生物工程學(xué)報(bào) 25 August 2009, 25(8):1151～1159
顆粒狀甲烷單加氧酶異源表達(dá)方法
韓冰1*, 蘇濤2*, 楊程1, 江皓1, 吳昊1, 張翀1, 李信2, 邢新會(huì)1
1 清華大學(xué)化工系生物化工研究所, 北京 100084
2 中國(guó)農(nóng)業(yè)科學(xué)院研究生院, 北京 100081
摘 要: 由于甲烷氧化菌只能利用甲烷作為唯一的碳源和能源, 存在生長(zhǎng)緩慢,、細(xì)胞密度低,、培養(yǎng)困難等問(wèn)題, 限制了其工業(yè)應(yīng)用。解決該問(wèn)題的有效途徑之一是在容易實(shí)現(xiàn)高密度培養(yǎng)的異源宿主菌中表達(dá)甲烷單加氧酶(Methane monooxygenase, MMO),。本實(shí)驗(yàn)室前期首次在一種紅球菌中成功地表達(dá)了來(lái)自于甲烷氧化菌(Methylosinus trichosporium)OB3b的pMMO(顆粒狀甲烷單加氧酶), 但比酶活較原始菌低很多,。本實(shí)驗(yàn)在該結(jié)果的基礎(chǔ)上, 通過(guò)選用不同的啟動(dòng)子和宿主細(xì)胞探索表達(dá)pMMO的可能性, 結(jié)果得到了具有氧化甲烷活性的重組菌, 但是產(chǎn)物檢測(cè)到乙醇的生成, 且該重組菌的pMMO活性不穩(wěn)定, 暗示pMMO的催化特性可能發(fā)生了變化。另外, 很多重組菌檢測(cè)到pMMO蛋白的表達(dá), 但沒(méi)有催化活性, 說(shuō)明pMMO在宿主細(xì)胞中的正確組裝是其功能表達(dá)的關(guān)鍵,。
關(guān)鍵詞: 甲烷氧化菌, 顆粒狀甲烷單加氧酶, 異源表達(dá), 生物催化
Heterologous expression of particulate methane monooxygenase in different host cells
Bing Han1*, Tao Su2*, Cheng Yang1, Hao Jiang1, Hao Wu1, Chong Zhang1, Xin Li2, and Xinhui Xing1
1 Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China
Abstract: Methanotrophs use methane as the sole carbon and energy source, which cause slow growth, low cell density and hinder its industrial applications. One promising solution is to heterologously express methane monooxygenase (MMO) in other host cells that can be easily cultivated at high cell density. We systematically exploited the possibility of functional expression of pMMO by choosing different promoters and different host cells. The results showed that the recombinants could oxidize methane to methanol. In particular, ethanol could also be detected in the oxidized products, but the enzyme activity was instable, implying that some changes of pMMO expressed in the host cells might have occurred. In addition, SDS-PAGE analysis showed that many recombinants could express the subunits of pMMO, but the enzyme activity could not be detected. In conclusion, correct fold of pMMO in the host cells is important for its functional expression.
Keywords: methanotroph, particulate methane monooxygenase (pMMO), heterologous expression, biocatalysis
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