漆斑菌属胆红素氧化酶的纯化、分析及基因克隆
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摘要
胆红素氧化酶(BOD)是一种催化胆红素生成胆绿素和其它物质的一种酶。一些胆红素氧化酶已从疣孢漆斑菌和微紫青霉等生物体中被分离纯化出来。
本研究对漆斑菌属的疣孢漆斑菌和三种露湿漆斑菌进行培养，培养过程中发现，加入26×10~(-4)％的诱导剂(CuSO_4)可很大程度地提高这四种菌BOD的活性。我们从漆斑菌属的四种菌中分离纯化出了胆红素氧化酶，纯化过程中阳离子交换树脂代替活性碳、低温、甘油都有助于酶活的保持。
与疣孢漆斑菌BOD相比较，露湿漆斑菌3.1967BOD的分子量、胆红素-聚丙烯酰胺凝胶电泳和聚丙烯酰胺凝胶电泳条带、糖染结果、等电点、纯化条件等都不相同。这说明露湿漆斑菌3.1967BOD是不同于疣孢漆斑菌BOD的一种酶。在BOD的鉴定过程中胆红素平板、胆红素-聚丙烯酰胺凝胶电泳、糖染与银染结合法是未被报导过的新方法，这些方法的应用对于BOD的鉴定很有帮助。
本实验对BOD的测定方法也有所改进，以露湿漆斑菌3.1967BOD各种性质作根据，在酶的最适反应条件下(37℃、pH8.1)进行反应，胆红素有最大吸收峰的波长440nm下测定OD值。以抗坏血酸和EDTA作为反应终止剂将反应终止在特定的时间，使酶活测定不用恒温分光光度计，而只用普通的分光光度计就可测定，这也将有助于胆红素酶法测定的推广和应用。
根据Genebank中疣孢漆斑菌胆红素氧化酶(MV BOD)基因的编码序列设计引物，利用PCR从疣孢漆斑菌的染色体DNA中扩增得到1937bp的基因，将其克隆到T载体中，构建成重组质粒，并对其测序，通过测序验证了本实验所合成引物的正确性，为下一步的基因工程操作打下了基础。
Bilirubin oxidases (BOD) are enzymes catalyzing the oxidation of bilirubin to biliverdin and other substrates. Some kinds of bilirubin oxidases have been purified from Myrothecium uerrucuria and Peniciilium jan thinellum.
In this study, we cultured Myrothecium verrucaria and three kinds of Myrothecium roridum. During the culture, we observed that adding 26 X 10-4% CuS04 into the medium can greatly promote the enzymatic activity of BOD. We have purified BOD from all of them and some factors, such as low temperature, glycerin and so on, are helpful to remain the enzymatic activity.
Comparing with BOD from Myrothecium verrucaria(MV BOD), BOD from Myrothecium roridum (MR BOD) shows difference in the molecular weight, the result of electrophoresis, isoelectric point and the condition of purification. It can be concluded that.BOD from Myrothecium roridum is a new type of BOD which is different from MV BOD. Agarose plate with bilirubin, Bilirubin-PAGE and Double Staining of schiff agent and sliver, are new methods that will be used in identification of BOD activities.
Based on the properties of MR BOD, the measure method is developed. The ordinary methods of measuring the BOD' s activity have to be performed under constant temperature. We improved the method by using Vitamin C and EDTA as a terminating agent, which made the method more convenient to carry
out.
According to the encoding sequence of the MV BOD gene in the Genebank,
the primers is designed and the 1937bp DNA fragment is obtained by PCR amplification. Then, this fragment is cloned into the plasmid pGEM-T-easy vector and sequenced. The result shows that the primers that I designed
Abstract
are corrected.
