造纸废水纸浆沉淀物宏基因组文库的构建及纤维素酶基因的克隆和鉴定
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摘要
分别采用间接提取法和直接提取法从造纸废水纸浆沉淀物中提取宏基因组DNA，使用含2％酸洗PVPP的Sephadex G200凝胶柱和电洗脱两步法进行纯化。以直接提取法获得并纯化的DNA为模板，PCR扩增该环境中细菌的16S rDNA并构建文库。随机挑取文库克隆进行测序，BlastN对比表明该环境中存在大量的未培养细菌，并具有种类的多样性，系统发育分析显示这些未培养细菌可分为螺旋体、变形杆菌、拟杆菌和厚壁菌四个群落。分别回收间接提取法和直接提取法获得的宏基因组DNA中30～50kb的片段并补平末端，以柯斯质粒pWEB∷TNC为载体构建了两个宏基因组文库PS1和PS2。以间接提取法提取的DNA构建的文库PS1含8，000个克隆，外源总DNA容量为2.43×10~8bp；以直接提取法提取的DNA构建的文库PS2含10，000个克隆，外源总DNA容量为3.53×10~8bp。
经活性筛选从文库PS1中得到一个表达内切葡聚糖酶活性的克隆；从文库PS2中得到2个表达内切葡聚糖酶活性的克隆，3个表达外切葡聚糖酶活性的克隆及2个表达β-葡萄糖苷酶活性的克隆。选择文库PS1中的活性克隆PS1-C64，文库PS2中表达不同活性克隆中活性最强的PS2-C2、PS2-M6和PS2-B1作为进一步研究的对象。经亚克隆、测序和表达鉴定了四个新的纤维素酶基因umcel5F、umcel5L、umcel5M和umbgl3D。
umcel5F全长1，110bp，编码一个含369个氨基酸残基的内切葡聚糖酶，该酶与一个来自嗜酸细菌的内切葡聚糖酶(GenBank索引号：AF40690)的同源性最高，具有38％的一致性和54％的相似性。umcel5L全长1，521bp，编码一个含506个氨基酸残基的内切葡聚糖酶，该酶与大豆慢生根瘤菌的一个内切葡聚糖酶(GenBank索引号：BAC48632)的同源性最高，具有43％的一致性和59％的相似性。umcel5M全长1，053bp，编码一个含350个氨基酸残基的纤维糊精酶，该酶与产琥珀酸丝状杆菌的一个纤维糊精酶(GenBank索引号：AAA50210)的同源性最高，具有48％的一致性和69％的相似性。umbgl3D全长2，382bp，编码一个含793个氨基酸残基的β-葡萄糖苷酶，该酶与海栖热袍菌的一个β-葡萄糖苷酶(GenBank索引号：AAD35119)的同源性最高，具有46％的一致性和61％的相似性。
在(?)KTA explorer 100蛋白纯化仪上使用Source 15Q阴离子交换柱对重组蛋白Umcel5F进行纯化。以羧甲基纤维素(CMC)为底物对纯化蛋白Umcel5F进行酶学特性分析，该酶的最适反应pH值为6.5，最适反应温度为55℃，Km为16.44mg／ml，Vmax为433.7U／mg蛋白，该酶可在pH 5.5-10.0之间及温度低于50℃保持活力稳定。以各种寡糖作为底物来分析Umcel5F的底物特异性，薄层层析显示Umcel5F对纤维二糖和纤维三糖没有水解作用，纤维四糖被完全水解为纤维二糖，纤维五糖被水解为纤维二糖和纤维三糖。对Umcel5F降解CMC的产物进行粘度测定，结果表明随着反应时间的推移，反应产物的粘度缓慢下降，同时伴随着还原糖量的不断上升。这说明Umcel5F是一个具有外切作用方式的内切葡聚糖酶。
这是第一次采用未培养方法对造纸废水纸浆沉淀物的细菌多样性进行分析并从中克隆新的纤维素酶基因的研究报告。
The metagenomic DNA of pulp sediments from paper mill effluent was extracted by indirect extraction method and direct extraction method respectively, the crude DNA was first purified by Sephadex G200 column containing 2% acid-washed polyvinylpolypyrrolidone (PVPP) and then by electroelution. A 16S rDNA library was prepared by cloning the amplified 16S rDNA using purified metagenomic DNA obtained by direct extraction method as template. Clones from the 16S rDNA library were randomly selected to be sequenced, BlastN showed that diverse of uncultured bacteria inhabit in this environment, and phylogenetic analysis revealed these bacteria can be classified into 4 clusters as Spirochaetes , Proteobacteria , Bacteroidetes and Firmicutes. The purified metagenomic DNA fragment between 30-50 kb was recovered and end-repaired, one metagenomic library PS1 harbored the DNA derived from indirect extraction method and another metagenomic library PS2 acquiring the DNA through direct extraction method were constructed with pWEB::TNC cosmid vector. The library PS1 contained 8,000 clones and the capacity of foreign DNA was 2.43×10~8 bp, the library PS2 contained 10,000 clones and the capacity of which was 3.53×10~8 bp.Functional screening of the library PS 1 resulted in isolation of one clone expressing endoglucanase activity, moreover, the same screening strategy applied to library PS2 brought on two independent clones expressing endoglucanase activity, three independent clones expressing exoglucanase activity and two independent clones expressingβ-glucosidase activity. The positive clone PS1-C64 from the library PS1 and one clone expressing strongest enzyme activity from each activity category in the library PS2 designated as PS2-C2, PS2-M6, PS2-B1 were chosen to be further analyzed. Four novel cellulase genes named umcel5F, umcel5L, umcel5M and umbgl3D were identified by subcloning, sequencing and expressing in Escherichia. coli.
