大蜡螟幼虫肠道微生物聚乙烯降解酶基因的表达及性质分析

doi:10.13304/j.nykjdb.2022.0880

摘要/Abstract

摘要：

聚乙烯（polyethylene， PE）塑料结构稳定，难以自然降解，对环境造成严重污染。大蜡螟（Galleria mellonella L.）幼虫取食聚乙烯后，排泄物出现羰基（C-O）和羟基（R-OH）官能团，能够使PE膜长链结构发生解聚。为研究其原理，基于免培养技术，对大蜡螟幼虫肠道内容物进行宏基因组和宏蛋白质组分析，挖掘得到2个潜在新PE降解酶基因POD8747和AhpC5341，并在大肠杆菌表达系统中进行了异源表达、纯化和初步性质分析。结果表明，重组POD8747和AhpC5341的最适温度均为50 ℃，最适pH均为8.0。经POD8747和AhpC5341处理后的PE膜亲水性增加，且其接触角从100?分别降低到（90.07±3.45）和（91.73±1.70）?。以上结果表明，POD8747和AhpC5341在PE降解中能够发挥表面氧化能力，从而促进其他酶进一步降解PE。

关键词: 聚乙烯, 大蜡螟, 肠道微生物, 过氧化物酶, 生物降解

Abstract:

Polyethylene （PE） is one of the most widely used plastic， which has stable structure， difficult to degrade and causes great pollution to the environment. In this study， the changes of PE in gut of Galleria mellonella L. larvae were investigated， the appearance of carbonyl （C-O） and hydroxyl （R-OH） functional groups indicated that the long chain structure in PE plastic film was depolymerized. Based on culture-independent method， high-throughput metagenomic and metaproteomic analysis of the microorganism in the intestinal contents of Galleria mellonella L.， two PE degrading enzyme encoding genes， POD8747 and Ahpc5341， were identified. Heterologous expression， purification and characterization were performed for both genes in Escherichia coli. The results showed the optimal temperature and optimal pH for both POD8747 and AhpC5341 were 50 ℃ and 8.0， respectively. The purified enzyme was evaluated for biodegradation of PE， after treated by POD8747 and AhpC5341， the PE film’s hydrophilicity increased and its contact angle of the PE film decreased from 100° to （90.07±3.45） and （91.73±1.70）°， respectively. These results indicated that POD8747 and AhpC5341 could exert surface oxidation ability in PE degradation， and could be used for PE degradation along with other enzymes in future.

Key words: polyethylene, Galleria mellonella L., gut microbiota, peroxidase, biodegradation

中图分类号:

Q933

柳春雨, 门丽娜, 连雅琴, 张宇宏, 张志伟, 张伟. 大蜡螟幼虫肠道微生物聚乙烯降解酶基因的表达及性质分析[J]. 中国农业科技导报, 2023, 25(3): 132-139.

Chunyu LIU, Lina MEN, Yaqin LIAN, Yuhong ZHANG, Zhiwei ZHANG, Wei ZHANG. Expression and Characterization of Enzymes for Polyethylene Degradation in the Gut of Galleria mellonella L. larvae[J]. Journal of Agricultural Science and Technology, 2023, 25(3): 132-139.

图/表 6

图1 大蜡螟幼虫排泄物的傅里叶变换红外光谱分析

Fig. 1 Fourier transform infrared spectroscopy analysis of Galleria mellonella L. larvae frass

表1 聚乙烯降解酶候选基因库

Table 1 Candidate gene library of polyethylene degrading enzyme

酶名称

Enzyme name

酶种类

Enzyme type

数量

Quantity

单加氧酶

Monooxygenase

单加氧酶、烷醛单加氧酶、链烷磺酸盐单加氧酶、4-乙基3-单加氧酶、α链烷醛单加氧酶、抗生素生物合成单加氧酶家族蛋白、对苯二酚单加氧酶

Monooxygenase， alkanal monooxygenase， alkanesulfonate monooxygenase， 4-hydroxyphenylacetate 3-monooxygenase， alkanal monooxygenase alpha chain， antibiotic biosynthesis monooxygenase family protein， quinol monooxygenase

双加氧酶

Dioxygenase

双功能一氧化氮、双加氧酶/二氢吡啶还原酶2、依赖于α-酮戊二酸的双加氧酶、槲皮素2，3-双加氧酶、顺式还原酮加双氧酶、牛磺酸加双氧酶、3，4-双乙基2，3-双加氧酶、LigB家族双加氧酶、萘- 1，2-双加氧酶、3-苯丙酸双加氧酶

Bifunctional nitric oxide， dioxygenase/dihydropyridine reductase 2， α-ketoglutarate-dependent dioxygenase， quercetin 2，3-dioxygenase， acireductone dioxygenase， taurine dioxygenase， 3，4-dihydroxyphenylacetate 2，3-dioxygenase， LigB family dioxygenase，

naphthalene 1，2-dioxygenase， 3-phenylpropionate dioxygenase

超氧化物歧化酶

Superoxide dismutase

超氧化物歧化酶Superoxide dismutase

脂肪酸氧化酶

Fatty acid oxidase

脂肪酸氧化蛋白亚单位、长链脂肪酸辅酶A连接酶

Fatty-acid oxidation protein subunit alpha， long-chain-fatty-acid-CoA ligase

过氧化氢酶

Catalase

过氧化氢酶Catalase

烷基过氧化氢还原酶

Alkyl hydroperoxide reductase

烷基过氧化氢还原酶Alkyl hydroperoxide reductase

假定氧化还原酶

Putative oxidoreductase

假定氧化还原酶Putative oxidoreductase

图2 PCR扩增目的基因核酸电泳分析注：M—DNA标准分子量marker DM8000。

Fig. 2 DNA electrophoresis analysis of PCR amplified target geneNote： M—DNA standard ladder DM8000.

图3 聚乙烯降解酶蛋白重组表达和纯化注：M—蛋白标准Marker；POD8747中泳道1～4分别为超声破碎上清、蛋白纯化穿透液、蛋白纯化洗脱液、超声破碎沉淀；AhpC 5341中，泳道1～4分别为超声破碎上清、超声破碎沉淀、蛋白纯化穿透液、蛋白纯化洗脱液。

Fig. 3 Recombinant expression and purification of polyethylene degrading enzyme proteinNote： M—Protein standard marker； In POD8747， lanes 1~4 are supernatant after ultrasonic crushing， penetrating of protein purification， eluent of protein purification， sedimentation after ultrasonic crushing， respectively； In AhpC5341， lanes 1~4 are supernatant after ultrasonic crushing， sedimentation after ultrasonic crushing， penetrating of protein purification， eluent of protein purification， respectively.

图4 POD8747和AhpC5341最适温度和最适pHA： POD8747酶蛋白最适温度；B：POD8747酶蛋白最适pH；C：AhpC5341酶蛋白最适温度；D：AhpC5341酶蛋白最适pH

Fig. 4 Optimal temperature and optimal pH of POD8747 and AhpC5341A： Optimum temperature of POD8747； B： Optimum pH of POD8747； C： Optimum temperature of AhpC5341； D： Optimum pH of AhpC5341

图5 PE膜接触角分析

Fig. 5 Polyethylene contact angle test

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