深海细菌 Pseudomonas sp．bIp-2黑色素生成条件研究及其 hppD基因的克隆

摘要

A melanin-producing strain was isolated from deep-sea sediment of the Atlantic Ocean and identified as Pseudomonas sp.bIp-2 by 16S rRNA gene sequencing.The study of growth and melanin production with varied culture conditions show that the melanin production in strain bIp-2 is growth-associated.The maximum yield of pig-ment was obtained after the culture attained the stationary growth phase.The temperature range for growth is 4~37℃ with optimal growth at 37℃,and the maximum melanin production was observed at 28℃.The pH range for growth is 6.0~9.0,with both optimal growth and melanin formation both being at pH 7.0.Strain bIp-2 grew in 1%~9%NaCl with a maximum melanin production at 5%NaCl.Low concentration of Fe(II)(0.05 mmol/dm3 ) can improve the production of melanin.Fe2+of more than 0.20 mmol/dm3 suppressed the melanin production and Fe2+of more than 1 .00 mmol/dm3 completely inhibited the growth of the strain.Cu2+of 0.1 mmol/dm3 had no effect to the melanin production and Cu2+of more than 0.5 mmol/dm3 inhibited the formation of melanin.Cu2+over 1 .0 mmol/dm3 completely inhibited the growth of the strain.A 1 087 bp fragment of the full-length hppD gene was amplified from the strain bIp-2.The hppD gene encoded an amino acid sequence sharing a 97%similarity with the HPPD from Pseudomonas stutzeri RCH2.These results laid a foundation to the study of the synthesis,applica-tion and ecological effect of the melanin from Pseudomonas sp.bIp-2.%从大西洋深海沉积物中分离得到1株产黑色素细菌bIp-2，16S rRNA基因序列分析表明该菌隶属于假单胞菌属（Pseudomonas），与施氏假单胞菌（P．stutzeri）有最高的16S rRNA基因序列相似性（99％）．进一步对该菌的生长及产黑色素条件进行了初步研究，结果表明bIp-2在稳定生长期后期才开始大量积累黑色素；bIp-2可在4～37℃的温度范围下生长，最适生长温度为37℃，在28℃具有最高的黑色素产量；bIp-2生长的pH范围偏碱性，在pH＝7的最适生长条件下黑色素产量最高，同时碱性环境有利于黑色素的快速形成；bIp-2可以在1％～9％的NaCl浓度范围中生长，在5％的NaCl浓度下具有最高的黑色素产量；低浓度的 Fe2＋（0．05 mmol/dm3）可以提高黑色素的产量， Fe2＋浓度≥0．20 mmol/dm3时明显抑制黑色素的生成，而Fe2＋浓度为1．00 mmol/dm3时可完全抑制菌株的生长；低浓度的 Cu2＋（0．1 mmol/dm3）对黑色素的产量无明显影响，Cu2＋浓度≥0．5 mmol/dm3时可抑制黑色素的生成，而Cu2＋浓度超过1．0 mmol/dm3时可完全抑制菌株的生长．通过PCR扩增获得菌株bIp-2的对羟苯基丙酮酸双加氧酶（hydroxyphenylpyruvic acid dioxygenase， HPPD）全长基因（1087 bp ），分析发现其编码的 HPPD 蛋白序列（含361氨基酸）与来自菌株Pseudomonas stutzeri RCH2的HPPD蛋白序列具有最高的同源性（序列相似性为97％）．这些结果为深入研究该细菌所产黑色素的合成，应用及生态作用奠定了基础．