黑曲霉脂肪酶的酶学性质、基因克隆与表达及结构预测
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摘要
在利用sn-1,3位置选择性的脂肪酶催化植物油脂转变为质构脂质的同时,还可以获得绿色新型生物能源——生物柴油。开发此工艺路线,对于植物油脂深加工和提高产品附加值具有重要意义,但目前我国关于sn-1,3位置选择性脂肪酶和质构脂质的研究开发尚处于起步阶段。本实验重点立足于研究开发具有sn-1,3位置选择性的脂肪酶,以期为发展质构脂质奠定酶学基础。本论文的主要内容和结果如下:
筛选到一株热稳定、耐酸和高比活力脂肪酶产生菌株。以罗丹明B作为指示剂,利用选择性培养基,从油污土壤中筛选到一株胞外脂肪酶高产菌株,经形态学和ITS序列鉴定为黑曲霉,该菌株编号为F044。黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAE Sepharose Fast Flow阴离子交换层析和Sephadex G-75凝胶过滤层析得到电泳纯的脂肪酶,纯化倍数为73.71倍,活性回收率为33.99%。对纯化脂肪酶酶学性质的研究表明:该脂肪酶分子量为35～40 kDa,水解橄榄油的最适温度和最适pH分别为45℃和7.0,在60℃以下和pH2.0～9.0之间有很好的稳定性。该脂肪酶的水解活性对Ca2+表现出明显的依赖性,而Mn2+、Fe2+和Zn2+对水解活性则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7.37 mmol/l和25.91μmol/min.mg。其N-端的15个氨基酸残基序列为Ser(Glu/His)-Val-Ser-Thr-Ser-Thr- Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。
首次克隆了黑曲霉脂肪酶基因。根据黑曲霉F044脂肪酶N-端氨基酸残基序列,结合丝状真菌脂肪酶分子活性中心和第一个氧阴离子洞区的保守氨基酸残基序列,运用生物信息学方法,从NCBI核酸数据库中找到一个与黑曲霉脂肪酶基因同源的侯选基因A84689。根据该基因序列,设计引物直接PCR扩增得到黑曲霉脂肪酶全长基因anl。anl全长1044 bp,含3个内含子,分别为54 bp,45 bp和51 bp,编码297个氨基酸(含信号肽27个氨基酸)。在基因水平上,anl与其它脂肪酶基因核苷酸序列没有任何同源性;在蛋白质水平上,anl编码的氨基酸序列与A. flavus, T. lanuginosus, P. allii脂肪酶氨基酸序列分别有40%、50%和40%的同源性。
首次实现了黑曲霉脂肪酶基因的异源表达。将黑曲霉FO44脂肪酶基因克隆到大肠杆菌表达载体pET-28a上,转化E. coli BL21(De3)。经IPTG诱导后,成熟黑曲霉F044脂肪酶的编码基因在宿主菌中大量表达,表达量为50 mg/g湿细胞,但均以无活性的包含体形式存在。利用Ni-NTA金属亲和色谱柱,一步纯化出N-端携带6×His标签的重组脂肪酶蛋白。通过大量稀释法和DEAE Sepharose Fast Flow柱层析法相结合的复性方法,变性后的纯化重组脂肪酶蛋白在体外实现再折叠复性。复活后的重组脂肪酶比活力为221.49 U/mg。
将N-端融合有6×His编码序列的成熟黑曲霉F044脂肪酶基因克隆到pPIC9K载体上,转化P. pastoris GS115。经甲醇诱导表达后,黑曲霉F044脂肪酶基因在P. pastoris GS115中实现了功能性表达,发酵上清液脂肪酶酶活为15.50 U/ml。利用Ni-NTA金属亲和色谱柱,一步纯化出N-端携带6×His标签的重组脂肪酶蛋白。SDS-PAGE电泳显示,P. pastoris GS115产生两种分子大小不同的重组脂肪酶蛋白。Sephadex G-75凝胶过滤分离纯化出这两种重组脂肪酶蛋白,均有脂肪酶活性,其中分子量较小的重组脂肪酶蛋白水解橄榄油的比活力为812 U/mg。
首次尝试利用BioEdit、PSIPRED和SwissModel等软件或服务器对黑曲霉脂肪酶的一级结构、二级结构和三级结构进行了分析。分析结果显示,无论是在一级结构、二级结构还是三级结构上,预测的黑曲霉脂肪酶结构与丝氨酸水解酶结构均相吻合。预测的黑曲霉脂肪酶三级结构与黑曲霉阿魏酸酯酶三级结构在“盖子”结构区存在显著差异。
Biodiesel, a novel alternative energy, can be attained as by-product in the structured lipids synthesis when the transesterification reaction is catalyzed by lipases with sn-1, 3-regionselectivity. It’s important for deeply processing of agriculture products and enhancing value of the oils and fats. However, there are very few reports on the lipases with sn-1, 3-regionselectivity and structured lipids synthesis in China. In this study, a lipase with sn-1, 3-regionselectivity from Aspergillus niger 404 was purified and biochemically characterized, and the lipase gene was cloned and expressed in E. coli and P. pastoris, respectively, for the first time in China. Furthermore, based on the homology of its amino acid sequence with T. lanuginosus lipase, the 3D-model of the A. niger lipase was predicted. The main results were as follows:
A novel filamentous fungi strain producing lipase with acid-tolerant, thermostable and high-specific activity characters was screened. On the selective media with rhodamine B as the indicator, a high-level lipase-producing strain was isolated from oil-polluted soil samples. The strain was identified and named as A. niger F044 based on the colony morphology characters and the ITS sequence analysis. The lipase from A. niger F044 was purified to homogeneity using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography, and Sephadex G-75 gel filtration chromatography. This protocol resulted in a 73.71-fold purification with 33.99 % final yield, and the relative