本研究对漆斑菌属的疣孢漆斑菌和三种露湿漆斑菌进行培养，培养过程中发现，加入26×10~(-4)％的诱导剂(CuSO_4)可很大程度地提高这四种菌BOD的活性。我们从漆斑菌属的四种菌中分离纯化出了胆红素氧化酶，纯化过程中阳离子交换树脂代替活性碳、低温、甘油都有助于酶活的保持。
与疣孢漆斑菌BOD相比较，露湿漆斑菌3.1967BOD的分子量、胆红素-聚丙烯酰胺凝胶电泳和聚丙烯酰胺凝胶电泳条带、糖染结果、等电点、纯化条件等都不相同。这说明露湿漆斑菌3.1967BOD是不同于疣孢漆斑菌BOD的一种酶。在BOD的鉴定过程中胆红素平板、胆红素-聚丙烯酰胺凝胶电泳、糖染与银染结合法是未被报导过的新方法，这些方法的应用对于BOD的鉴定很有帮助。
本实验对BOD的测定方法也有所改进，以露湿漆斑菌3.1967BOD各种性质作根据，在酶的最适反应条件下(37℃、pH8.1)进行反应，胆红素有最大吸收峰的波长440nm下测定OD值。以抗坏血酸和EDTA作为反应终止剂将反应终止在特定的时间，使酶活测定不用恒温分光光度计，而只用普通的分光光度计就可测定，这也将有助于胆红素酶法测定的推广和应用。
根据Genebank中疣孢漆斑菌胆红素氧化酶(MV BOD)基因的编码序列设计引物，利用PCR从疣孢漆斑菌的染色体DNA中扩增得到1937bp的基因，将其克隆到T载体中，构建成重组质粒，并对其测序，通过测序验证了本实验所合成引物的正确性，为下一步的基因工程操作打下了基础。
Bilirubin oxidases (BOD) are enzymes catalyzing the oxidation of bilirubin to biliverdin and other substrates. Some kinds of bilirubin oxidases have been purified from Myrothecium uerrucuria and Peniciilium jan thinellum.
In this study, we cultured Myrothecium verrucaria and three kinds of Myrothecium roridum. During the culture, we observed that adding 26 X 10-4% CuS04 into the medium can greatly promote the enzymatic activity of BOD. We have purified BOD from all of them and some factors, such as low temperature, glycerin and so on, are helpful to remain the enzymatic activity.
Comparing with BOD from Myrothecium verrucaria(MV BOD), BOD from Myrothecium roridum (MR BOD) shows difference in the molecular weight, the result of electrophoresis, isoelectric point and the condition of purification. It can be concluded that.BOD from Myrothecium roridum is a new type of BOD which is different from MV BOD. Agarose plate with bilirubin, Bilirubin-PAGE and Double Staining of schiff agent and sliver, are new methods that will be used in identification of BOD activities.
Based on the properties of MR BOD, the measure method is developed. The ordinary methods of measuring the BOD' s activity have to be performed under constant temperature. We improved the method by using Vitamin C and EDTA as a terminating agent, which made the method more convenient to carry
out.
According to the encoding sequence of the MV BOD gene in the Genebank,
the primers is designed and the 1937bp DNA fragment is obtained by PCR amplification. Then, this fragment is cloned into the plasmid pGEM-T-easy vector and sequenced. The result shows that the primers that I designed
Abstract
are corrected.
引文
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志，2000，18(6)：345-346.
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[27] Arvan D, Shirey TL. Conjugated bilirubin: a better indicator of impaired hepatobiliary excretion than direct bilirubin. Ann Clin Lab Sci, 1985, 15: 252-259.
[28] Doumas B T, Wu T W. The measurement of bilirubin fraction in serum. Crit Rev Clin Lab Sci, 1991, 28: 415-445.
[29] Kurosaka K, Senba S, Tsubota H, et al. A new enzymatic assay for selectively measuring conjugated bilirubin concentrationin serum with use of bilirubin oxidase. Clinica Chimica Acta, 1998, 269: 125-136.
[30] Kimura S, Lyama S, Yamaguchi Y, et al. Enzymatic assay for conjugated bilirubin(Bc) in serum using bilirubin oxidase(BOD). J Clin Lab Anal, 1999, 13: 219-223.
[31] 赵建华，王毓三，结合胆红素酶法测定的一种新方法，中华检验医学杂志，2003，26(4)：207-210．
[32] Brodersen, R. and Bartels, P., Enzymatic oxidation of bilirubin, Eur J Biochem 1969, 10: 468-473.
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[2] marutto P Ricca G.S etal. The conformation of bilirubin and its esters. Gazz. Chim, Ital., 1974, 104, 633.
[3] Kaplan D. Navon G. On the assignment of the carbon-13 NMR spectrum of bilirubin. Spectroscopy Lett, 1980, 13-59.
[4] Kuenzle C. C et al. Structrue and conformation of bilirubin, Biochemitry, 1983, 1330, 364.
