Kocuria sp.3-3来源木聚糖酶基因筛选、克隆、表达、性质研究及定点突变研究
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摘要
木聚糖是半纤维素的主要组成部分,是自然界中第二大生物质资源。木聚糖酶是指将木聚糖水解成低聚木糖或木糖的酶系总称,其中内切p-1,4-D-木聚糖酶[EC 3.2.1.8]是其中最关键的酶。木聚糖酶在饲料、食品、造纸和纺织等领域都有广泛的应用前景。近年来,随着酶法造纸术的发展,碱性木聚糖酶引起了人们木聚糖酶的高度关注。本研究基于已经得到的一株能够产碱性木聚糖酶的放线菌,通过构建该菌株的基因组文库,筛选出木聚糖酶基因,明确了基因的结构,并将其在大肠杆菌表达系统中进行表达。在对酶进行纯化后,深入研究了该酶的酶学性质。
1)对3-3进行菌种鉴定,包括形态观察、生理生化检测和系统发育树分析,确定菌株的分类；
2)提取3-3的基因组DNA,对基因组DNA进行酶切,将4-9 kb范围内的酶切片段与载体相连并转化入大肠杆菌感受态中,从而构建了3-3的基因组文库,并筛选获得了1个含木聚糖酶基因Kxg的阳性转化子X-27,经测序后确定了Kxg的序列,从而得到了木聚糖酶Kxp的序列。对Kxp一系列的分析结果表明,Kxp含有389个氨基酸残基,分子量为43806.4,属于F/10家族糖苷水解酶。
3)根据Kxg序列设计引物,以X-27提取的质粒为模板,进行PCR扩增得到木聚糖酶基因Kxg。将Kxg以正确的阅读框架连接到大肠杆菌表达载体pET-Duet上,并转化入E.coli rosetta,获得重组工程菌R32。经过IPTG诱导,在培养基上清液中得到了Kxp。
4)采用离子交换和亲和层析的方法对得到的Kxp粗酶液进行纯化,经过两步纯化后,得到了纯度很高的终产物。对Kxp进行酶学性质分析,酶Km和Vmax分别为5.41mg/mL和202.3μmol·min-1·mg-1。Kxp的最适反应pH为8.5,在pH7-10之间保持最高活力的50%以上。Kxp具有很好的pH稳定性,在pH8-11内4℃过夜放置后,酶活残留仍在80%以上。Kxp的最适反应温度为55℃,在反应温度低于40℃C或高于60℃C时酶活性较低。Kxp具有良好的热稳定性,70℃C处理1h后酶活仍有30%残留。在反应体系中加入5 mM的各种金属离子或化合物,观察其对酶活的影响,结果表明酶活受重金属离子影响较大,而轻金属离子对酶活几乎没有影响。
5)对木聚糖酶Kxp的H131、W135、E176、N179、H259、E288、W332、W340和H102等位点进行定点突变,并对各突变蛋白的性质进行初步研究。结果表明,前八个位点对于维持酶活具有重要作用,而H131、W135和H259也对酶和底物的结合起一定作用。为提高木聚糖酶Kxp的热稳定性,将H102用Cys残基替代。对突变蛋白KxpH102C的热稳定性研究结果表明,突变蛋白的热稳定性较之Kxp并未有提高。
Xylan is the major component of hemicellulose, and the second most abundant bioresource in the nature. Xylanolytic enzymes are a system of hydrolytic enzymes, which act cooperatively to hydrolysis xylan to its constituent sugars. Among all the enzymes, endo-β-1,4-D-xylanase [EC3.2.1.8] plays a crucial role. It has great potential applications in feed industry, food industry, pulp and paper industry and textile industry. Since the development of using xylanase to bleach alki-treated wood pulp instead of using usual environmentally damaging chemical reagents, the research for alkaline xylanase has been extensively studied. In this research, we constructed a genomic library of a strain of actinomyce (strain 3-3) capable of producing xylanase (Kxp) and screened the gene (Kxg) encoding Kxp. Thus we determined the structure of the gene, and expressed it in the E.coli expression system. The properties of the xylanase was determined after purification.
