转氨酶(transglutaminase,TGase)与菌体分化之间的关系,以及TGase的生理功能。方法:以S. mobaraensis为出发菌株,通过向培养基中添加乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)抑制TGase激活关键蛋白酶的活性从而调控成熟TGase的合成,同时利用激光共聚焦显微镜和扫描电镜观察发酵过程中菌体的活力和形态变化。结果:对照组随着TGase产量的升高,菌体生存活力呈先上升后下降的趋势,并且菌体表现出复杂的形态变化;实验组自加入EDTA后TGase产量不再升高,菌体生存活力也逐渐减弱,菌体形态分化滞后。结论:TGase的合成会影响茂原链霉菌生存活力及其形态分化。
The purposes of the present study were to clarify the relationship between the morphological differentiation and transglutaminase(TGase) production Streptomyces mobaraensis, and to explore the physiological role of TGase in S. mobaraensis development. EDTA was added into the fermentation medium to inhibit the activities of the key proteases for TGase biosynthesis, and then the viability and morphological differentiation of the pellets were investigated using a confocal laser scanning microscope(CLSM) and a scanning electron microscope(SEM). The viability of S. mobaraensis pellets in the control group increased initially and decreased afterwards as the TGase activity increased, and the mycelia showed complex morphological changes. In the experimental group, a significant inhibition effect of EDTA was observed on TGase production, and the viability of S. mobaraensis pellets was decreased upon the addition of EDTA. In addition, mycelial morphological differentiation was delayed. These results suggest that TGase biosynthesis affects the viability of pellets and mycelial morphological differentiation of S. mobaraensis.
The purposes of the present study were to clarify the relationship between the morphological differentiation and transglutaminase(TGase) production Streptomyces mobaraensis, and to explore the physiological role of TGase in S. mobaraensis development. EDTA was added into the fermentation medium to inhibit the activities of the key proteases for TGase biosynthesis, and then the viability and morphological differentiation of the pellets were investigated using a confocal laser scanning microscope(CLSM) and a scanning electron microscope(SEM). The viability of S. mobaraensis pellets in the control group increased initially and decreased afterwards as the TGase activity increased, and the mycelia showed complex morphological changes. In the experimental group, a significant inhibition effect of EDTA was observed on TGase production, and the viability of S. mobaraensis pellets was decreased upon the addition of EDTA. In addition, mycelial morphological differentiation was delayed. These results suggest that TGase biosynthesis affects the viability of pellets and mycelial morphological differentiation of S. mobaraensis.
引文
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[7] DEWEID L, AVRUTINA O, KOLMAR H. Microbial transglutaminase for biotechnological and biomedical engineering[J]. Biological Chemistry, 2019, 400(3): 257. DOI:10.1515/hsz-2018-0335.
[8] TESFAW A, ASSEFA F. Applications of transglutaminase in textile, wool, and leather processing[J]. International Journal of Textile Science, 2014, 3(4): 64-69. DOI:10.5923/j.textile.20140304.02.
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[10] ZHANG Y, HE S, SIMPSON B K. A cold active transglutaminase from Antarctic krill (Euphausia superba): purification, characterization and application in the modification of cold-set gelatin gel[J]. Food Chemistry, 2017, 232: 155-162. DOI:10.1016/j.foodchem.2017.03.135.
[11] LI H, CUI Y, ZHANG L, et al. Heterologous expression and purification of Zea mays transglutaminase in Pichia pastoris[J]. Food Science and Biotechnology, 2014, 23(5): 1507-1513. DOI:10.1007/s10068-014-0206-1.
[12]宋小平,王雅洁,王迎新,等.谷氨酰胺转氨酶菌株的筛选及其产酶条件研究[J].食品研究与开发, 2015, 36(1): 114-117. DOI:10.3969/j.issn.1005-6521.2015.01.030.
[13]张莹莹,石楠,杜萍萍,等.一株产谷氨酰胺转氨酶菌株的分离、鉴定及其tgl的克隆表达[J].食品工业科技, 2015, 36(11): 141-146. DOI:10.13386/j.issn1002-0306.2015.11.020.
