中性纤维素内切酶的发掘、表征及其应用于生物石洗的基础研究
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摘要
中性纤维素内切酶(中性EG)在牛仔布生物石洗中具有很高的应用价值,但国内中性EG不具有自主知识产权,因此,发掘全新中性EG,对于打破国外中性EG的垄断地位具有重要意义。完整中性EG和截短中性EG在牛仔布生物石洗上有不同表现,这与纤维素酶的纤维素结合区相关,但深层次原因还尚未发现。本文主要研究中性EG的生产菌株筛选、发掘、异源表达以及酶学性质表征,初步研究中性EG在牛仔布生物石洗上的应用,具体研究内容和结论如下。
第一部分,Bacillus subtilis LH中性EG的基因发掘、异源高效表达、表征及其应用于生物石洗的基础研究。
1)从鲁华生物技术研究所菌库中,筛选到产中性EG的菌株B. subtilis LH,获得B. subtilis LH的中性EG基因。Pichia pastoris GS115和Escherichia.coli Rosetta (DE3)分别表达了成熟中性EG(分别命名为MBSEG-1和MBSEG-2),结果表明MBSEG-2表达水平远高于MBSEG-1。采用最优产酶条件,未浓缩MBSEG-2的EG酶活可达25686U/mL,是目前所有报道的最高水平。
2)MBSEG-1和MBSEG-2的酶学性质相似,最适温度均为65℃,最适pH值均为6.86,既有内切酶活性又有外切酶活性,N-糖基化的有无引起了酶在热稳定性、木瓜蛋白酶有限水解上的差异。
3)基于截短MBSEG-2以及完整MBSEG-1和MBSEG-2水洗牛仔布实验,并结合蛋白3D模型分析,发现具有高比例的表面疏水氨基酸和芳香氨基酸的EG有助于提高牛仔布的除毛率和脱色率,而与EG(但必须有催化域)的完整性无关。
第二部分,真菌Petriella setifera LH的鉴定,中性EG的表征及其应用于生物石洗的基础研究。
从鲁华生物技术研究所的保存土壤中,筛选到一株产中性EG的真菌,基于延伸因子基因(EF-la)部分序列的系统发育树的分析,并结合真菌形态学特征,鉴定该真菌为Pe. Setifera,命名为Pe. setifera LH。Pe. setifera LH的中性EG于40℃时可保持80%以上的相对酶活,最适pH为6-7,水洗牛仔布的效果与商业化中性EG相当,揭示了它在牛仔布生物石洗上具有潜在的应用价值。
第三部分,真菌Phaeosphaeria sp. LH21两个中性EG的基因发掘、序列分析及酶学性质表征。
1)从极端环境的深海淤泥中,筛选到一株产中性EG的真菌,基于EF-1α部分序列和18S rDNA部分序列的比对分析,鉴定该真菌为Phaeosphaeria sp.,命名为Phaeosphaeria sp. LH21。
2)采用基因组走读技术和RT-PCR方法,首次克隆了Phaeosphaeria sp. LH的两个全新EG(GH45EG-1和GH45EG-2)基因。GH45EG-1和GH45EG-2全长分别为235和233个氨基酸。通过NCBI BLAST对氨基酸序列进行比对分析,表明GH45EG-1和GH45EG-2属于糖苷水解酶第45家族,仅有催化域无纤维素结合区,其氨基酸序列和已知EG氨基酸序列分别拥有71%和63%的最大一致性。
3)基于两个成熟EG (mGH45EG-1和mGH45EG-2)和Humicola insolens EG V氨基酸序列之间的比对分析,再根据蛋白3D结构模型的分析,推测nGH45EG-1其中个活性中心位点为Asp16,推测mGH45EG-2活性中心位点为Asp122和Asp11,推测它们催化机制为反转机制。
4)mGH45EG-1和mGH45EG-2分别于P. pastohs GS115中表达。重组酶mGH45EG-1和mGH45EG-2的底物谱证实它们属于内切酶类型,对羧甲基纤维素钠具有最高降解活性,对结晶纤维素合滤纸降解的相对酶活仅为0.1-0.3%。10mM Ca2+和Mg2+提高了8-27%重组酶mGH45EG-1和nGH45EG-2的相对酶活,多数金属离子对它们有抑制作用。重组酶mGH45EG-1和mGH45EG-2最适pH值均为8,在pH5-10时相对酶活均在75%以上,于50℃及pH3-10保温1h历相对残余酶活均在90%以上,最适温度分别为65℃和60℃。
Neutral (3-1,4-endoglucanase (EG) has a high application value in biostoning of denim, but domestic neutral EG doesn't have independent intellectual property rights. As a result, it is significantly meaningful to dig new neutral EG in order to break the monopoly of foreign neutral EG. Complete neutral EGs and truncated neutral EGs have different performances in biostoning of denim, which is concerned with cellulose-binding domain of cellulase, but the deep-seated reason has not been found. The paper focuses on digging, heterologous expression and characterization of neutral EG, and fundamentally explores the application of denim in biostoning.