1) Determine the classification of the strain 3-3 by means of morphologic observation, performing physiological and biochemical tests and anlysis of
phylogenetic tress;
2) In order to construct the genomic library of 3-3, the genomic DNA was extracted and cut. The fragments within the range of 4-9 kb was ligated to vectors and transfered into competence E.coli. A positive clon carrying Kxg, named X-27, was screened and Kxg was sequenced. The sequence of Kxp was determined by translating the sequence of Kxg. The analysis of Kxg shows that it has 389 amino acid residues and its molecular weight is 43146.4. Kxg is a member of hydrolase F/10.
3) Kxg was obtained from by PCR amplification, using the plasmid extracted from X-27 as the template. After several steps of treatments, it was ligated into the E.coli expression vector pET-Duet with the correct open reading frame, transferred into E.coli rosetta (DE3), and finally the recombinant R32 was obtained. After induced by IPTG, Kxg was successfully expressed in R32 and Kxp was found in the supernatant.
4) The Kxp crude enzyme was well purified by means of anion exchange. Then its properties were studied. Its optimal pH was 8.5, and it sustained above 50%of the enzyme activity between pH 7-10. Kxp showed high pH stability in 4℃overnight between pH 7.5-11.5, with more than 80%of the enzyme activity remained. Its optimal temperature was 55℃, and it showed low enzyme activity below 30℃Cor over 60℃. Kxp had high thermal stability, with 30%of the enzyme activity remaining after incubated at 70℃for 1 h. The research on the effect of 5 mM metallic ions on the enzyme activity showed that the enzyme activity was greatly affected by heavy metallic ions, while light metallic ions almost had no effects on the enzyme activity.
5) Nine sites (H131, W135, E176, N179, H259, E288, W332, W340 and H102) were chosen for site-directed mutagenesis, and the properties of the mutanted proteins were studied. The results showed that the former eight sites palyed important roles in maintaining enzyme activity, while H131, W135 and H259 also effected the combination between the enzyme and xylan. In order to enhance the thermostability of Kxp, H102 was replaced by cysteine. The thermostability of KxpH102C was studied, and the resulted showed that the thermostability of KxpH102C wasn't enhanced compared to Kxp.
1)对3-3进行菌种鉴定,包括形态观察、生理生化检测和系统发育树分析,确定菌株的分类；
2)提取3-3的基因组DNA,对基因组DNA进行酶切,将4-9 kb范围内的酶切片段与载体相连并转化入大肠杆菌感受态中,从而构建了3-3的基因组文库,并筛选获得了1个含木聚糖酶基因Kxg的阳性转化子X-27,经测序后确定了Kxg的序列,从而得到了木聚糖酶Kxp的序列。对Kxp一系列的分析结果表明,Kxp含有389个氨基酸残基,分子量为43806.4,属于F/10家族糖苷水解酶。
3)根据Kxg序列设计引物,以X-27提取的质粒为模板,进行PCR扩增得到木聚糖酶基因Kxg。将Kxg以正确的阅读框架连接到大肠杆菌表达载体pET-Duet上,并转化入E.coli rosetta,获得重组工程菌R32。经过IPTG诱导,在培养基上清液中得到了Kxp。
4)采用离子交换和亲和层析的方法对得到的Kxp粗酶液进行纯化,经过两步纯化后,得到了纯度很高的终产物。对Kxp进行酶学性质分析,酶Km和Vmax分别为5.41mg/mL和202.3μmol·min-1·mg-1。Kxp的最适反应pH为8.5,在pH7-10之间保持最高活力的50%以上。Kxp具有很好的pH稳定性,在pH8-11内4℃过夜放置后,酶活残留仍在80%以上。Kxp的最适反应温度为55℃,在反应温度低于40℃C或高于60℃C时酶活性较低。Kxp具有良好的热稳定性,70℃C处理1h后酶活仍有30%残留。在反应体系中加入5 mM的各种金属离子或化合物,观察其对酶活的影响,结果表明酶活受重金属离子影响较大,而轻金属离子对酶活几乎没有影响。
5)对木聚糖酶Kxp的H131、W135、E176、N179、H259、E288、W332、W340和H102等位点进行定点突变,并对各突变蛋白的性质进行初步研究。结果表明,前八个位点对于维持酶活具有重要作用,而H131、W135和H259也对酶和底物的结合起一定作用。为提高木聚糖酶Kxp的热稳定性,将H102用Cys残基替代。对突变蛋白KxpH102C的热稳定性研究结果表明,突变蛋白的热稳定性较之Kxp并未有提高。
