羽毛角蛋白质降解菌的分离、鉴定与角蛋白酶基因的克隆、表达
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摘要
从长期堆积羽毛废弃物的土壤及羽毛处理场的污泥中分离到能降解羽毛角蛋白质的嗜麦芽窄食单胞菌属(Stenotrophomonas sp.)、微杆菌属(Microbacterium sp.)、芽孢杆菌属(Bacillus sp.)、金黄杆菌属(Chryseobacterium sp.)4种微生物。对其中能高效降解羽毛角蛋白质的Stenotrophomonas sp.菌株进行了生理生化鉴定和系统发育树分析及产酶性能研究,并克隆与表达了角蛋白酶基因。
克隆了Stenotrophomonas sp.菌株的16S rDAN序列。测序分析表明,其16S rDAN序列全长1509bp(GenBank序列号:FJ765513)。BLAST显示该菌株与嗜麦芽窄食单胞菌(S. maltophilia)同源。以同源性为基础构建了相关属种的系统发育树,该菌株在进化关系上也与嗜麦芽窄食单胞菌聚成一族;特别是与S. maltophilia strain 1.22的同源性最高,其序列相似性达99.8%。结合形态学、生理生化特征及16S rDNA分子鉴定,将其鉴定为嗜麦芽窄食单胞菌,命名为Stenotrophomonas maltophilia YHYJ-1。
利用PCR技术克隆了编码S. maltopilia YHYJ-1的角蛋白酶基因。该基因序列全长1905bp,(G+C)含量为68.9%,编码634个氨基酸,含信号肽序列。蛋白序列比对结果显示,含PA家族蛋白保守的结构域,该序列与碱性丝氨酸蛋白酶同源。通过序列比对分析,kerD与已报道的其它来源的角蛋白酶的序列相似性都较低,在28-35%之间。其中与预测的嗜麦芽窄食单胞菌S. maltophilia R551-3的丝氨酸蛋白酶类基因序列同源性较高。
为了验证kerD基因的功能,构建了重组原核表达质粒pET-kerD,转化大肠杆菌BL21(DE3)诱导表达,并对原核表达的角蛋白酶进行了酶学性质初步分析。结果表明,该酶在大肠杆菌中获得了高效表达,在重组菌超声破碎上清液中检测到角蛋白酶活性,诱导4h酶活最高为10.5U/mL,这说明kerD具有角蛋白酶活性。酶学性质分析显示,kerD的最适反应温度为50℃,最适pH为7.8。因此确定kerD为新的角蛋白酶基因。
本研究分离到多株能降解角蛋白质微生物,为研究微生物降解羽毛角蛋白质提供了新材料。首次报道从嗜麦芽窄食单胞菌中克隆到角蛋白酶基因。本实验结果为进一步开展S.maltopilia YHYJ-1角蛋白酶的基础理论研究和应用研究奠定了良好的基础;为探讨角蛋白酶基因结构与功能的关系提供了一些有价值的数据;对从分子水平上研究羽毛降解机制提供了理论依据。
Four feather-degrading strain bacteria, isolated and screened from soil and sludge of long-time deposit of waste feather and feather processing plants, were classified as Stenotrophomonas sp, Microbacterium sp, Bacillus sp and Chryseobacterium sp. Stenotrophomonas strain showed the strongest feather-drgrading ability among the isolated microorganisms. Phylogenetic analysis, cloning and expression of keratinase gene were studied of the strain.
The 16S rDAN gene sequences for Stenotrophomonas strain was amplified and sequenced. The results showed that the complete 16S rDAN gene sequence was 1509bp. A phylogenetic tree was constructed by comparing the 16S rDAN sequence of the strain (GenBank number: FJ765513) with other relaive bacteria species in the GenBank database. In the phylogenetic tree, the studied strain, Stenotrophomonas maltophilia strain 1.22 constitute a branch with the similarity value 99.8%. According to its morphological, physiological and biochemical characteristics, and phylogenetic analysis, the microorganism was identified as Stenitrophomonas maltophilia strain YHYJ-1.
The gene kerD encoding keratinase was cloned by PCR. Sequences analysis reveals that the sequences with the length of 1905bp encoding a polypeptide of 634 amino acid residues. (G+C)% was 68.9%, and with a signal pepetide. The alignment result of kerD protein indicates that it belongs to PA superfamily with the homology to serine protease. Via blast, the deduced amino acid sequence of this keratinase showed 28-35% identities to the previously reported keratinase gene, which shared high homology with the deduced keratinase sequence from S. maltophilia R551-3. The gene kerD is a novely kind of keratianse firstly reported from S.maltophilia.
Rrecombinant plasmid(pET-kerD) was constructed and transformed into E.coli BL21 to express in order to verify the function of the gene kerD. A preliminary analysis of keratinase in E.coli BL21 showed that this keratinse was highly expressed in E.coli BL21 with the endocellular enzyme activity (10.5U/mL) induced after 4 hour, which proved this kerD had the activity of keratinase. Its optimal reaction temperature and pH were 50℃and 7.8, respectively.