In the first section, the study about gene digging, efficient heterologous expression and characterization of a neutral EG from Bacillus subtilis LH, and the fundamental study on application of it in biostoning of denim were carried out.
1) B. subtilis LH producing the neutral EG was screened from the strain bank of Newworld Institute of Biotechnology, and the neutral EG gene from B. subtilis LH was amplified. The mature EG was expressed in Escherichia.coli Rosetta (DE3) and Pichia pastoris GS115(named as MBSEG-1and MBSEG-2, respectively), respectively, and the result showed that the expression level of MBSEG-2is much higher than that of MBSEG-1. By using the optimized condition, the EG activity of MBSEG-2without concentration is25686U/mL, which is the highest expression level having been reported at present.
2) MBSEG-1and MBSEG-2have some similar properties such as the optimum temperature of65℃and the optimum pH of6.86, and possess β-1,4-endoglucanase and β-1,4-exoglucanase activities, but N-glycosylation produces the differences in thermal stability and the limited papain hydrolysis of the two enzymes.
3) On the basis of the experiment about treating denim with the truncated MBSEG-2and the complete MBSEG-1and MBSEG-2, and according to the analysis of their3D models, it was found out that neutral EGs with the higher percentage of surface hydrophobic amino acids and aromatic amino acids regardless of whether neutral EGs (must have catalytic domain) help denim to enhance the indigo dye removal and the weight loss.
In the second section, identification of Petriella setifera LH, characterization of neutral EG, and the fundamental study on application of neutral EG in biostoning of denim were carried out.
The fungus producing the neutral EG was screened from the preserved soil of Newworld Institute of Biotechnology. The fungus was identified as Pe. Setifera and named as Pe. setifera LH on the basis of the analysis of the phylogenetic tree of the partial elongation factor-1a gene (EF-la) combined with its morphological characteristics. The EG from Pe. setifera LH has an above80%relative activity at40℃and the optimum pH of6-7. Compared with denim treatment by a commercial neutral cellulase, the EG exhibited a similar fabric weight loss and indigo dye removal, revealing that the enzyme has a potential application in denim biostoning.
In the third section, gene digging, sequence analysis and characterization of two HGs from Phaeosphaeria sp LH21were carried out.
1) The fungus was screened from the extreme environment deep-sea mud, identified as Phaeosphaeria sp. and named as Phueosphaeria sp. LH21on the basis of the alignment analysis of the partial sequences of EF-1a and18S rDNA.
2) The two new EG (GH45EG-1and EG45EG-2) genes from Phaeosphaeria sp. LH21were first cloned by genome walking and RT-PCR. GH45EG-1and GH45EG-2consists of235and233amino acids, respectively. The alignment analysis of the amino acid sequences was performed by NCBI BLAST, indicating that GH45EG-1and GH45EG-2belong to glycoside hydrolase family45, possess catalytic domain without cellulose-binding domain and share the maximum amino acid sequence identities of71%and63%with the known EGs. respectively.
3) On the basis of the amino acid sequences from the two mature EGs (mGH45EG-1and mGH45EG-2) and Humicola insolens EG V, and according to their3D models, it was speculated that one of the active central sites of mGH45EG-1could be Asp16, that the active central sites of mGH45EG-2could be Asp122and Aspl1and that their catalytic mechanisms could be the inversion mechanism.
4) mGH45EG-1and mGH45EG-2were expressed in P. pastoris GS115, respectively. The substrate spectra of the recombinant enzymes mGH45EG-1and mGH45EG-2confirmed that they belong to endonuclease. They have the highest activities on CMC, and the relative activities on crystalline cellulose and filter paper are0.1-0.3%.10mM Ca2+and Mg2+increase8-27%of relative activities of the recombinant enzymes mGH45EG-1and mGH45EG-2, and most metal ions inactivate them. The recombinant enzymes mGH45EG-1and mGH45EG-2have the optimum pH of8, above75%relative activities at pH (?)10, above90%relative residual activities after incubating at50℃and pH3-10for1h, and the optimum temperatures of65℃and60℃, respectively.