Xylan is the major component of hemicellulose, and the second most abundant bioresource in the nature. Xylanolytic enzymes are a system of hydrolytic enzymes, which act cooperatively to hydrolysis xylan to its constituent sugars. Among all the enzymes, endo-β-1,4-D-xylanase [EC3.2.1.8] plays a crucial role. It has great potential applications in feed industry, food industry, pulp and paper industry and textile industry. Since the development of using xylanase to bleach alki-treated wood pulp instead of using usual environmentally damaging chemical reagents, the research for alkaline xylanase has been extensively studied. In this research, we constructed a genomic library of a strain of actinomyce (strain 3-3) capable of producing xylanase (Kxp) and screened the gene (Kxg) encoding Kxp. Thus we determined the structure of the gene, and expressed it in the E.coli expression system. The properties of the xylanase was determined after purification.
1) Determine the classification of the strain 3-3 by means of morphologic observation, performing physiological and biochemical tests and anlysis of
phylogenetic tress;
2) In order to construct the genomic library of 3-3, the genomic DNA was extracted and cut. The fragments within the range of 4-9 kb was ligated to vectors and transfered into competence E.coli. A positive clon carrying Kxg, named X-27, was screened and Kxg was sequenced. The sequence of Kxp was determined by translating the sequence of Kxg. The analysis of Kxg shows that it has 389 amino acid residues and its molecular weight is 43146.4. Kxg is a member of hydrolase F/10.
3) Kxg was obtained from by PCR amplification, using the plasmid extracted from X-27 as the template. After several steps of treatments, it was ligated into the E.coli expression vector pET-Duet with the correct open reading frame, transferred into E.coli rosetta (DE3), and finally the recombinant R32 was obtained. After induced by IPTG, Kxg was successfully expressed in R32 and Kxp was found in the supernatant.
4) The Kxp crude enzyme was well purified by means of anion exchange. Then its properties were studied. Its optimal pH was 8.5, and it sustained above 50%of the enzyme activity between pH 7-10. Kxp showed high pH stability in 4℃overnight between pH 7.5-11.5, with more than 80%of the enzyme activity remained. Its optimal temperature was 55℃, and it showed low enzyme activity below 30℃Cor over 60℃. Kxp had high thermal stability, with 30%of the enzyme activity remaining after incubated at 70℃for 1 h. The research on the effect of 5 mM metallic ions on the enzyme activity showed that the enzyme activity was greatly affected by heavy metallic ions, while light metallic ions almost had no effects on the enzyme activity.
5) Nine sites (H131, W135, E176, N179, H259, E288, W332, W340 and H102) were chosen for site-directed mutagenesis, and the properties of the mutanted proteins were studied. The results showed that the former eight sites palyed important roles in maintaining enzyme activity, while H131, W135 and H259 also effected the combination between the enzyme and xylan. In order to enhance the thermostability of Kxp, H102 was replaced by cysteine. The thermostability of KxpH102C was studied, and the resulted showed that the thermostability of KxpH102C wasn't enhanced compared to Kxp.
引文
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