荧光假单胞菌脂肪酶的克隆表达、酶学性质及其在手性拆分中的应用研究
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摘要
假单胞菌脂肪酶在催化酯水解、酯合成和酯交换反应中常常表现出很高的选择性,因此广泛应用于制药和农药等许多领域中。多年来的研究表明,假单胞菌脂肪酶基因在E. coli中的表达大多以没有活性的包涵体形式存在,这极大地限制了经济而高效的E. coli表达系统在其表达上的应用。本论文中我们采用PCR的方法,从可降解有机溶剂的Pseudomonas fluorescens JCM5963菌株中成功克隆了一个新的脂肪酶基因PFL296,并以有活性的聚集体的形式在E. coli中实现了高水平的表达。通过对重组酶性质表征发现,PFL296编码的酶蛋白PFL的分子量是32 426 kD,最适温度为55℃,最适pH值为8.8,具有较好的低温水解能力和高温活性,具有较宽范围的pH稳定性以及碱性作用条件,因此具有作为洗涤剂用酶的应用潜力。重组PFL的另一个重要性质是在多种水溶性有机溶剂的存在下得到激活并保持稳定,对一些水不溶性的有机溶剂也具有一定的耐受性,这些特点使其具有很好的应用于有机合成的潜力。在此基础上,本文对重组PFL在立体选择性拆分(R, S)-2-[(2-甲基-6-乙基)苯基氨基]丙酸((R, S)-NEMPA)和(R, S)-2-辛醇中的应用进行了研究。结果表明,重组酶的粗酶聚集体和全细胞酶制剂形式都分别表现出了较好的实际应用价值。
Lipases catalyze ester synthesis and transesterification reactions with high regional and stereo selectivity in nonaqueous solvent systems. These features make lipases play an important role in the function of detergent additives, fine chemicals synthesis, pharmaceutical and agrochemical productions. Among various bacterial lipases, those from Pseudomonas (Ps.) genus and Burkholderia (B., formerly Pseudomonas) genus are the most widely used members in biotechnological applications. In recent years, a variety of lipase-encoding genes from different Ps. species have been cloned and sequenced, and the corresponding proteins have been expressed in homologous or heterogeneous hosts. Unfortunately, the heterogeneous expressions of subfamily I.1 and I.2 Ps. lipases are hampered by the fact that a lipase chaperone is necessary for correctly folding to an enzymatically active form. Therefore, to increase lipase productivity in a biochemically safe and economic expression system such as E. coli is always causing a tremendous interest among scientists and industrialists. Several previous researches have reported the overexpression of Ps. lipases and their chaperon proteins in E. coli as inactive inclusion bodies, and subsequently denaturing and refolding are necessary in order to obtain enzymatically active recombinant lipases the high-level expression of active Ps. lipases in E. coli has not yet been reported up to now. The present thesis describes the cloning, heterogeneous expression, purification and characterization of an organic solvent tolerant lipase of this strain. The recombinant lipase possessed the various advantages existed in many other Ps. lipases, and high expression level and satisfactory recovery were achieved through designed well-connected experiments. This work may be of great significance to the increased biotechnological application in organic synthesis by supplying efficient and stable biocatalyst and to the improved production of family I bacterial lipases. In this thesis, a novel lipase-encoding gene PFL296 was successfully amplified from chromosomal DNA of the organic degradable strain of Pseudomonas fluorescens JCM5963 by PCR using a pair of degenerate oligonucleotide primers. We also focus on the enantioselectivity resolution of (R, S)-2-(2-ethyl-6-methylphenyl) alanine ((R, S)-NEMPA) and (R, S)-2-octanol by recombinant PFL at various conditions. The results are as follows:
1. Cloning and expression of the gene of PFL296
On the basis of search and alignment of the homologous Ps. lipases, three pair of degenerated oligonucleotide primers was designed based on the nucleotide sequences immediately upstream and downstream the known coding sequences of lipase. Using these primers, a band of about 900 bp length was amplified from the chromosomal DNA of Ps. fluorescens 5963. Sequencing and gene analysis indicated an ORF of 891 bp, a full-length lipase gene motif encoding a polypeptide of 296aa residues. The nucleotide sequence data reported here were submitted to GenBank with the accession number EU310372. The translated amino acid sequence was submitted to Esther and NCBI database with the accession number ABY26520.