第一部分,Bacillus subtilis LH中性EG的基因发掘、异源高效表达、表征及其应用于生物石洗的基础研究。
1)从鲁华生物技术研究所菌库中,筛选到产中性EG的菌株B. subtilis LH,获得B. subtilis LH的中性EG基因。Pichia pastoris GS115和Escherichia.coli Rosetta (DE3)分别表达了成熟中性EG(分别命名为MBSEG-1和MBSEG-2),结果表明MBSEG-2表达水平远高于MBSEG-1。采用最优产酶条件,未浓缩MBSEG-2的EG酶活可达25686U/mL,是目前所有报道的最高水平。
2)MBSEG-1和MBSEG-2的酶学性质相似,最适温度均为65℃,最适pH值均为6.86,既有内切酶活性又有外切酶活性,N-糖基化的有无引起了酶在热稳定性、木瓜蛋白酶有限水解上的差异。
3)基于截短MBSEG-2以及完整MBSEG-1和MBSEG-2水洗牛仔布实验,并结合蛋白3D模型分析,发现具有高比例的表面疏水氨基酸和芳香氨基酸的EG有助于提高牛仔布的除毛率和脱色率,而与EG(但必须有催化域)的完整性无关。
第二部分,真菌Petriella setifera LH的鉴定,中性EG的表征及其应用于生物石洗的基础研究。
从鲁华生物技术研究所的保存土壤中,筛选到一株产中性EG的真菌,基于延伸因子基因(EF-la)部分序列的系统发育树的分析,并结合真菌形态学特征,鉴定该真菌为Pe. Setifera,命名为Pe. setifera LH。Pe. setifera LH的中性EG于40℃时可保持80%以上的相对酶活,最适pH为6-7,水洗牛仔布的效果与商业化中性EG相当,揭示了它在牛仔布生物石洗上具有潜在的应用价值。
第三部分,真菌Phaeosphaeria sp. LH21两个中性EG的基因发掘、序列分析及酶学性质表征。
1)从极端环境的深海淤泥中,筛选到一株产中性EG的真菌,基于EF-1α部分序列和18S rDNA部分序列的比对分析,鉴定该真菌为Phaeosphaeria sp.,命名为Phaeosphaeria sp. LH21。
2)采用基因组走读技术和RT-PCR方法,首次克隆了Phaeosphaeria sp. LH的两个全新EG(GH45EG-1和GH45EG-2)基因。GH45EG-1和GH45EG-2全长分别为235和233个氨基酸。通过NCBI BLAST对氨基酸序列进行比对分析,表明GH45EG-1和GH45EG-2属于糖苷水解酶第45家族,仅有催化域无纤维素结合区,其氨基酸序列和已知EG氨基酸序列分别拥有71%和63%的最大一致性。
3)基于两个成熟EG (mGH45EG-1和mGH45EG-2)和Humicola insolens EG V氨基酸序列之间的比对分析,再根据蛋白3D结构模型的分析,推测nGH45EG-1其中个活性中心位点为Asp16,推测mGH45EG-2活性中心位点为Asp122和Asp11,推测它们催化机制为反转机制。
4)mGH45EG-1和mGH45EG-2分别于P. pastohs GS115中表达。重组酶mGH45EG-1和mGH45EG-2的底物谱证实它们属于内切酶类型,对羧甲基纤维素钠具有最高降解活性,对结晶纤维素合滤纸降解的相对酶活仅为0.1-0.3%。10mM Ca2+和Mg2+提高了8-27%重组酶mGH45EG-1和nGH45EG-2的相对酶活,多数金属离子对它们有抑制作用。重组酶mGH45EG-1和mGH45EG-2最适pH值均为8,在pH5-10时相对酶活均在75%以上,于50℃及pH3-10保温1h历相对残余酶活均在90%以上,最适温度分别为65℃和60℃。
Neutral (3-1,4-endoglucanase (EG) has a high application value in biostoning of denim, but domestic neutral EG doesn't have independent intellectual property rights. As a result, it is significantly meaningful to dig new neutral EG in order to break the monopoly of foreign neutral EG. Complete neutral EGs and truncated neutral EGs have different performances in biostoning of denim, which is concerned with cellulose-binding domain of cellulase, but the deep-seated reason has not been found. The paper focuses on digging, heterologous expression and characterization of neutral EG, and fundamentally explores the application of denim in biostoning.