[14] ECKERT R L, KAARTINEN M T, NURMINSKAYA M, et al. Transglutaminase regulation of cell function[J]. Physiological Reviews, 2014, 94(2): 383-417. DOI:10.1152/physrev.00019.2013.
[15] ZHU Y, RINZEMA A, TRAMPER J, et al. Fed-batch fermentation dealing with nitrogen limitation in microbial transglutaminase production by Streptoverticillium mobaraense[J]. Applied Microbiology and Biotechnology, 1998, 49(3): 251-257. DOI:10.1007/s002530051165.
[16] FOLK J E, COLE P W. Structural requirements of specific substrates for Guinea pig liver transglutaminase[J]. Journal of Biological Chemistry, 1965, 240(7): 2951-2960.
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[19] GAVET O, PINES J. Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis[J]. Developmental Cell, 2010, 18(4): 533-543. DOI:10.1016/j.devcel.2010.02.013
[20] YUAN W M, CRAWFORD D L. Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots[J]. Applied and Environmental Microbiology, 1995, 61(8): 3119-3128.
[21] ZOTZEL J, KELLER P, FUCHSBAUER H L. Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease[J]. European Journal of Biochemistry, 2003, 270(15): 3214-3222. DOI:10.1046/j.1432-1033.2003.03703.x.
[22] ZOTZEL J, PASTERNACK R, PELZER C, et al. Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step[J]. European Journal of Biochemistry, 2003, 270(20): 4149-4155. DOI:10.1046/j.1432-1033.2003.03809.x.
[23]张莉丽,韩雪,张兰威.影响茂原链霉菌TGase产量关键蛋白酶的确定[J].食品与发酵工业, 2014, 40(2): 6-9. DOI:10.13995/j.cnki.11-1802/ts.2014.02.010.
[24] BEITES T, OLIVEIRA P, RIOSERAS B, et al. Streptomyces natalensis programmed cell death and morphological differentiation are dependent on oxidative stress[J]. Scientific Reports, 2015, 5(1): 12887. DOI:10.1038/srep12887.
[25] GODINEZ O, DYSON P, DEL SOL R, et al. Targeting the osmotic stress response for strain improvement of an industrial producer of secondary metabolites[J]. Journal of Microbiology and Biotechnology, 2015, 25(11): 1787-1795. DOI:10.4014/jmb.1503.03042.
[26] ZHANG L, ZHANG L, HAN X, et al. Enhancement of transglutaminase production in Streptomyces mobaraensis as achieved by treatment with excessive MgCl2[J]. Applied Microbiology and Biotechnology, 2012, 93(6): 2335-2343. DOI:10.1007/s00253-011-3790-5.
[27] CHEN K, ZHANG D, LIU S, et al. Improvement of transglutaminase production by extending differentiation phase of Streptomyces hygroscopicus: mechanism and application[J]. Applied Microbiology and Biotechnology, 2013, 97(17): 7711-7719. DOI:10.1007/s00253-012-4614-y.
[28]陈康康,刘松,鞠晓辉,等.吸水链霉菌谷氨酰胺转胺酶基因的阻断及其对细胞分化的影响[J].微生物学报, 2010, 50(12): 1626-1632. DOI:10.13343/j.cnki.wsxb.2010.12.004.
[29] HENRIQUES A O, MORAN C P. Structure and assembly of the bacterial endospore coat[J]. Methods, 2000, 20(1): 95-110. DOI:10.1006/meth.1999.0909.
[30] HENRIQUES A O, MORAN C P. Structure, assembly, and function of the spore surface layers[J]. Annual Review of Microbiology, 2007, 2007, 61(1): 555-588. DOI:10.1146/annurev.micro.61.080706.093224.
[31] TAO W, DONG H, ZHANG Y, et al. Introducing transglutaminase with its precursor region into Clostridium acetobutylicum,improves its tolerance to oxidative stress and solvent production[J]. Process Biochemistry, 2015, 50(1): 111-118. DOI:10.1016/j.procbio.2014.10.016.