We got a prediction of the properties and structural informations of the coding protein by the methods of bioinformatics. It is indicated that the protein shares high identities with the subfamily I.1 lipases. Sequence alignment and Homology- modelling reveal that the protein had aα/βhydrolase fold and the residues forming the catalytic triad were Ser83,Asp241and His263. Moreover, the prediction of hydrophobicity of the protein indicates that there might exist a transmembrane region near its N terminus.
The amplified fragments were inserted in pET28b vector according to the manufacturer’s instructions, and the recombinant plasmid was transformed into E. coli BLP for protein expression. The transformed strains were grown in a modified 2YT medium (adding 1% glucose) at 37℃until the OD600 reached 1.2. Protein expression was induced by adding IPTG to a final concentration of 0.05 mM, and then the culture was shaken for 8-10 h at 20℃and the cells were harvested by centrifugation. The results indicate that the recombinant protein reached to near 20% of the total protein in the cells, and the activity reaches 10 U/g wet cells using p-Nitrophenyl caprylate as substrate under 50℃.
2. Purification and characterizition of recombinant PFL (rPFL)
During the course of purification, an important phenomenon was observed, that was, rPFL was unstable in solution and easy to form aggregates like other family I lipases, we found that only no more than 30% of the total expression protein was solved in supernatant of crude extract, and most of them existed in cell debris as active aggregates. This discovery is of great significance for the further expression research of Ps. lipases in an efficient and economic expression system, for this is the first time that a Ps. lipase was successfully expressed E. coli as active form at such a high level. Considering all the results above, we conclude that these lipases exhibit an intrinsic folding capability in vitro without chaperon protein, which support the hypothesis put forward by I. Beacham et al., that is, these subfamily I.1 lipases might be secreted via a signal peptide-independent pathway. The aggregates were only partially dissolved in Tris-HCl buffer (pH 8.0), but almost completely dissolved in Tris-HCl buffer (pH 8.0) containing 0.2% DOC. Considering the property of forming large molecular mass aggregates, a reverse procedure of purification was taken, The easily soluble fractions existed in the precipitate derived from saturated ammonium sulfate fractionations and cell debris were removed by washing twice with small amount buffer, and this treatment greatly simplified the purification. Finally, the next Ni-NTA affinity chromatography gave a production of 8.0 mg rPFL per g of wet cells, with a purity of over 95% target protein contain and a specific activity of 435 U/mg. Apparent molecular mass of purified enzyme estimated by native polyacrylamide gel electrophoresis and gel filtration chromatography also indicates that there is nearly no monomer found in the enzyme solution even at a low concentration.
The results of characterization of purified enzyme indicate that the optimal temperature is 55℃, and the optima pH value is 9.0, as well as the highest hydrolytic activity of rPFL is obtained to the substrate of pNPC8. Activity detection using olive oil emulsion as substrate demonstrates rPFL exhibits hydrolysis activity of 156 U/mg, which suggests that rPFL can be defined as a true lipase. Ca2+ or Sn2+ increases the activity of rPFL by 10% or 5% respectively. On the contrary, Zn2+ inhabits nearly 70% activity especially. EDTA inhibits the activity of rPFL by 40%, while non-ionic surfactants Triton X-100 and Tween 20 at 0.1% (v/v) increase its activity by 8% and 9%, respectively. Anionic surfactant SDS completely inhibits the hydrolytic activity of rPFL. The purified recombinant lipase not only exhibits high activity in wide ranges of temperatures and pH values, but also behaves quite stable under alkaline, moderate temperature conditions and even in the presence of some surfactants. The most important feature of rPFL is the high activity and extreme stability against a variety of organic solvents. The rPFL was activated in the presence of almost all the water-miscible organic solvents investigated after it was incubated for 1 h, and remained stable after it was incubated for 24 h in the presence of short carbon chain alcohols including isopropanol, methanol and ethanol, as well as acetone and glycerol. The observed highest activities relative to that of the control were 142% in isoamyl alcohol after incubation for 1 h and 120% in isopropanol after incubation for 24 h. All these results provide sufficient advantages to make rPFL a promising candidate for application in nonaqueous biocatalysis and in the field of detergent additives.
3. Resolution reactions catalyzed by rPFL
On the basis of the former work, we prepared three kind of preparations of rPFL (Purified rPFL, P-rPFL; Crude enzyme aggregates rPFL, CEA-rPFL and Whole cell rPFL, WC-rPFL), with which resolution reactions were performed to (R, S)-2-(2-ethyl-6-methylphenyl) alanine ((R, S)-NEMPA) and (R, S)-2-octanol. Seven kind of permeabilization methods were taken in order to enhance the activity when the whole cell enzymes were prepared.
