一株产1-氨基环丙烷-1-羧酸脱氨酶的氢氧化细菌的分离鉴定及酶活力测定

Isolation and identification of hydrogen-oxidizing bacteria producing 1-aminocyclopropane-1-carboxylate deaminase and the determination of enzymatic activity

【目的】以结瘤豆科植物紫花苜蓿根际土壤为研究材料,筛选具有ACC脱氨酶活力的氢氧化细菌,探索氢氧化细菌植物促生作用机制。【方法】利用持续通H2的气体循环培养体系、矿质盐固体培养基,分离、培养氢氧化细菌,观察菌株形态并测定生理生化特征;16SrDNA序列分析法构建系统发育树;采用薄层层析法筛选ACC脱氨酶阳性菌株,茚三酮显色法测定ACC脱氨酶活力。【结果】分离的37株细菌中有8株菌氧化氢和自养生长能力较强,初步确定为氢氧化细菌,从中筛选出1株ACC脱氨酶阳性菌株WMQ-7。菌株WMQ-7的形态特征、生理生化特征与恶臭假单胞菌(Pseudomonas putida)的特征基本一致;16SrDNA序列(GenBank登录号为EU807744)在系统发育树中与恶臭假单胞菌同属一个类群,序列同源性99%。鉴定菌株WMQ-7为恶臭假单胞菌,其ACC脱氨酶活力为0.671U/μg。【结论】采用气体循环培养体系分离氢氧化细菌,克服了传统配气法的局限。ACC脱氨酶阳性菌株的筛选,为深入研究氢氧化细菌作为植物根际促生菌的菌株特性和促生机制提供理论依据。

[Objective] We used Medicago sativa rhizosphere in Shaanxi province of China to isolate and identify hydrogen- oxidizing bacteria that produced ACC （1-aminocyclopropane-l-carboxylate） deaminase, and then studied the mechanism why they can promote the growth of plants.[Methods] Hydrogen-oxidizing bacteria were isolated by gas-cycle incubation system. We studied the morphological character, physiological characteristics, 16S rDNA sequence analysis and built the phylogenic tree. Thin layer chromatography was used to isolate the strain that produced ACC deaminase. Ninhydrin reaction was used to test the enzyme activity.[Results] In total 37 strains were isolated, 8 of which could oxidize H2 strongly and grow chemolithoautotrophically. We initially identified them as hydrogen-oxidizing bacteria. Only strain WMQ-7 produced ACC deaminase among these 8 strains. Morphological and physiological characteristics analysis showed that strain WMQ-7 was essentially consistent with Pseudomonas putida. The 16S rDNA sequence analysis （GenBank accession number EU807744） suggested that strain WMQ-7 was clustered together with Pseudomonas putida in phylogenetic tree, with the sequence identity of 99% . Based on all these results, strain WMQ-7 was identified as Pseudomonas putida. The enzyme activity of strain WMQ-7 was 0.671U/μg. [Conclusion] A strain producing ACC deaminase was identified and tested.