[5] 许国荣，赵建华，王毓三，血清结合胆红素的酶法测定，临床检验学杂志 2000，18(6)：325-326．
[6] 上海第一医学院主编．医用生物化学．北京：人民卫生出版社，1979
[7] 曹稳根，李卫华．蛋白质与胆红素相互作用的研究．信阳师范学院学报(自然科学版)，1998，11(2)：148-150．
[8] Dalton J etal, Luminecence of bilirubin. Chem. Phys.Lett. 1979, 61-242
[9] Mcdonagh A F, Assisi F. The ready isomerization of bilirubin in aqueous solution. Biochem J, 1972, 129: 797-800.
[10] Mcdonagh A F, The role of singlet oxygen in bilirubin photo-oxidation. Biochem Biophys Res Commun, 1971, 44: 1306-1311.
[11] Fog J, Bugge-Asperberm B. Stability of bilirubin. Nature, 1964, 203: 756.
[12] Mcdonagh A F, Thermal and photochemical reactions of bilirubin. Ann N Y Acad Sci, 1975, 244, 553-569,
[13] 曾百肇，王琰，王丰，胆红素和胆绿素稳定性的研究，武汉大学学报(自然科学版)，1994，5：75—79．
[14] 许顺姬，张淑芳，金幸，陈兵，钒酸氧化法在测定血清总胆红素及直接胆红素值中的应用，延边大学医学学报，2003，26(1)：27-29．
[15] 邱谷，高碘酸钠．氧化法测定血清总胆红素，临床检验杂
志，2000，18(6)：345-346.
[16] 吕邦泰，硝酸氧化法测定血清总胆红素和直接胆红素，现代实用医学，2002，14(11)：618-619．
[17] Domuas B T, Perry B, Tendrzcjzak B, etal. Measurement of direct bilirubin by use of bilirubin oxidase[J]. Chin Chem, 1987, 33(8): 1349-1353.
[18] 蔡金堞，胆红素两种检测方法的比较．福建化工．1995，3：21-33．
[19] 宋克征，沈瑛，胆红素及检测方法研究的进展，国外医学临床生物化学与检验学分册，2002，23(5)：283-284．
[20] Perry B. Measurement of total bilirubin by use of bilirubin oxidase.Clin Chem, 1986, 32(2): 329.
[21] 顾国宝，陈洁，杨卫华等，化学氧化法测定血清直接胆红素试剂的研制，上海医学检验杂志，2000，15(4)：244-245．
[22] Tokuda K. Kanimoto K. A new method of measuring bilirubinin serum by Vanadicacid. Japan J Clin Chem (in Japanese with English abstract), 1993;22: 116～122
[23] 陈薇，胡汉宁，贺小玲，血清胆红素3种测定方法的比较．医学新知杂志，2003，13(2)：83-85．
[24] 王永明，寿建兵，毛小勇，二甲亚砜双试剂法测定血清胆红素，临床检验杂志，2001，19(2)：110．
[25] 李祥坤，4种胆红素测定方法的比较，重庆医学，2002，31(12)：1279-1280．
[26] 章秀兰，直接胆红素检测方法可靠性的评估，上海医学检验杂志，2003，18(5)：304-306．
[27] Arvan D, Shirey TL. Conjugated bilirubin: a better indicator of impaired hepatobiliary excretion than direct bilirubin. Ann Clin Lab Sci, 1985, 15: 252-259.
[28] Doumas B T, Wu T W. The measurement of bilirubin fraction in serum. Crit Rev Clin Lab Sci, 1991, 28: 415-445.
[29] Kurosaka K, Senba S, Tsubota H, et al. A new enzymatic assay for selectively measuring conjugated bilirubin concentrationin serum with use of bilirubin oxidase. Clinica Chimica Acta, 1998, 269: 125-136.
[30] Kimura S, Lyama S, Yamaguchi Y, et al. Enzymatic assay for conjugated bilirubin(Bc) in serum using bilirubin oxidase(BOD). J Clin Lab Anal, 1999, 13: 219-223.
[31] 赵建华，王毓三，结合胆红素酶法测定的一种新方法，中华检验医学杂志，2003，26(4)：207-210．
[32] Brodersen, R. and Bartels, P., Enzymatic oxidation of bilirubin, Eur J Biochem 1969, 10: 468-473.
[33] Wu TW, Bio Chem, 1988, 66(4): 1248.
[34] Murao, S., and Tanaka, N. A new enzyme bilirubin oxidase produced by Myrothecium verrucaria MT-1, Agric. Biol. Chem., 1981, 45: 2383-2384.
[35] Takahashi M, Eur Pat Appl EP, 1988, 295: 101.
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