When used in resolution of (R, S)-NEMPA from the corresponding racemic methyl ester through an enantioselective hydrolysis reaction, enhanced activity and stability were achieved for the CEA-rPFL compared with P-rPFL and WC-rPFL. The effects of reaction conditions, such as enzyme loading, temperature, pH, organic solvents were investigated. It is found that addition of 20% (v/v) of isoamyl alcohol in the reaction mixture greatly increase the catalytic activity by 35% and without any decrease on enantioselectivity. Under the optimum conditions, that was in 100mM phosphate buffer solution (pH 8.0), under 50℃,enzyme loading 10 mg/ml and 200 rpm for16 h, the enantiomeric excess value of the produced (R)-NEMPA reached 98% at the conversion of 48.3%, corresponding to an enantiomeric ratio (E) value of above 100. It could compete with the Ac-CLEA-PSL, which was prepared from the commercial PSL by cross linking the aggregates by glutaraldehyde, as far as the activity and enantioselectivity were concerned. The operational stability experiment results also indicate that the CEA-rPFL can be readily recycled, and maintained nearly 60% of its initial activity even after being reused for eight times.
CEA-rPFL enzyme preparation was a novel form of enzyme attempted by us, and we confirmed its availability through experiments. It was prepared from the engineering recombinant strains, through ultrasonication, wash and lyophilization, and its major components were active aggregates formed in the procedure of expression. Moreover, we considered it as a kind a natural immobilization enzyme because the rPFL might associate with lipopolysaccharides according to the description of the literature and its exhibition in solution. It was the hydrophobic adsorption action that rendered rPFL higher activity and stability, as well as better enantioselectivity. Thus, the results demonstrated that rPFL was a kind of easy-making, cheap, efficient and practical enzyme preparation.
At the same time, the whole cell biocatalyst of recombinant lipase (WC-rPFL) exhibited excellent catalytic activity and operational stability compared with CEA-rPFL and P-rPFL in the resolution of (R, S)-2-octanol by a transesteration reaction using vinyl acetate as acyl donor. The effects of reaction conditions, such as permeabilizer, enzyme loading, temperature, pH, water activity and organic solvents were investigated. It is found that isopropanol was the most efficient permeabilizer. At the same time, n-Heptane was found to be the optimum organic solvent. Under the optimum conditions, the enantiomeric excess value of the produced (S)-2-octanol reached 99% at the conversion of 60.0%, corresponding to an E value of 25. The operational stability experiment results also indicated that the WC-rPFL can also be readily recycled, and maintained 90% of its initial activity even after being reused for ten times.
The WC-rPFL preparation can be obtained easily and cheaply without the complicated and expensive separation and purification steps. Furthermore, the rPFL existed inside the cell was more stable under the protection of cell membrane. However, the enantioselectivity needed to be improved. At present, we have docked the molecule of (R, S)-acetate-2-octanol ester in the homologous modelling three dimension structure of PFL, and planned to enhance the enantioselectivity by rational design method, so that to obtain a practical biocatalyst in resolution of (R, S)-2-octanol.
Lipases catalyze ester synthesis and transesterification reactions with high regional and stereo selectivity in nonaqueous solvent systems. These features make lipases play an important role in the function of detergent additives, fine chemicals synthesis, pharmaceutical and agrochemical productions. Among various bacterial lipases, those from Pseudomonas (Ps.) genus and Burkholderia (B., formerly Pseudomonas) genus are the most widely used members in biotechnological applications. In recent years, a variety of lipase-encoding genes from different Ps. species have been cloned and sequenced, and the corresponding proteins have been expressed in homologous or heterogeneous hosts. Unfortunately, the heterogeneous expressions of subfamily I.1 and I.2 Ps. lipases are hampered by the fact that a lipase chaperone is necessary for correctly folding to an enzymatically active form. Therefore, to increase lipase productivity in a biochemically safe and economic expression system such as E. coli is always causing a tremendous interest among scientists and industrialists. Several previous researches have reported the overexpression of Ps. lipases and their chaperon proteins in E. coli as inactive inclusion bodies, and subsequently denaturing and refolding are necessary in order to obtain enzymatically active recombinant lipases the high-level expression of active Ps. lipases in E. coli has not yet been reported up to now. The present thesis describes the cloning, heterogeneous expression, purification and characterization of an organic solvent tolerant lipase of this strain. The recombinant lipase possessed the various advantages existed in many other Ps. lipases, and high expression level and satisfactory recovery were achieved through designed well-connected experiments. This work may be of great significance to the increased biotechnological application in organic synthesis by supplying efficient and stable biocatalyst and to the improved production of family I bacterial lipases. In this thesis, a novel lipase-encoding gene PFL296 was successfully amplified from chromosomal DNA of the organic degradable strain of Pseudomonas fluorescens JCM5963 by PCR using a pair of degenerate oligonucleotide primers. We also focus on the enantioselectivity resolution of (R, S)-2-(2-ethyl-6-methylphenyl) alanine ((R, S)-NEMPA) and (R, S)-2-octanol by recombinant PFL at various conditions. The results are as follows:
1. Cloning and expression of the gene of PFL296
On the basis of search and alignment of the homologous Ps. lipases, three pair of degenerated oligonucleotide primers was designed based on the nucleotide sequences immediately upstream and downstream the known coding sequences of lipase. Using these primers, a band of about 900 bp length was amplified from the chromosomal DNA of Ps. fluorescens 5963. Sequencing and gene analysis indicated an ORF of 891 bp, a full-length lipase gene motif encoding a polypeptide of 296aa residues. The nucleotide sequence data reported here were submitted to GenBank with the accession number EU310372. The translated amino acid sequence was submitted to Esther and NCBI database with the accession number ABY26520.