[2] CHATER K F. Genetics of differentiation in Streptomyces[J]. Annual Reviews in Microbiology, 1993, 47(1): 685-711. DOI:10.1146/annurev.mi.47.100193.003345.
[3] RIOSERAS B, LóPEZ-GARCíA M T, YAGüE P, et al. Mycelium differentiation and development of Streptomyces coelicolor in labscale bioreactors: programmed cell death, differentiation, and lysis are closely linked to undecylprodigiosin and actinorhodin production[J]. Bioresource Technology, 2014, 151: 191-198. DOI:10.1016/j.biortech.2013.10.068.
[4] RIOSERAS B, SLIAHA P V, GORSHKOV V, et al. Quantitative proteome and phosphoproteome analyses of Streptomyces coelicolor reveal proteins and phosphoproteins modulating differentiation and secondary metabolism[J]. Molecular & Cellular Proteomics, 2018, 17(8): 1591-1611. DOI:10.1074/mcp.RA117.000515.
[5] GASPAR A L C, GóES-FAVONI S P D. Action of microbial transglutaminase (MTGase) in the modification of food proteins: a review[J]. Food Chemistry, 2015, 171: 315-322. DOI:10.1016/j.foodchem.2014.09.019.
[6] KIELISZEK M, MISIEWICZ A. Microbial transglutaminase and its application in the food industry. A review[J]. Folia Microbiologica, 2014, 59(3): 241-250. DOI:10.1007/s12223-013-0287-x.
[7] DEWEID L, AVRUTINA O, KOLMAR H. Microbial transglutaminase for biotechnological and biomedical engineering[J]. Biological Chemistry, 2019, 400(3): 257. DOI:10.1515/hsz-2018-0335.
[8] TESFAW A, ASSEFA F. Applications of transglutaminase in textile, wool, and leather processing[J]. International Journal of Textile Science, 2014, 3(4): 64-69. DOI:10.5923/j.textile.20140304.02.
[9] ROY I, SMITH O, CLOUTHIER C M, et al. Expression, purification and kinetic characterisation of human tissue transglutaminase[J]. Protein Expression and Purification, 2013, 87(1): 41-46. DOI:10.1016/j.pep.2012.10.002.
[10] ZHANG Y, HE S, SIMPSON B K. A cold active transglutaminase from Antarctic krill (Euphausia superba): purification, characterization and application in the modification of cold-set gelatin gel[J]. Food Chemistry, 2017, 232: 155-162. DOI:10.1016/j.foodchem.2017.03.135.
[11] LI H, CUI Y, ZHANG L, et al. Heterologous expression and purification of Zea mays transglutaminase in Pichia pastoris[J]. Food Science and Biotechnology, 2014, 23(5): 1507-1513. DOI:10.1007/s10068-014-0206-1.
[12]宋小平,王雅洁,王迎新,等.谷氨酰胺转氨酶菌株的筛选及其产酶条件研究[J].食品研究与开发, 2015, 36(1): 114-117. DOI:10.3969/j.issn.1005-6521.2015.01.030.
[13]张莹莹,石楠,杜萍萍,等.一株产谷氨酰胺转氨酶菌株的分离、鉴定及其tgl的克隆表达[J].食品工业科技, 2015, 36(11): 141-146. DOI:10.13386/j.issn1002-0306.2015.11.020.
[14] ECKERT R L, KAARTINEN M T, NURMINSKAYA M, et al. Transglutaminase regulation of cell function[J]. Physiological Reviews, 2014, 94(2): 383-417. DOI:10.1152/physrev.00019.2013.
[15] ZHU Y, RINZEMA A, TRAMPER J, et al. Fed-batch fermentation dealing with nitrogen limitation in microbial transglutaminase production by Streptoverticillium mobaraense[J]. Applied Microbiology and Biotechnology, 1998, 49(3): 251-257. DOI:10.1007/s002530051165.
