Inquilinus sp. P6-4菌株的生理生化特征及萘降解特性

doi:10.13560/j.cnki.biotech.bull.1985.2020-1392

摘要/Abstract

摘要：

以从尾矿中筛选出的 P6-4菌株为研究对象,基于16S rDNA测序确定其分类地位,并研究其生理生化特征和萘降解特性,同时分析该菌株对萘的最佳降解条件和降解途径。结果表明:P6-4菌株为Inquilinus属革兰氏阴性好氧菌,可在含1%-3%盐度的R₂A培养基和柠檬酸盐培养基上生长,并产过氧化氢酶、IAA和铁载体,同时具有溶磷、固氮功能;该菌株不产淀粉酶,分解葡萄糖时不生成乙酰甲基甲醇。菌株在萘初始质量浓度400 mg/L、30℃、pH 9.0、150 r/min条件下生长最佳。在最适降解条件下培养7 d后,以正己烷做萃取剂,利用紫外-可见分光光度法测得降解率为93.22%。分子生物学发现该菌株含有PAH(多环芳烃环羟基化双加氧酶基因)、HBHA(反式-o-羟基苯脱萘丙酮酸水合醛缩酶基因)、nahU(水杨酸羟化酶基因)、S5H(水杨酸5-羟化酶基因)、CATA(儿茶酚1,2-双加氧酶基因)、C230(儿茶酚2,3-双加氧酶基因)和GDO(龙胆酸1,2-双加氧酶基因)基因,推测P6-4菌株对萘的降解存在水杨酸和龙胆酸两种途径。

关键词: Inquilinus sp., 萘, 生理生化特征, 降解特性

Abstract:

The P6-4 strain screened from tailings was taken as the research object,its taxonomic status was determined based on 16S rDNA sequencing,and its physiological and biochemical characteristics and degradation characteristics for naphthalene were studied. Concurrently,the optimal degradation conditions and degradation pathways of the strain for naphthalene were analyzed. The results showed that P6-4 strain was a gram-negative aerobic bacterium of the genus Inquilinus,grew on R₂A medium with a salinity of 1%-3% and citrate medium,and produced catalase,IAA and iron carriers. It had the function of dissolving phosphorus and fixing nitrogen. It did not produce amylase and acetyl methyl methanol while decomposing glucose. The strain grew the best under the conditions of initial naphthalene mass concentration of 400 mg/L,30℃,pH 9.0,and 150 r/min. After 7 d of culture under the optimal degradation conditions,using n-hexane as the extractant,the degradation rate measured by UV-V is spectrophotometry was 93.22%. The study of molecular biology revealed that the strain contained PAH(PAH-ring hydroxylating dioxygenase gene),HBHA(trans-o-hydroxybenzylidenepyruvate hydratase-aldolase gene),nahU(salicylate hydroxylase gene),S5H(salicylate 5-hydroxylase gene),CATA(catechol 1,2-dioxygenase gene),C230(catechol 2,3-dioxygenase gene),and GDO(gentisate 1,2-dioxygenase gene),and it was speculated that the P6-4 strain degraded naphthalene in two pathways:salicylic acid and and gentianic acid.

Key words: Inquilinus sp., naphthalene, physiological and biochemical characteristics, degradation characteristics

汪颖, 陈永静, 孙庆业, 杨梦瑶, 吴盾. Inquilinus sp. P6-4菌株的生理生化特征及萘降解特性[J]. 生物技术通报, 2021, 37(6): 117-126.

WANG Ying, CHEN Yong-jing, SUN Qing-ye, YANG Meng-yao, WU Dun. Physiological and Biochemical Characteristics of Inquilinus sp. P6-4 Strain and Its Degradation Characteristics for Naphthalene[J]. Biotechnology Bulletin, 2021, 37(6): 117-126.

图/表 7

图1 萘的降解途径 A:龙胆酸途径;B:水杨酸途径;C:邻苯二甲酸途径

Fig.1 Degradation pathways for naphthalene A:Gentisate pathway. B:Salicylate pathway. C:Phthalandione pathway

表1 关键酶基因引物

Table 1 Primers of key enzyme gene

基因名称 Gene name	引物序列 Primer sequence(5'-3')	基因长度 Gene length/bp
PAH HBHA nahU S5H CATA C230 GDO	GAGATGCATACCACGTKGGTTGGA AGCTGTTGTTCGGGAAGAYWGTGCMGTT ATGTGGGTGAAGATGGACCTGCC GCATATCATCGGAGATGGCTTTGGC CAAATACCTCGGTTGCAGCG CCCGAACTGGGCAATACCTT TGATCGACTTCAAAGTGTATTTCGA CAAGCGGTGCAACATCGACC ACVCCVCGHACCATYGAAGG CGSGTNGCAWANGCAAAGT AAGAGGCATGGGGGCGCACCGGTTCGATCA CCAGCAAACACCTCGTTGCGGTTGCC ACGAACGGGATGTCGAGG ACGAAACCGATCAGCCGA	306 400 112 490 470 298 489

图2 P6-4降解菌株的16S rDNA的PCR产物电泳图 M:分子量标准

Fig.2 Electrophoretogram of 16S rDNA PCR products ofP6-4 degrading strain M:Molecular weight standard

表2 P6-4降解菌株的生理生化特征

Table 2 Physiological and biochemical characteristics of P6-4 degrading strain

生化反应 Physiological and biochemical reaction	结果 Result	生化反应 Physiological and biochemical reaction	结果 Result
1%、2%、3% NaCl生长 1%,2%,3% NaCl growth	+	固氮 Nitrogen fixation	+
过氧化氢酶 Catalase	+	柠檬酸盐 Utilization of citrate	+
吲哚乙酸 Indole acetic acid	+	VP试验 VP test	-
有机磷 Organophosphorus	+	厌氧生长 Anaerobic growth	-
无机磷 Inorganic phosphorus	+	淀粉酶 Amylase	-
革兰氏染色 Gram staining	-	铁载体 Iron carrier	+

图3 不同因素对P6-4菌株降解萘生长的影响

Fig.3 Effects of different factors on the growth of P6-4 degrading strain for degradate naphthalene

图4 P6-4降解菌株的生长曲线

Fig.4 Growth curve of P6-4 degrading strain

图5 关键酶基因的PCR扩增电泳图 M:分子量标准;1:PAH基因片段;2:HBHA基因片段;3:nahU基因片段;4:S5H基因片段;5:CATA基因片段;6:C230基因片段;7:GDO基因片段

Fig.5 PCR amplified electrophoretic map of key enzyme genes M:Molecular weight standard. 1:PAH gene fragment. 2:HBHA gene fragment. 3:nahU gene fragment. 4:S5H gene fragment. 5:CATA gene fragment. 6:C230 gene fragment. 7:GDO gene fragment

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