Gibberella intermedia腈水解酶的克隆、鉴定及分子改造
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摘要
腈水解酶是腈水解酶超级家族中的一种重要催化剂，它能将腈类化合物中的腈基直接转化为羧基。在有机酸的生物合成、聚合物的表面修饰、腈污染物的生物除污中具有重要应用。然而，腈水解酶的应用开发仍受到诸多因素限制，如酶品种较少、催化活力不够高、稳定性较差等问题，此外，目前多数研究主要围绕细菌腈水解酶展开，真菌腈水解酶的潜力远未得到开发。这些因素阻碍了腈水解酶在工业生物催化领域的发展。为此，本文以真菌腈水解酶为研究对象，开展了从理论到应用研究的一系列工作。
本研究首先分别以3-氰基吡啶和甘氨腈为唯一氮源从环境样品中进行产腈水解酶真菌的筛选，通过施加选择性压力的两轮富集培养以及简单快速的苯酚-次氯酸钠法初筛及精确的HPLC方法最终筛选，最后从初筛得到的81株腈转化酶产生菌中获得一株具有最高3-氰基吡啶转化活力以及较高热稳定性的真菌CA3-1用于后续研究。采用分子生物学手段对该真菌菌株进行鉴定和系统进化分析，确认该菌株为Gibberellaintermedia CA3-1。该菌株现保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.4903。对G. intermedia CA3-1的腈化合物催化特征进行了初步考察。结果证明该真菌拥有较广泛的底物谱和良好的热稳定性，能转化利用各类腈化合物尤其3-氰基吡啶和脂肪族腈化合物表现出极高的特异性；在30°C，40°C和50°C下的半衰期分别为231.1h,72.9h和6.4h；在5%(v/v)的丙醇中仍能保留较高酶活；50mmol·L-13-氰基吡啶能在30min内被转化完全，同时产生约＜5%的烟酰胺。
鉴于筛选获得的G. intermedia CA3-1对腈类化合物的生物转化表现出较大潜力，利用基因重组技术对该菌株中所包含的真菌腈水解酶基因进行克隆和异源表达。采用反转录PCR的方法，从G. intermedia CA3-1野生菌的RNA通过反转录PCR扩增cDNA序列，并进一步PCR获得真菌腈水解酶的编码基因。开放读码框由963bp碱基所组成，共编码320个氨基酸，理论分子量为35.94kDa。该真菌腈水解酶基因与G. moniliformis(ABF83489)的假定腈水解酶基因具有97%的同源性，与其他各类已报道的细菌及真菌腈水解酶同源性均低于40%。将目的基因转入表达宿主Escherichia coli Rosetta-gami(DE3)，成功构建产真菌腈水解酶的重组菌株E. coli Rosetta-gami (DE3)/pET28a(+)-Nit，重组菌表现出较好的腈化合物水解活力，以3-氰基吡啶为底物时游离菌体的比酶活能达到0.5U·mg-1，游离细胞在30°C、40°C和50°C下的半衰期t1/2分别为24.75h，2.55h和1.34h。
对重组酶进行分离纯化和酶学表征。通过Ni-NTA琼脂糖凝胶柱对重组酶进行分离纯化，得到了电泳纯的腈水解酶蛋白，实际分子量约为37.0kDa；并对纯化酶的酶学性质进行了研究。其最适反应条件为温度45°C和pH7.8；重组真菌腈水解酶能水解脂肪族和芳香族腈化合物，尤其对3-氰基吡啶和4-氰基吡啶特异性较高；以3-氰基吡啶为底物的动力学参数Vmax和Km分别为0.81μmol·min-1·mg-1和12.11mmol·L-1。采用重叠延伸PCR技术确立Glu-45，Lys-127和Cys-162为G. intermedia CA3-1腈水解酶的催化活性中心。
为提高G. intermedia腈水解酶的应用性能，通过基因工程手段对该酶进行分子改造。采用点饱和突变的方式对G. intermedia CA3-1腈水解酶活性中心附近的128位异亮氨酸及161位天冬酰胺残基进行饱和突变改造，构建了这两个位点的饱和突变库，筛选获得多株比酶活提高、酰胺生成量降低的突变株；选取其中酶活提高和酰胺降低幅度较为显著的2株突变株I128V和N161Q，进行进一步的组合突变，成功构建了组合突变株I128L-N161Q，组合突变株的酶活进一步提高、酰胺生成相应减少；研究了3株突变株游离细胞的热稳定性，结果表明，单点突变株的稳定性均有明显提高，尤其是在30°C和40°C情况下，然而，组合突变并未导致稳定性的进一步提高，突变导致酶在酸性pH条件下的酶活降低，碱性pH条件下的酶活则有一定提升，突变酶I128L和N161Q依然拥有广泛的作用pH范围，而I128L-N161Q作用范围相比野生型则相对变窄；I128L和N161Q的最适反应温度有所提高均为50°C，突变酶I128L-N161Q的最适反应温度仍不变；突变酶的底物谱发生了一定改变，尤其是对4-氰基吡啶的活性大幅提高，达到40%以上；突变导致酶的催化效率也有不同程度提高。