Several kinds of feather-degrading microorganisms were isolated in this study,which provided experimental materials for feather degrading.It was first reported that keratinase gene was cloned from S.maltophilia. The result we got laid solid base for the further research on keratinase from S. maltophilia YHYJ-1 and provided valuable figures for research on the relationship between structure and function of keratinase and helped to clarify the catalytic mechanisim of keratinase from the molecular level.
克隆了Stenotrophomonas sp.菌株的16S rDAN序列。测序分析表明,其16S rDAN序列全长1509bp(GenBank序列号:FJ765513)。BLAST显示该菌株与嗜麦芽窄食单胞菌(S. maltophilia)同源。以同源性为基础构建了相关属种的系统发育树,该菌株在进化关系上也与嗜麦芽窄食单胞菌聚成一族;特别是与S. maltophilia strain 1.22的同源性最高,其序列相似性达99.8%。结合形态学、生理生化特征及16S rDNA分子鉴定,将其鉴定为嗜麦芽窄食单胞菌,命名为Stenotrophomonas maltophilia YHYJ-1。
利用PCR技术克隆了编码S. maltopilia YHYJ-1的角蛋白酶基因。该基因序列全长1905bp,(G+C)含量为68.9%,编码634个氨基酸,含信号肽序列。蛋白序列比对结果显示,含PA家族蛋白保守的结构域,该序列与碱性丝氨酸蛋白酶同源。通过序列比对分析,kerD与已报道的其它来源的角蛋白酶的序列相似性都较低,在28-35%之间。其中与预测的嗜麦芽窄食单胞菌S. maltophilia R551-3的丝氨酸蛋白酶类基因序列同源性较高。
为了验证kerD基因的功能,构建了重组原核表达质粒pET-kerD,转化大肠杆菌BL21(DE3)诱导表达,并对原核表达的角蛋白酶进行了酶学性质初步分析。结果表明,该酶在大肠杆菌中获得了高效表达,在重组菌超声破碎上清液中检测到角蛋白酶活性,诱导4h酶活最高为10.5U/mL,这说明kerD具有角蛋白酶活性。酶学性质分析显示,kerD的最适反应温度为50℃,最适pH为7.8。因此确定kerD为新的角蛋白酶基因。
本研究分离到多株能降解角蛋白质微生物,为研究微生物降解羽毛角蛋白质提供了新材料。首次报道从嗜麦芽窄食单胞菌中克隆到角蛋白酶基因。本实验结果为进一步开展S.maltopilia YHYJ-1角蛋白酶的基础理论研究和应用研究奠定了良好的基础;为探讨角蛋白酶基因结构与功能的关系提供了一些有价值的数据;对从分子水平上研究羽毛降解机制提供了理论依据。
Four feather-degrading strain bacteria, isolated and screened from soil and sludge of long-time deposit of waste feather and feather processing plants, were classified as Stenotrophomonas sp, Microbacterium sp, Bacillus sp and Chryseobacterium sp. Stenotrophomonas strain showed the strongest feather-drgrading ability among the isolated microorganisms. Phylogenetic analysis, cloning and expression of keratinase gene were studied of the strain.
The 16S rDAN gene sequences for Stenotrophomonas strain was amplified and sequenced. The results showed that the complete 16S rDAN gene sequence was 1509bp. A phylogenetic tree was constructed by comparing the 16S rDAN sequence of the strain (GenBank number: FJ765513) with other relaive bacteria species in the GenBank database. In the phylogenetic tree, the studied strain, Stenotrophomonas maltophilia strain 1.22 constitute a branch with the similarity value 99.8%. According to its morphological, physiological and biochemical characteristics, and phylogenetic analysis, the microorganism was identified as Stenitrophomonas maltophilia strain YHYJ-1.
The gene kerD encoding keratinase was cloned by PCR. Sequences analysis reveals that the sequences with the length of 1905bp encoding a polypeptide of 634 amino acid residues. (G+C)% was 68.9%, and with a signal pepetide. The alignment result of kerD protein indicates that it belongs to PA superfamily with the homology to serine protease. Via blast, the deduced amino acid sequence of this keratinase showed 28-35% identities to the previously reported keratinase gene, which shared high homology with the deduced keratinase sequence from S. maltophilia R551-3. The gene kerD is a novely kind of keratianse firstly reported from S.maltophilia.
Rrecombinant plasmid(pET-kerD) was constructed and transformed into E.coli BL21 to express in order to verify the function of the gene kerD. A preliminary analysis of keratinase in E.coli BL21 showed that this keratinse was highly expressed in E.coli BL21 with the endocellular enzyme activity (10.5U/mL) induced after 4 hour, which proved this kerD had the activity of keratinase. Its optimal reaction temperature and pH were 50℃and 7.8, respectively.
Several kinds of feather-degrading microorganisms were isolated in this study,which provided experimental materials for feather degrading.It was first reported that keratinase gene was cloned from S.maltophilia. The result we got laid solid base for the further research on keratinase from S. maltophilia YHYJ-1 and provided valuable figures for research on the relationship between structure and function of keratinase and helped to clarify the catalytic mechanisim of keratinase from the molecular level.
引文
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