海藻酸裂解酶和海藻糖合成酶基因工程研究
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摘要
随着生物技术的迅速发展,使得生物催化剂成为获得许多活性物质的有效手段。通过生物催化剂获得的寡糖和多糖降解物,被证明具有多种生物活性。在食品工业中,寡糖和多糖可以作为添加剂,提高食品的营养价值和保健活性;在饲料工业中,饲用酶制剂和寡糖的应用是生物技术在饲料工业中应用最广、而且最有前途的一个方面。它们的应用可以改善动物胃肠道环境,提高抗病力,提高家畜生产力,降低饲养成本,并且为开发新的饲料资源提供了可能。目前大部分的工业用酶均来源于微生物,其中尤以细菌最为丰富。因此,对细菌来源酶的性质特点进行研究,并筛选发现新的酶蛋白是现在各种酶制剂研究的重要方向。
本研究针对海藻多糖降解酶和海藻糖生成酶,这两类能够产生活性寡糖物质的细菌来源的酶进行了基因工程研究。通过同源克隆,分别从褐球固氮菌(Azotobacter chroococcum)和铜绿假单胞菌(Pseudomonas aeruginosa)中扩增了海藻酸裂解酶成熟肽编码序列,并在毕赤酵母中进行了表达和酶学性质分析。此外,通过对细菌来源的海藻糖合成酶蛋白的保守序列分析,设计了简并引物,从土壤中筛选并克隆到了肠杆菌科微生物(Enterobacter hormaechei)的海藻糖合成酶基因片段,利用TAIL-PCR技术,得到了完整的编码序列,将其在大肠杆菌中进行了表达,得到了具有生物学活性的重组酶。
克隆到的褐球固氮菌海藻酸裂解酶成熟蛋白编码序列AcalgL为1056kb,将其插入改造过的表达载体pPIC9K-6HISEn5’中,重组质粒线性化后用聚乙二醇(PEG)法导入毕赤酵母菌株GS115中,获得高效分泌表达海藻酸裂解酶的毕赤酵母工程菌株。用甲醇诱导培养基进行摇瓶发酵,表达得到40kDa的目的蛋白,酶比活力为2968U/mg。经测定,该重组酶的最适反应pH为8.5,最适反应温度为40°C,并且在20～55°C,pH5～11具有较高的反应活性和稳定性。另外,10mM的Cu~(2+)、Fe~(2+)、Co~(2+)、Mn~(2+)和Ca~(2+)对酶有不同程度的抑制作用。
克隆到了1023bp的铜绿假单胞菌海藻酸裂解酶基因PaalgL的成熟蛋白编码序列。通过原生质体法导入毕赤酵母菌株GS115中表达。甲醇诱导表达产物分子量约为39kDa的目的蛋白。纯化酶比活力为2440U/mg。经测定,该重组酶的最适反应pH为8.5,最适反应温度为40°C,并且在20～45°C,pH3～12都具有较高的反应活性,并且稳定性较好。50mM的Co~(2+)和Ca~(2+)对酶活力均有显著的促进作用,Zn~(2+)、Cu~(2+)和Fe~(2+)在不同浓度下对酶活力均有不同程度的抑制作用。在重组酶的辅助作用下,铜绿假单胞菌胞外海藻酸盐保护层被破坏,从而使抗生素对致病铜绿假单胞菌的抑菌效果明显提高。
本试验根据蛋白序列保守区,设计了一系列简并引物,用于海藻糖合成酶产生菌和基因的筛选和克隆。使用简并PCR从土壤富集的菌株中筛选到了一些海藻糖合成酶产酶菌。其中霍氏肠杆菌海藻糖合成酶为新基因,利用TAIL-PCR获得了基因全长序列,Genbank登录号:FJ215664。将该基因连入大肠杆菌pET-30a(+)表达载体中,构建成pET-30a-EhtreS,转化表达宿主菌后,诱导表达得到了65kDa的重组EhTreS蛋白。纯化的重组TreS最适反应条件为37°C、pH6,转化率为40%以上。Hg~(2+)、Zn~(2+)、Cu~(2+)和SDS对酶活力有不同程度的抑制作用,Mn~(2+)可以将酶活力提高10%。
在本研究中首次报道了在毕赤酵母中表达海藻酸裂解酶及其酶学性质研究。酶比活力均高于国内外其它报道,并且发现了这两个重组酶酸性pH稳定性较高,这些特点有利于该酶的应用。另外,本研究首次提出了海藻糖合成酶的保守序列,这对新基因的克隆具有较强的指导意义,并且通过对霍氏肠杆菌海藻糖合成酶基因的表达与分析,证明了这种直接利用PCR进行产酶菌的筛选方法的有效性,同时也为海藻糖的生物转化增添了新的工具酶。
With the development of genetic engineering, recombinant biocatalyst becomes one of the powerful pathways for obtaining substances. It has been confirmed that a lot of oligosaccharides and digested polysaccharides had some biological functions which are interesting in industrial and agricultural applications. In food industry, some oligosaccharides and polysaccharides have been used as additives to improve nutritional and healthy value. In feed industry, polysaccharide-hydrolytic enzymes and oligosaccharides are widely used as dietary additives. Feed enzymes could improve animal performance and feed conversion efficiency and afford opportunities for new feed resources discovery. Presently, the majority of functional enzymes come from microorganisms, especially bacteria, thus we focus on the genetic engineering study of alginate lyases and trehalose synthase.