We got a prediction of the properties and structural informations of the coding protein by the methods of bioinformatics. It is indicated that the protein shares high identities with the subfamily I.1 lipases. Sequence alignment and Homology- modelling reveal that the protein had aα/βhydrolase fold and the residues forming the catalytic triad were Ser83,Asp241and His263. Moreover, the prediction of hydrophobicity of the protein indicates that there might exist a transmembrane region near its N terminus.
The amplified fragments were inserted in pET28b vector according to the manufacturer’s instructions, and the recombinant plasmid was transformed into E. coli BLP for protein expression. The transformed strains were grown in a modified 2YT medium (adding 1% glucose) at 37℃until the OD600 reached 1.2. Protein expression was induced by adding IPTG to a final concentration of 0.05 mM, and then the culture was shaken for 8-10 h at 20℃and the cells were harvested by centrifugation. The results indicate that the recombinant protein reached to near 20% of the total protein in the cells, and the activity reaches 10 U/g wet cells using p-Nitrophenyl caprylate as substrate under 50℃.
2. Purification and characterizition of recombinant PFL (rPFL)
During the course of purification, an important phenomenon was observed, that was, rPFL was unstable in solution and easy to form aggregates like other family I lipases, we found that only no more than 30% of the total expression protein was solved in supernatant of crude extract, and most of them existed in cell debris as active aggregates. This discovery is of great significance for the further expression research of Ps. lipases in an efficient and economic expression system, for this is the first time that a Ps. lipase was successfully expressed E. coli as active form at such a high level. Considering all the results above, we conclude that these lipases exhibit an intrinsic folding capability in vitro without chaperon protein, which support the hypothesis put forward by I. Beacham et al., that is, these subfamily I.1 lipases might be secreted via a signal peptide-independent pathway. The aggregates were only partially dissolved in Tris-HCl buffer (pH 8.0), but almost completely dissolved in Tris-HCl buffer (pH 8.0) containing 0.2% DOC. Considering the property of forming large molecular mass aggregates, a reverse procedure of purification was taken, The easily soluble fractions existed in the precipitate derived from saturated ammonium sulfate fractionations and cell debris were removed by washing twice with small amount buffer, and this treatment greatly simplified the purification. Finally, the next Ni-NTA affinity chromatography gave a production of 8.0 mg rPFL per g of wet cells, with a purity of over 95% target protein contain and a specific activity of 435 U/mg. Apparent molecular mass of purified enzyme estimated by native polyacrylamide gel electrophoresis and gel filtration chromatography also indicates that there is nearly no monomer found in the enzyme solution even at a low concentration.