In the first section, the study about gene digging, efficient heterologous expression and characterization of a neutral EG from Bacillus subtilis LH, and the fundamental study on application of it in biostoning of denim were carried out.
1) B. subtilis LH producing the neutral EG was screened from the strain bank of Newworld Institute of Biotechnology, and the neutral EG gene from B. subtilis LH was amplified. The mature EG was expressed in Escherichia.coli Rosetta (DE3) and Pichia pastoris GS115(named as MBSEG-1and MBSEG-2, respectively), respectively, and the result showed that the expression level of MBSEG-2is much higher than that of MBSEG-1. By using the optimized condition, the EG activity of MBSEG-2without concentration is25686U/mL, which is the highest expression level having been reported at present.
2) MBSEG-1and MBSEG-2have some similar properties such as the optimum temperature of65℃and the optimum pH of6.86, and possess β-1,4-endoglucanase and β-1,4-exoglucanase activities, but N-glycosylation produces the differences in thermal stability and the limited papain hydrolysis of the two enzymes.
3) On the basis of the experiment about treating denim with the truncated MBSEG-2and the complete MBSEG-1and MBSEG-2, and according to the analysis of their3D models, it was found out that neutral EGs with the higher percentage of surface hydrophobic amino acids and aromatic amino acids regardless of whether neutral EGs (must have catalytic domain) help denim to enhance the indigo dye removal and the weight loss.
In the second section, identification of Petriella setifera LH, characterization of neutral EG, and the fundamental study on application of neutral EG in biostoning of denim were carried out.
The fungus producing the neutral EG was screened from the preserved soil of Newworld Institute of Biotechnology. The fungus was identified as Pe. Setifera and named as Pe. setifera LH on the basis of the analysis of the phylogenetic tree of the partial elongation factor-1a gene (EF-la) combined with its morphological characteristics. The EG from Pe. setifera LH has an above80%relative activity at40℃and the optimum pH of6-7. Compared with denim treatment by a commercial neutral cellulase, the EG exhibited a similar fabric weight loss and indigo dye removal, revealing that the enzyme has a potential application in denim biostoning.
In the third section, gene digging, sequence analysis and characterization of two HGs from Phaeosphaeria sp LH21were carried out.
1) The fungus was screened from the extreme environment deep-sea mud, identified as Phaeosphaeria sp. and named as Phueosphaeria sp. LH21on the basis of the alignment analysis of the partial sequences of EF-1a and18S rDNA.
2) The two new EG (GH45EG-1and EG45EG-2) genes from Phaeosphaeria sp. LH21were first cloned by genome walking and RT-PCR. GH45EG-1and GH45EG-2consists of235and233amino acids, respectively. The alignment analysis of the amino acid sequences was performed by NCBI BLAST, indicating that GH45EG-1and GH45EG-2belong to glycoside hydrolase family45, possess catalytic domain without cellulose-binding domain and share the maximum amino acid sequence identities of71%and63%with the known EGs. respectively.
3) On the basis of the amino acid sequences from the two mature EGs (mGH45EG-1and mGH45EG-2) and Humicola insolens EG V, and according to their3D models, it was speculated that one of the active central sites of mGH45EG-1could be Asp16, that the active central sites of mGH45EG-2could be Asp122and Aspl1and that their catalytic mechanisms could be the inversion mechanism.
4) mGH45EG-1and mGH45EG-2were expressed in P. pastoris GS115, respectively. The substrate spectra of the recombinant enzymes mGH45EG-1and mGH45EG-2confirmed that they belong to endonuclease. They have the highest activities on CMC, and the relative activities on crystalline cellulose and filter paper are0.1-0.3%.10mM Ca2+and Mg2+increase8-27%of relative activities of the recombinant enzymes mGH45EG-1and mGH45EG-2, and most metal ions inactivate them. The recombinant enzymes mGH45EG-1and mGH45EG-2have the optimum pH of8, above75%relative activities at pH (?)10, above90%relative residual activities after incubating at50℃and pH3-10for1h, and the optimum temperatures of65℃and60℃, respectively.
引文
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