In this thesis, two sorts of enzymes, alginate lyase with hydrolytic activity with algae polysaccharides as substrate and trehalose synthase with activity to convert maltose to trehalose, were studied. The mature alginate lyase coding sequences from Azotobacter chroococcum and Pseudomonas aeruginosa were cloned, expressed and the enzymes were characterized. In addition, the six trehalose synthase (TreS) producing strains were screened and isolated from a soil specimen obtained from the Tibetan plateau by degenerate PCR. A novel treS gene from Enterobacter hormaechei was amplified and expressed. These main results are formulated as follows:
The sequence encoding the mature peptide of alginate lyase of Azotobacter chroococcum, called AcalgL, was cloned. This 1,056bp sequence was inserted into the modified Pichia pastoris vector pPIC9K-6HISEn5’and introduced into the host Pichia pastoris GS115 by PEG method. The purified recombinant AcAlgL had a specific activity of 2,968U/mg and a Mr of 41kDa. The recombinant AcAlgL displayed a maximum activity at 40°C and pH8.5. It was stable over the range of pH5～11. The activity of recombinant AcAlgL decreased by over 50% in presence of 10mM Cu~(2+) and Fe~(2+), furthermore, 10mM Co~(2+), Mn~(2+), Ca~(2+) and 1mM Fe~(2+) decreased it by 20%. The other cations including K+, Na+ and Zn~(2+) did not significantly affect its enzymatic activity.
The mature alginate lyase coding sequences of P. aeruginosa, 1,023bp, was amplified and inserted into the modified pPIC9K expression vector. The positive construct was transformed to Pichia pastoris GS115 cells by spheroplasting method. The purified recombinant alginate lyase showed a Mr of 40kDa and a specific activity of 2,440U/mg at an optimal temperature 40°C and pH8.5, respectively. The recombinant PaAlgL was stable below 45°C at a wide range of pH3～12. The recombinant PaAlgL was sensitive to some metal cations negatively including Zn~(2+), Cu~(2+) and Fe~(2+), but Co~(2+) and Ca~(2+) promoted it dramatically. The antimicrobial activity of ampicilin and kanamycin agaist P. aeruginosa was increased by the help of PaAlgL.
The gene of Enterobacter hormaechei TreS, containing a 1,626bp ORF encoding 541 amino acids, was cloned. The nucleotide sequence had been deposited in Genbank under No. FJ215664. The recombinant EhTreS had a Mr of 65kDa and a specific activity of 18.5U/mg. The EhTreS displayed a converting rate of 40% at the optimum condition of pH6 and 40°C. Hg~(2+), Zn~(2+), Cu~(2+)and SDS inhibited the enzyme activity at different levels whereas Mn~(2+) shows an enhancing effect by 10%.