The ORF of umcel5F was 1,110 bp, which encoded an endoglucanase with 369 amino acid residues. The Umcel5F shares highest homology with an endoglucanase (Accession number: AF40690) from Acidobacterium bacterium Ellin345 at 38% identity and 54% similarity. The ORF of umcel5L was 1,521 bp, which encoded an endoglucanase with 506 amino acid residues. The Umcel5L is most related to an endoglucanase (Accession number: BAC48632) from Bradyrhizobiumjaponicum at 43% identity and 59% similarity. The ORF of umcel5M was 1,053 bp, which encoded a cellodextrinase with 350 amino acid residues. The Umcel5M is most similar to a cellodextrinase (Accession number: AAA50210) from Fibrobacter succinogenes at 48% identity and 69% similarity. The ORF of umbgl3D was 2,382 bp, which encoded a putativeβ-glucosidase with 793 amino acid residues. The Umbgl3D shares highest homology with aβ-glucosidase (Accession number: AAD35119) from Thermotoga maritima at 46% identity and 61% similarity.
The recombinant enzyme Umcel5F was purified in (?)KTA explorer 100 HPLC with Source 15Q anion-exchange column. The purified Umcel5F was characterized with carboxymethyl cellulose sodium salt (CMC) as template. The optimal pH and temperature of the enzyme against CMC were pH6.5 and 55℃, respectively. Km and Vmax of the recombinant enzyme toward CMC were 16.44 mg/ml and 433.7 U/mg protein. The enzyme was stable at temperature below 50℃and pH 5.5～10.0. To investigate the activities of Umcel5F with various cello-oligosaccharides, the hydrolysis products obtained from the substrates were qualitatively analyzed by TLC. Cellobiose and cellotriose were not hydrolysed by Umcel5F, cellotetraose was mostly hydrolysed to cellobiose, and degradation of cellopentaose produced both cellobiose and cellotriose. The viscosity assay showed that hydrolysis of CMC was accompanied by a slow decrease in the specific viscosity of the reaction solution and a gradual increase in the level of reducing sugar. These results indicated that the recombinant endoglucanase Umcel5F is a processive endoglucanase.
This is the first report on the bacterial diversity of pulp sediments from paper mill effluent and cloning novel cellulase genes from the bacteria by culture-independent method.
经活性筛选从文库PS1中得到一个表达内切葡聚糖酶活性的克隆；从文库PS2中得到2个表达内切葡聚糖酶活性的克隆，3个表达外切葡聚糖酶活性的克隆及2个表达β-葡萄糖苷酶活性的克隆。选择文库PS1中的活性克隆PS1-C64，文库PS2中表达不同活性克隆中活性最强的PS2-C2、PS2-M6和PS2-B1作为进一步研究的对象。经亚克隆、测序和表达鉴定了四个新的纤维素酶基因umcel5F、umcel5L、umcel5M和umbgl3D。
umcel5F全长1，110bp，编码一个含369个氨基酸残基的内切葡聚糖酶，该酶与一个来自嗜酸细菌的内切葡聚糖酶(GenBank索引号：AF40690)的同源性最高，具有38％的一致性和54％的相似性。umcel5L全长1，521bp，编码一个含506个氨基酸残基的内切葡聚糖酶，该酶与大豆慢生根瘤菌的一个内切葡聚糖酶(GenBank索引号：BAC48632)的同源性最高，具有43％的一致性和59％的相似性。umcel5M全长1，053bp，编码一个含350个氨基酸残基的纤维糊精酶，该酶与产琥珀酸丝状杆菌的一个纤维糊精酶(GenBank索引号：AAA50210)的同源性最高，具有48％的一致性和69％的相似性。umbgl3D全长2，382bp，编码一个含793个氨基酸残基的β-葡萄糖苷酶，该酶与海栖热袍菌的一个β-葡萄糖苷酶(GenBank索引号：AAD35119)的同源性最高，具有46％的一致性和61％的相似性。
在(?)KTA explorer 100蛋白纯化仪上使用Source 15Q阴离子交换柱对重组蛋白Umcel5F进行纯化。以羧甲基纤维素(CMC)为底物对纯化蛋白Umcel5F进行酶学特性分析，该酶的最适反应pH值为6.5，最适反应温度为55℃，Km为16.44mg／ml，Vmax为433.7U／mg蛋白，该酶可在pH 5.5-10.0之间及温度低于50℃保持活力稳定。以各种寡糖作为底物来分析Umcel5F的底物特异性，薄层层析显示Umcel5F对纤维二糖和纤维三糖没有水解作用，纤维四糖被完全水解为纤维二糖，纤维五糖被水解为纤维二糖和纤维三糖。对Umcel5F降解CMC的产物进行粘度测定，结果表明随着反应时间的推移，反应产物的粘度缓慢下降，同时伴随着还原糖量的不断上升。这说明Umcel5F是一个具有外切作用方式的内切葡聚糖酶。
这是第一次采用未培养方法对造纸废水纸浆沉淀物的细菌多样性进行分析并从中克隆新的纤维素酶基因的研究报告。
