Characterization and functional expression of a rubber degradation gene of a Nocardia degrader from a rubber-processing factory
Characterization and functional expression of a rubber degradation gene of a Nocardia degrader from a rubber-processing factory
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DOI：
10.1016/j.jbiosc.2016.11.012
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发表时间：
2017-04-01
影响因子：
2.8
通讯作者：
Fukuda, Masao
中科院分区：
文献类型：
--
作者：
Dao Viet Linh;Nguyen Lan Huong;Fukuda, Masao
利用天然胶乳和越南橡胶加工厂废料进行浓缩培养，获得了一个名为H2DA的橡胶降解菌联合体。凝胶渗透色谱分析表明，在发现的三株菌株中，只有NVL3菌株能将合成的聚顺-1，4-异戊二烯降解成低分子量的中间体。NVL3的16SRRNA基因序列与法氏诺卡氏菌DSM 43665(T)的同源性最高。Schiff‘s染色表明，合成的聚顺-1，4-异戊二烯的NVL3在橡胶覆盖板上积累了醛中间体。NVL3还能将脱蛋白天然橡胶降解成低分子量醛中间体。在NVL3的基因组序列中发现了一个胶乳清除蛋白基因的同源基因，它与已报道的橡胶降解菌的胶乳清除蛋白基因具有中等的氨基酸同源性(54-75%)。NVL3蛋白在大肠杆菌BL21(DE3)中的异源表达使我们能够纯化出46.8 kDa的His标记的ICP基因产物(His-LCP)。His-LCP降解合成的聚顺-1，4-异戊二烯，并从脱蛋白的天然橡胶中积累醛中间体，表明诺卡氏菌降解者的icp基因在大肠杆菌中得到了功能表达。定量逆转录聚合酶链式反应分析表明，在人工合成的聚顺-1，4-异戊二烯存在下，LCP基因在NVL3中有很强的转录诱导作用。这些结果表明LCP基因参与了橡胶在NVL3中的降解。
A rubber-degrading bacterial consortium named H2DA was obtained from an enrichment culture with natural rubber latex and rubber-processing factory waste in Vietnam. Gel permeation chromatography analysis revealed that only the strain NVL3 degraded synthetic poly(cis-1,4-isoprene) into low-molecular-weight intermediates among the three strains found in the H2DA. The 16S-rRNA gene sequence of NVL3 showed the highest identity with that of Nocardia farcinica DSM 43665(T). NVL3 accumulated aldehyde intermediates from synthetic poly(cis-1,4-isoprene) on a rubber-overlay plate as indicated by Schiff's staining. NVL3 also degraded deproteinized natural rubber into low-molecular-weight aldehyde intermediates. A latex-clearing protein (Icp) gene ortholog was identified within the genome sequence of NVL3, and it showed a moderate amino-acid identity (54-75%) with the Icp genes from previously reported rubber degraders. The heterologous expression of the NVL3 Icp in Escherichia coli BL21(DE3) allowed us to purify the 46.8-kDa His-tagged Icp gene product (His-Lcp). His-Lcp degraded synthetic poly(cis-1,4-isoprene) and accumulated aldehyde intermediates from deproteinized natural rubber suggesting the functional expression of the Icp gene from a Nocardia degrader in E. coli. Quantitative reverse transcription PCR analysis indicated the strong transcriptional induction of the lcp gene in NVL3 in the presence of synthetic poly(cis-1,4-isoprene). These results suggest the involvement of the lcp gene in rubber degradation in NVL3.