molecular weight of the lipase was determined to be approximately 35-40 kDa using SDS-PAGE. The optimal pH and temperature for lipolytic activity of the lipase were 7.0 and 45℃, respectively. It was stable at temperature below 60℃and retained 98.70 % of its original activity for 30 min, while its activity declined rapidly as soon as the temperature rose over 65℃. The lipase was highly stable in the pH range from 2.0 to 9.0 for 4 h. Ca2+ ions stimulated its lipolytic activity, whereas Mn2+, Fe2+, and Zn2+ ions caused inhibition. The values of Km and Vmax calculated from the Lineweaver–Burk plot using p-nitrophenyl palmitate as hydrolysis substrate were 7.37 mmol/l and 25.91μmol·min-1·mg-1, respectively. The N-terminal amino acid sequence of the lipase was Ser/Glu/His-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala -Gln.
It is the first time that the lipase gene from A. niger was cloned and reported. Based on the N-terminal amino acid sequence of the lipase from A. niger F044 and the same conserved sequences with filamentous fungi lipases, a potential homologous gene A84689 to the anl (the gene encoding the lipase from A. niger F044) was identified in NCBI nucleotide database by means of bioinformatics. A pair of primers was designed according to the nucleotide sequence of A84689, and the anl was directly cloned by PCR. Nucleotide sequencing revealed that the anl has an ORF of 1, 044 bp, containing three introns of 54 bp, 45 bp and 51 bp. The deduced amino acid sequence corresponds to 297 amino acid residues including a signal sequence of 27 amino acid residues. Nucleotide sequence alignment through BLAST reveal that the anl had no sequence identity to any known lipase genes except for another A. niger lipase gene (GenBank access No. DQ680030). The overall amino-acid sequence identity of the ANL (the mature A. niger lipase) to those of A. flavus lipase, T. lanuginosus lipase, P. allii lipase was 40%, 50% and 40%, respectively.
It is also for the first time that the lipase gene from A. niger was expressed successfully in heterogeneous host. The cDNA coded for the ANL was cloned into pET28a vector and the recombinant plasmid was transformed into E. coli BL21 (De3). The anl was overexpressed in E. coli BL21 (De3) after induction by IPTG, and the rANL accumulated in the cells in an insoluble form as inclusion bodies. The production level of the anl in the cells was 50 mg rANL/g wet cells. The recombinant ANL with 6-His-tag was purified by Ni-NTA Agrose chromatography. The denatured recombinant ANL by 8mol/l urea was refolded in vitro by dilution and DEAE Sepharose Fast Flow chromatography. The specific activity of the renatured rANL was 221.49 U/mg.
The cDNA fragment coding the ANL with 6-His encoding sequence at N-terminus was cloned into the pPIC9K vector and the reconstructed expression plasmid was then transformed into P. pastoris GS115. The anl was actively expressed in P. pastoris GS115 after induction by mehanol and the rANL was secreted into the fermentation broth. The measured lipolytic activity of the fermentation broth was 15.50 U/ml. The rANL with 6-His-tag at N-terminus was purified by Ni-NTA Agrose chromatography. Two kinds of rANL, ANLⅠand ANLⅡ, were detected in the fermentation broth by SDS-PAGE analysis. After isolation by Sephadex G-75 gel filtration chromatography, the specific activity of the ANLⅡwas 812 U/mg.