This is the first report for expression of alginante lyases in Pichia pastoris system. It was also provided some detailed characters of Azotobacter chroococcum and Pseudomonas aeruginosa alginate lyases. Their activities are much higher than any other reports. In the research on TreS, the degenerate primers based on the conserved domains of trehalose synthases were proved to be effective for new gene screening. This result provides a prospective biochemical method for the trehalose production.
本研究针对海藻多糖降解酶和海藻糖生成酶,这两类能够产生活性寡糖物质的细菌来源的酶进行了基因工程研究。通过同源克隆,分别从褐球固氮菌(Azotobacter chroococcum)和铜绿假单胞菌(Pseudomonas aeruginosa)中扩增了海藻酸裂解酶成熟肽编码序列,并在毕赤酵母中进行了表达和酶学性质分析。此外,通过对细菌来源的海藻糖合成酶蛋白的保守序列分析,设计了简并引物,从土壤中筛选并克隆到了肠杆菌科微生物(Enterobacter hormaechei)的海藻糖合成酶基因片段,利用TAIL-PCR技术,得到了完整的编码序列,将其在大肠杆菌中进行了表达,得到了具有生物学活性的重组酶。
克隆到的褐球固氮菌海藻酸裂解酶成熟蛋白编码序列AcalgL为1056kb,将其插入改造过的表达载体pPIC9K-6HISEn5’中,重组质粒线性化后用聚乙二醇(PEG)法导入毕赤酵母菌株GS115中,获得高效分泌表达海藻酸裂解酶的毕赤酵母工程菌株。用甲醇诱导培养基进行摇瓶发酵,表达得到40kDa的目的蛋白,酶比活力为2968U/mg。经测定,该重组酶的最适反应pH为8.5,最适反应温度为40°C,并且在20～55°C,pH5～11具有较高的反应活性和稳定性。另外,10mM的Cu~(2+)、Fe~(2+)、Co~(2+)、Mn~(2+)和Ca~(2+)对酶有不同程度的抑制作用。
克隆到了1023bp的铜绿假单胞菌海藻酸裂解酶基因PaalgL的成熟蛋白编码序列。通过原生质体法导入毕赤酵母菌株GS115中表达。甲醇诱导表达产物分子量约为39kDa的目的蛋白。纯化酶比活力为2440U/mg。经测定,该重组酶的最适反应pH为8.5,最适反应温度为40°C,并且在20～45°C,pH3～12都具有较高的反应活性,并且稳定性较好。50mM的Co~(2+)和Ca~(2+)对酶活力均有显著的促进作用,Zn~(2+)、Cu~(2+)和Fe~(2+)在不同浓度下对酶活力均有不同程度的抑制作用。在重组酶的辅助作用下,铜绿假单胞菌胞外海藻酸盐保护层被破坏,从而使抗生素对致病铜绿假单胞菌的抑菌效果明显提高。
本试验根据蛋白序列保守区,设计了一系列简并引物,用于海藻糖合成酶产生菌和基因的筛选和克隆。使用简并PCR从土壤富集的菌株中筛选到了一些海藻糖合成酶产酶菌。其中霍氏肠杆菌海藻糖合成酶为新基因,利用TAIL-PCR获得了基因全长序列,Genbank登录号:FJ215664。将该基因连入大肠杆菌pET-30a(+)表达载体中,构建成pET-30a-EhtreS,转化表达宿主菌后,诱导表达得到了65kDa的重组EhTreS蛋白。纯化的重组TreS最适反应条件为37°C、pH6,转化率为40%以上。Hg~(2+)、Zn~(2+)、Cu~(2+)和SDS对酶活力有不同程度的抑制作用,Mn~(2+)可以将酶活力提高10%。
在本研究中首次报道了在毕赤酵母中表达海藻酸裂解酶及其酶学性质研究。酶比活力均高于国内外其它报道,并且发现了这两个重组酶酸性pH稳定性较高,这些特点有利于该酶的应用。另外,本研究首次提出了海藻糖合成酶的保守序列,这对新基因的克隆具有较强的指导意义,并且通过对霍氏肠杆菌海藻糖合成酶基因的表达与分析,证明了这种直接利用PCR进行产酶菌的筛选方法的有效性,同时也为海藻糖的生物转化增添了新的工具酶。
With the development of genetic engineering, recombinant biocatalyst becomes one of the powerful pathways for obtaining substances. It has been confirmed that a lot of oligosaccharides and digested polysaccharides had some biological functions which are interesting in industrial and agricultural applications. In food industry, some oligosaccharides and polysaccharides have been used as additives to improve nutritional and healthy value. In feed industry, polysaccharide-hydrolytic enzymes and oligosaccharides are widely used as dietary additives. Feed enzymes could improve animal performance and feed conversion efficiency and afford opportunities for new feed resources discovery. Presently, the majority of functional enzymes come from microorganisms, especially bacteria, thus we focus on the genetic engineering study of alginate lyases and trehalose synthase.