The results of characterization of purified enzyme indicate that the optimal temperature is 55℃, and the optima pH value is 9.0, as well as the highest hydrolytic activity of rPFL is obtained to the substrate of pNPC8. Activity detection using olive oil emulsion as substrate demonstrates rPFL exhibits hydrolysis activity of 156 U/mg, which suggests that rPFL can be defined as a true lipase. Ca2+ or Sn2+ increases the activity of rPFL by 10% or 5% respectively. On the contrary, Zn2+ inhabits nearly 70% activity especially. EDTA inhibits the activity of rPFL by 40%, while non-ionic surfactants Triton X-100 and Tween 20 at 0.1% (v/v) increase its activity by 8% and 9%, respectively. Anionic surfactant SDS completely inhibits the hydrolytic activity of rPFL. The purified recombinant lipase not only exhibits high activity in wide ranges of temperatures and pH values, but also behaves quite stable under alkaline, moderate temperature conditions and even in the presence of some surfactants. The most important feature of rPFL is the high activity and extreme stability against a variety of organic solvents. The rPFL was activated in the presence of almost all the water-miscible organic solvents investigated after it was incubated for 1 h, and remained stable after it was incubated for 24 h in the presence of short carbon chain alcohols including isopropanol, methanol and ethanol, as well as acetone and glycerol. The observed highest activities relative to that of the control were 142% in isoamyl alcohol after incubation for 1 h and 120% in isopropanol after incubation for 24 h. All these results provide sufficient advantages to make rPFL a promising candidate for application in nonaqueous biocatalysis and in the field of detergent additives.
3. Resolution reactions catalyzed by rPFL
On the basis of the former work, we prepared three kind of preparations of rPFL (Purified rPFL, P-rPFL; Crude enzyme aggregates rPFL, CEA-rPFL and Whole cell rPFL, WC-rPFL), with which resolution reactions were performed to (R, S)-2-(2-ethyl-6-methylphenyl) alanine ((R, S)-NEMPA) and (R, S)-2-octanol. Seven kind of permeabilization methods were taken in order to enhance the activity when the whole cell enzymes were prepared.
When used in resolution of (R, S)-NEMPA from the corresponding racemic methyl ester through an enantioselective hydrolysis reaction, enhanced activity and stability were achieved for the CEA-rPFL compared with P-rPFL and WC-rPFL. The effects of reaction conditions, such as enzyme loading, temperature, pH, organic solvents were investigated. It is found that addition of 20% (v/v) of isoamyl alcohol in the reaction mixture greatly increase the catalytic activity by 35% and without any decrease on enantioselectivity. Under the optimum conditions, that was in 100mM phosphate buffer solution (pH 8.0), under 50℃,enzyme loading 10 mg/ml and 200 rpm for16 h, the enantiomeric excess value of the produced (R)-NEMPA reached 98% at the conversion of 48.3%, corresponding to an enantiomeric ratio (E) value of above 100. It could compete with the Ac-CLEA-PSL, which was prepared from the commercial PSL by cross linking the aggregates by glutaraldehyde, as far as the activity and enantioselectivity were concerned. The operational stability experiment results also indicate that the CEA-rPFL can be readily recycled, and maintained nearly 60% of its initial activity even after being reused for eight times.
CEA-rPFL enzyme preparation was a novel form of enzyme attempted by us, and we confirmed its availability through experiments. It was prepared from the engineering recombinant strains, through ultrasonication, wash and lyophilization, and its major components were active aggregates formed in the procedure of expression. Moreover, we considered it as a kind a natural immobilization enzyme because the rPFL might associate with lipopolysaccharides according to the description of the literature and its exhibition in solution. It was the hydrophobic adsorption action that rendered rPFL higher activity and stability, as well as better enantioselectivity. Thus, the results demonstrated that rPFL was a kind of easy-making, cheap, efficient and practical enzyme preparation.
At the same time, the whole cell biocatalyst of recombinant lipase (WC-rPFL) exhibited excellent catalytic activity and operational stability compared with CEA-rPFL and P-rPFL in the resolution of (R, S)-2-octanol by a transesteration reaction using vinyl acetate as acyl donor. The effects of reaction conditions, such as permeabilizer, enzyme loading, temperature, pH, water activity and organic solvents were investigated. It is found that isopropanol was the most efficient permeabilizer. At the same time, n-Heptane was found to be the optimum organic solvent. Under the optimum conditions, the enantiomeric excess value of the produced (S)-2-octanol reached 99% at the conversion of 60.0%, corresponding to an E value of 25. The operational stability experiment results also indicated that the WC-rPFL can also be readily recycled, and maintained 90% of its initial activity even after being reused for ten times.
The WC-rPFL preparation can be obtained easily and cheaply without the complicated and expensive separation and purification steps. Furthermore, the rPFL existed inside the cell was more stable under the protection of cell membrane. However, the enantioselectivity needed to be improved. At present, we have docked the molecule of (R, S)-acetate-2-octanol ester in the homologous modelling three dimension structure of PFL, and planned to enhance the enantioselectivity by rational design method, so that to obtain a practical biocatalyst in resolution of (R, S)-2-octanol.
引文
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