We have tried for the first time to annotate the primary structure, the secondary structure, and the three-dimensional structure of the A. niger lipase through the homologous lipase with known structure in BioEdit software, PSIPRED server and SwissModel sever, respectively. The predicted structures of the A. niger lipase were consistent with those of the serine hydrolase. The molecular structure models of the A. niger lipase and the A. niger feruloyl esterase were superimposed on each other to study the structure difference, and the result suggested that the main structure difference lied in the lid structure covering the active site.
筛选到一株热稳定、耐酸和高比活力脂肪酶产生菌株。以罗丹明B作为指示剂,利用选择性培养基,从油污土壤中筛选到一株胞外脂肪酶高产菌株,经形态学和ITS序列鉴定为黑曲霉,该菌株编号为F044。黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAE Sepharose Fast Flow阴离子交换层析和Sephadex G-75凝胶过滤层析得到电泳纯的脂肪酶,纯化倍数为73.71倍,活性回收率为33.99%。对纯化脂肪酶酶学性质的研究表明:该脂肪酶分子量为35～40 kDa,水解橄榄油的最适温度和最适pH分别为45℃和7.0,在60℃以下和pH2.0～9.0之间有很好的稳定性。该脂肪酶的水解活性对Ca2+表现出明显的依赖性,而Mn2+、Fe2+和Zn2+对水解活性则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7.37 mmol/l和25.91μmol/min.mg。其N-端的15个氨基酸残基序列为Ser(Glu/His)-Val-Ser-Thr-Ser-Thr- Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。
首次克隆了黑曲霉脂肪酶基因。根据黑曲霉F044脂肪酶N-端氨基酸残基序列,结合丝状真菌脂肪酶分子活性中心和第一个氧阴离子洞区的保守氨基酸残基序列,运用生物信息学方法,从NCBI核酸数据库中找到一个与黑曲霉脂肪酶基因同源的侯选基因A84689。根据该基因序列,设计引物直接PCR扩增得到黑曲霉脂肪酶全长基因anl。anl全长1044 bp,含3个内含子,分别为54 bp,45 bp和51 bp,编码297个氨基酸(含信号肽27个氨基酸)。在基因水平上,anl与其它脂肪酶基因核苷酸序列没有任何同源性;在蛋白质水平上,anl编码的氨基酸序列与A. flavus, T. lanuginosus, P. allii脂肪酶氨基酸序列分别有40%、50%和40%的同源性。
首次实现了黑曲霉脂肪酶基因的异源表达。将黑曲霉FO44脂肪酶基因克隆到大肠杆菌表达载体pET-28a上,转化E. coli BL21(De3)。经IPTG诱导后,成熟黑曲霉F044脂肪酶的编码基因在宿主菌中大量表达,表达量为50 mg/g湿细胞,但均以无活性的包含体形式存在。利用Ni-NTA金属亲和色谱柱,一步纯化出N-端携带6×His标签的重组脂肪酶蛋白。通过大量稀释法和DEAE Sepharose Fast Flow柱层析法相结合的复性方法,变性后的纯化重组脂肪酶蛋白在体外实现再折叠复性。复活后的重组脂肪酶比活力为221.49 U/mg。
将N-端融合有6×His编码序列的成熟黑曲霉F044脂肪酶基因克隆到pPIC9K载体上,转化P. pastoris GS115。经甲醇诱导表达后,黑曲霉F044脂肪酶基因在P. pastoris GS115中实现了功能性表达,发酵上清液脂肪酶酶活为15.50 U/ml。利用Ni-NTA金属亲和色谱柱,一步纯化出N-端携带6×His标签的重组脂肪酶蛋白。SDS-PAGE电泳显示,P. pastoris GS115产生两种分子大小不同的重组脂肪酶蛋白。Sephadex G-75凝胶过滤分离纯化出这两种重组脂肪酶蛋白,均有脂肪酶活性,其中分子量较小的重组脂肪酶蛋白水解橄榄油的比活力为812 U/mg。
首次尝试利用BioEdit、PSIPRED和SwissModel等软件或服务器对黑曲霉脂肪酶的一级结构、二级结构和三级结构进行了分析。分析结果显示,无论是在一级结构、二级结构还是三级结构上,预测的黑曲霉脂肪酶结构与丝氨酸水解酶结构均相吻合。预测的黑曲霉脂肪酶三级结构与黑曲霉阿魏酸酯酶三级结构在“盖子”结构区存在显著差异。
Biodiesel, a novel alternative energy, can be attained as by-product in the structured lipids synthesis when the transesterification reaction is catalyzed by lipases with sn-1, 3-regionselectivity. It’s important for deeply processing of agriculture products and enhancing value of the oils and fats. However, there are very few reports on the lipases with sn-1, 3-regionselectivity and structured lipids synthesis in China. In this study, a lipase with sn-1, 3-regionselectivity from Aspergillus niger 404 was purified and biochemically characterized, and the lipase gene was cloned and expressed in E. coli and P. pastoris, respectively, for the first time in China. Furthermore, based on the homology of its amino acid sequence with T. lanuginosus lipase, the 3D-model of the A. niger lipase was predicted. The main results were as follows:
A novel filamentous fungi strain producing lipase with acid-tolerant, thermostable and high-specific activity characters was screened. On the selective media with rhodamine B as the indicator, a high-level lipase-producing strain was isolated from oil-polluted soil samples. The strain was identified and named as A. niger F044 based on the colony morphology characters and the ITS sequence analysis. The lipase from A. niger F044 was purified to homogeneity using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography, and Sephadex G-75 gel filtration chromatography. This protocol resulted in a 73.71-fold purification with 33.99 % final yield, and the relative molecular weight of the lipase was determined to be approximately 35-40 kDa using SDS-PAGE. The optimal pH and temperature for lipolytic activity of the lipase