【摘要】：Bacillus sp.HJ14和Bacillus sp.SD01、Bacillus sp.SD02分別篩選自云南和山東特殊環(huán)境土壤樣本。通過對三株菌進行全基因組測序及注釋分析,發(fā)現(xiàn)其基因組序列中含酯酶基因EstZ1、EstZ14和EstZ22。本研究成功克隆3段基因,并利用Escherichia coli BL21(DE3)進行異源表達,純化得到重組酯酶EstZ1、EstZ14和EstZ22,同時對他們的酶學(xué)性質(zhì)及對DBP、DEP、DiBP和DCHP的降解潛力進行了研究,具體研究結(jié)果如下:(1)重組酶Est Z1可以水解�；滈L度為2 10的人工合成pNP底物,最適底物是pNPC4;最適pH為9.0,在pH 5.0 9.5處理1 h酶活力仍剩余60%以上;最適溫度為50℃,在40℃ 70℃保持50%以上的酶活力,37℃和60℃時的熱穩(wěn)定性很好,80℃下半衰期為20 min;大多數(shù)金屬離子和化學(xué)試劑對其活性影響較小,部分有促進作用;能催化水解DEP和DiBP的一個酯鍵生成相應(yīng)的醇和單酯。(2)重組酶Est Z14可以水解酰基鏈長度為2 6的人工合成pNP底物,最適底物是pNPC4;最適pH為7.0,在pH 6.0 11.0處理1 h酶活力仍剩余60%以上;最適溫度為45℃,在低于25℃和高于50℃時酶活較低(≤20%),37℃時的熱穩(wěn)定性良好;大部分金屬離子及化學(xué)試劑對其活性無影響或影響微弱。(3)重組酶Est Z22可以水解�；滈L度為2 8的人工合成pNP底物,最適底物是pNPC4;最適pH為9.0,在pH 6.0 9.0處理1 h酶活仍保持60%以上;最適溫度為70℃,37℃時的熱穩(wěn)定性很好,60℃下半衰期為40 min,70℃下處理15 min酶活還剩余40%以上;大多數(shù)金屬離子和化學(xué)試劑對其活性均影響不大,少數(shù)有促進作用;對DEP、DiBP、DBP和DCHP有水解活性。本研究獲得3個酯酶EstZ1、EstZ14和EstZ22。其中,EstZ1和EstZ22均為嗜熱酶并且對DEP、DiBP、DBP和DCHP有水解活性。嗜熱酯酶包含了嗜熱酶和酯酶的雙重性質(zhì),在食品工業(yè)、生物醫(yī)療、環(huán)境治理等方面有著非常巨大的應(yīng)用潛力。
[Abstract]:Bacillus sp.HJ14 and Bacillus sp.SD01Bacillus sp.SD02 were screened from special environment soil samples of Yunnan and Shandong, respectively. By sequencing and annotating the whole genome of three strains of bacteria, it was found that the esterase genes EstZ1, EstZ14 and EstZ22 were found in the genomic sequence. In this study, three segments of genes were cloned and expressed heterogeneously by Escherichia coli BL21 (DE3). The recombinant esterases EstZ1, EstZ14 and EstZ22 were purified, and their enzymatic properties and biodegradation potential of DBP DEPDiBP and DCHP were studied. The results are as follows: (1) the recombinant enzyme Est Z1 can hydrolyze synthetic pNP substrate with acyl chain length of 2 ~ 10, the optimum substrate is pNPC4, the optimum pH is 9.0, and the enzyme activity remains above 60% at pH 5.0 for 1 h, and the optimum temperature is 50 鈩，

本文編號：2140906