The metagenomic DNA of pulp sediments from paper mill effluent was extracted by indirect extraction method and direct extraction method respectively, the crude DNA was first purified by Sephadex G200 column containing 2% acid-washed polyvinylpolypyrrolidone (PVPP) and then by electroelution. A 16S rDNA library was prepared by cloning the amplified 16S rDNA using purified metagenomic DNA obtained by direct extraction method as template. Clones from the 16S rDNA library were randomly selected to be sequenced, BlastN showed that diverse of uncultured bacteria inhabit in this environment, and phylogenetic analysis revealed these bacteria can be classified into 4 clusters as Spirochaetes , Proteobacteria , Bacteroidetes and Firmicutes. The purified metagenomic DNA fragment between 30-50 kb was recovered and end-repaired, one metagenomic library PS1 harbored the DNA derived from indirect extraction method and another metagenomic library PS2 acquiring the DNA through direct extraction method were constructed with pWEB::TNC cosmid vector. The library PS1 contained 8,000 clones and the capacity of foreign DNA was 2.43×10~8 bp, the library PS2 contained 10,000 clones and the capacity of which was 3.53×10~8 bp.Functional screening of the library PS 1 resulted in isolation of one clone expressing endoglucanase activity, moreover, the same screening strategy applied to library PS2 brought on two independent clones expressing endoglucanase activity, three independent clones expressing exoglucanase activity and two independent clones expressingβ-glucosidase activity. The positive clone PS1-C64 from the library PS1 and one clone expressing strongest enzyme activity from each activity category in the library PS2 designated as PS2-C2, PS2-M6, PS2-B1 were chosen to be further analyzed. Four novel cellulase genes named umcel5F, umcel5L, umcel5M and umbgl3D were identified by subcloning, sequencing and expressing in Escherichia. coli.
The ORF of umcel5F was 1,110 bp, which encoded an endoglucanase with 369 amino acid residues. The Umcel5F shares highest homology with an endoglucanase (Accession number: AF40690) from Acidobacterium bacterium Ellin345 at 38% identity and 54% similarity. The ORF of umcel5L was 1,521 bp, which encoded an endoglucanase with 506 amino acid residues. The Umcel5L is most related to an endoglucanase (Accession number: BAC48632) from Bradyrhizobiumjaponicum at 43% identity and 59% similarity. The ORF of umcel5M was 1,053 bp, which encoded a cellodextrinase with 350 amino acid residues. The Umcel5M is most similar to a cellodextrinase (Accession number: AAA50210) from Fibrobacter succinogenes at 48% identity and 69% similarity. The ORF of umbgl3D was 2,382 bp, which encoded a putativeβ-glucosidase with 793 amino acid residues. The Umbgl3D shares highest homology with aβ-glucosidase (Accession number: AAD35119) from Thermotoga maritima at 46% identity and 61% similarity.
The recombinant enzyme Umcel5F was purified in (?)KTA explorer 100 HPLC with Source 15Q anion-exchange column. The purified Umcel5F was characterized with carboxymethyl cellulose sodium salt (CMC) as template. The optimal pH and temperature of the enzyme against CMC were pH6.5 and 55℃, respectively. Km and Vmax of the recombinant enzyme toward CMC were 16.44 mg/ml and 433.7 U/mg protein. The enzyme was stable at temperature below 50℃and pH 5.5～10.0. To investigate the activities of Umcel5F with various cello-oligosaccharides, the hydrolysis products obtained from the substrates were qualitatively analyzed by TLC. Cellobiose and cellotriose were not hydrolysed by Umcel5F, cellotetraose was mostly hydrolysed to cellobiose, and degradation of cellopentaose produced both cellobiose and cellotriose. The viscosity assay showed that hydrolysis of CMC was accompanied by a slow decrease in the specific viscosity of the reaction solution and a gradual increase in the level of reducing sugar. These results indicated that the recombinant endoglucanase Umcel5F is a processive endoglucanase.
This is the first report on the bacterial diversity of pulp sediments from paper mill effluent and cloning novel cellulase genes from the bacteria by culture-independent method.
引文
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