In this thesis, two sorts of enzymes, alginate lyase with hydrolytic activity with algae polysaccharides as substrate and trehalose synthase with activity to convert maltose to trehalose, were studied. The mature alginate lyase coding sequences from Azotobacter chroococcum and Pseudomonas aeruginosa were cloned, expressed and the enzymes were characterized. In addition, the six trehalose synthase (TreS) producing strains were screened and isolated from a soil specimen obtained from the Tibetan plateau by degenerate PCR. A novel treS gene from Enterobacter hormaechei was amplified and expressed. These main results are formulated as follows:
The sequence encoding the mature peptide of alginate lyase of Azotobacter chroococcum, called AcalgL, was cloned. This 1,056bp sequence was inserted into the modified Pichia pastoris vector pPIC9K-6HISEn5’and introduced into the host Pichia pastoris GS115 by PEG method. The purified recombinant AcAlgL had a specific activity of 2,968U/mg and a Mr of 41kDa. The recombinant AcAlgL displayed a maximum activity at 40°C and pH8.5. It was stable over the range of pH5～11. The activity of recombinant AcAlgL decreased by over 50% in presence of 10mM Cu~(2+) and Fe~(2+), furthermore, 10mM Co~(2+), Mn~(2+), Ca~(2+) and 1mM Fe~(2+) decreased it by 20%. The other cations including K+, Na+ and Zn~(2+) did not significantly affect its enzymatic activity.
The mature alginate lyase coding sequences of P. aeruginosa, 1,023bp, was amplified and inserted into the modified pPIC9K expression vector. The positive construct was transformed to Pichia pastoris GS115 cells by spheroplasting method. The purified recombinant alginate lyase showed a Mr of 40kDa and a specific activity of 2,440U/mg at an optimal temperature 40°C and pH8.5, respectively. The recombinant PaAlgL was stable below 45°C at a wide range of pH3～12. The recombinant PaAlgL was sensitive to some metal cations negatively including Zn~(2+), Cu~(2+) and Fe~(2+), but Co~(2+) and Ca~(2+) promoted it dramatically. The antimicrobial activity of ampicilin and kanamycin agaist P. aeruginosa was increased by the help of PaAlgL.
The gene of Enterobacter hormaechei TreS, containing a 1,626bp ORF encoding 541 amino acids, was cloned. The nucleotide sequence had been deposited in Genbank under No. FJ215664. The recombinant EhTreS had a Mr of 65kDa and a specific activity of 18.5U/mg. The EhTreS displayed a converting rate of 40% at the optimum condition of pH6 and 40°C. Hg~(2+), Zn~(2+), Cu~(2+)and SDS inhibited the enzyme activity at different levels whereas Mn~(2+) shows an enhancing effect by 10%.
This is the first report for expression of alginante lyases in Pichia pastoris system. It was also provided some detailed characters of Azotobacter chroococcum and Pseudomonas aeruginosa alginate lyases. Their activities are much higher than any other reports. In the research on TreS, the degenerate primers based on the conserved domains of trehalose synthases were proved to be effective for new gene screening. This result provides a prospective biochemical method for the trehalose production.
引文
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