[16] FOLK J E, COLE P W. Structural requirements of specific substrates for Guinea pig liver transglutaminase[J]. Journal of Biological Chemistry, 1965, 240(7): 2951-2960.
[17] FERNANDEZ M, SANCHEZ J. Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling of the mycelium in submerged cultures of Streptomyces antibioticus ETH7451[J]. Journal of Microbiological Methods, 2001, 47(3): 293-298. DOI:10.1016/S0167-7012(01)00332-3.
[18] FERNANDEZ M, SANCHEZ J. Corrigendum to “Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling of the mycelium in submerged cultures of Streptomyces antibioticus, ETH7451”(J. Microbiol. Method 47 (2001) 293-298)[J]. Journal of Microbiological Methods, 2002, 49(2): 207-208. DOI:10.1016/S0167-7012(01)00381-5.
[19] GAVET O, PINES J. Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis[J]. Developmental Cell, 2010, 18(4): 533-543. DOI:10.1016/j.devcel.2010.02.013
[20] YUAN W M, CRAWFORD D L. Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots[J]. Applied and Environmental Microbiology, 1995, 61(8): 3119-3128.
[21] ZOTZEL J, KELLER P, FUCHSBAUER H L. Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease[J]. European Journal of Biochemistry, 2003, 270(15): 3214-3222. DOI:10.1046/j.1432-1033.2003.03703.x.
[22] ZOTZEL J, PASTERNACK R, PELZER C, et al. Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step[J]. European Journal of Biochemistry, 2003, 270(20): 4149-4155. DOI:10.1046/j.1432-1033.2003.03809.x.
[23]张莉丽,韩雪,张兰威.影响茂原链霉菌TGase产量关键蛋白酶的确定[J].食品与发酵工业, 2014, 40(2): 6-9. DOI:10.13995/j.cnki.11-1802/ts.2014.02.010.
[24] BEITES T, OLIVEIRA P, RIOSERAS B, et al. Streptomyces natalensis programmed cell death and morphological differentiation are dependent on oxidative stress[J]. Scientific Reports, 2015, 5(1): 12887. DOI:10.1038/srep12887.
[25] GODINEZ O, DYSON P, DEL SOL R, et al. Targeting the osmotic stress response for strain improvement of an industrial producer of secondary metabolites[J]. Journal of Microbiology and Biotechnology, 2015, 25(11): 1787-1795. DOI:10.4014/jmb.1503.03042.
[26] ZHANG L, ZHANG L, HAN X, et al. Enhancement of transglutaminase production in Streptomyces mobaraensis as achieved by treatment with excessive MgCl2[J]. Applied Microbiology and Biotechnology, 2012, 93(6): 2335-2343. DOI:10.1007/s00253-011-3790-5.
[27] CHEN K, ZHANG D, LIU S, et al. Improvement of transglutaminase production by extending differentiation phase of Streptomyces hygroscopicus: mechanism and application[J]. Applied Microbiology and Biotechnology, 2013, 97(17): 7711-7719. DOI:10.1007/s00253-012-4614-y.
[28]陈康康,刘松,鞠晓辉,等.吸水链霉菌谷氨酰胺转胺酶基因的阻断及其对细胞分化的影响[J].微生物学报, 2010, 50(12): 1626-1632. DOI:10.13343/j.cnki.wsxb.2010.12.004.
[29] HENRIQUES A O, MORAN C P. Structure and assembly of the bacterial endospore coat[J]. Methods, 2000, 20(1): 95-110. DOI:10.1006/meth.1999.0909.
[30] HENRIQUES A O, MORAN C P. Structure, assembly, and function of the spore surface layers[J]. Annual Review of Microbiology, 2007, 2007, 61(1): 555-588. DOI:10.1146/annurev.micro.61.080706.093224.
[31] TAO W, DONG H, ZHANG Y, et al. Introducing transglutaminase with its precursor region into Clostridium acetobutylicum,improves its tolerance to oxidative stress and solvent production[J]. Process Biochemistry, 2015, 50(1): 111-118. DOI:10.1016/j.procbio.2014.10.016.