were 7.0 and 45℃, respectively. It was stable at temperature below 60℃and retained 98.70 % of its original activity for 30 min, while its activity declined rapidly as soon as the temperature rose over 65℃. The lipase was highly stable in the pH range from 2.0 to 9.0 for 4 h. Ca2+ ions stimulated its lipolytic activity, whereas Mn2+, Fe2+, and Zn2+ ions caused inhibition. The values of Km and Vmax calculated from the Lineweaver–Burk plot using p-nitrophenyl palmitate as hydrolysis substrate were 7.37 mmol/l and 25.91μmol·min-1·mg-1, respectively. The N-terminal amino acid sequence of the lipase was Ser/Glu/His-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala -Gln.
It is the first time that the lipase gene from A. niger was cloned and reported. Based on the N-terminal amino acid sequence of the lipase from A. niger F044 and the same conserved sequences with filamentous fungi lipases, a potential homologous gene A84689 to the anl (the gene encoding the lipase from A. niger F044) was identified in NCBI nucleotide database by means of bioinformatics. A pair of primers was designed according to the nucleotide sequence of A84689, and the anl was directly cloned by PCR. Nucleotide sequencing revealed that the anl has an ORF of 1, 044 bp, containing three introns of 54 bp, 45 bp and 51 bp. The deduced amino acid sequence corresponds to 297 amino acid residues including a signal sequence of 27 amino acid residues. Nucleotide sequence alignment through BLAST reveal that the anl had no sequence identity to any known lipase genes except for another A. niger lipase gene (GenBank access No. DQ680030). The overall amino-acid sequence identity of the ANL (the mature A. niger lipase) to those of A. flavus lipase, T. lanuginosus lipase, P. allii lipase was 40%, 50% and 40%, respectively.
It is also for the first time that the lipase gene from A. niger was expressed successfully in heterogeneous host. The cDNA coded for the ANL was cloned into pET28a vector and the recombinant plasmid was transformed into E. coli BL21 (De3). The anl was overexpressed in E. coli BL21 (De3) after induction by IPTG, and the rANL accumulated in the cells in an insoluble form as inclusion bodies. The production level of the anl in the cells was 50 mg rANL/g wet cells. The recombinant ANL with 6-His-tag was purified by Ni-NTA Agrose chromatography. The denatured recombinant ANL by 8mol/l urea was refolded in vitro by dilution and DEAE Sepharose Fast Flow chromatography. The specific activity of the renatured rANL was 221.49 U/mg.
The cDNA fragment coding the ANL with 6-His encoding sequence at N-terminus was cloned into the pPIC9K vector and the reconstructed expression plasmid was then transformed into P. pastoris GS115. The anl was actively expressed in P. pastoris GS115 after induction by mehanol and the rANL was secreted into the fermentation broth. The measured lipolytic activity of the fermentation broth was 15.50 U/ml. The rANL with 6-His-tag at N-terminus was purified by Ni-NTA Agrose chromatography. Two kinds of rANL, ANLⅠand ANLⅡ, were detected in the fermentation broth by SDS-PAGE analysis. After isolation by Sephadex G-75 gel filtration chromatography, the specific activity of the ANLⅡwas 812 U/mg.
We have tried for the first time to annotate the primary structure, the secondary structure, and the three-dimensional structure of the A. niger lipase through the homologous lipase with known structure in BioEdit software, PSIPRED server and SwissModel sever, respectively. The predicted structures of the A. niger lipase were consistent with those of the serine hydrolase. The molecular structure models of the A. niger lipase and the A. niger feruloyl esterase were superimposed on each other to study the structure difference, and the result suggested that the main structure difference lied in the lid structure covering the active site.
引文
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