为充分挖掘其应用潜能，进行了G. intermedia腈水解酶突变体的应用研究。采用复合固定化的方式对腈水解酶游离细胞进行固定化以考察其应用潜力。选取了壳聚糖和聚乙烯醇这两种材料进行包埋固定能获得较佳的酶活回收和机械强度，固定条件为在8%的PVA浓度、4%的壳聚糖浓度下进行固定，固化操作在6%的三聚磷酸钠的饱和硼酸溶液中进行。复合固定化细胞在30°C和40°C下的热稳定性相比游离细胞提高了2倍以上；固定化还导致催化剂在4°C和-20°C的贮藏稳定性显著提高。转化不同浓度3-氰基吡啶的结果表明，以100mmol·L-13-氰基吡啶为底物时表现出最高酶活力。采用底物流加模式，通过固定化细胞进行生物转化，经18批次的100mmol·L-1底物流加，最终在525min-1转化时间内累积产物浓度达到208g·L。通过分批转化模式能进一步促进烟酸产量，利用批次提升至22次。
Nitrilases are an important biocatalyst in the nitrilase superfamily, which can directlyconvert a wide range of nitrile compounds into corresponding carboxylic acids. Thus theenzyme is well applied in organic acid biosynthesis, surface modification of polyacrylonitrileand bioremediation of high toxic nitrile wastes. However, its application might be hindered byseveral limitations, including less variety, low catalytic activity, poor operational stability, andso on. On the other hand, conventional studies mainly focused on the bacterial nitrilase andthe potential of fungal nitrilase has been far from being fully explored. These factors becamethe barrier to the utilization of nitrilase in industrial biocatalysis. In this study, fungal nitrilaseis selected as the research object and a series of relevant works are carried out as follows.
Isolation of nitrilase-producing fungus from environmental samples was firstly performedwith3-cyanopyridine and glycinonitrile as the sole nitrogen source in the present study. Afungus CA3-1displaying high3-cyanopyridine hydrolyzing activity and good thermostabilitywas isolated from81strains through two rounds of enrichment culture, subsequent primaryscreening with phenol-hypochlorite method and final screening with HPLC method. Thisfungal strain was designated as Gibberella intermedia CA3-1using molecular identification,and phylogenetic analysis of this strain was also performed. The strain was deposited in theChina General Microbiological Culture Collection Center and the accession number isCGMCC4903. The catalytic properties of G. intermedia resting cells were determined.Results showed that this fungus showed a wide substrate spectrum with high specificity forheterocyclic and aliphatic nitriles. It also displayed good thermostability and the half-lives at30°C,40°C, and50°C were231.1h,72.9h, and6.4h, respectively. It remained extremelyactive in5%(v/v) propanol.3-Cyanopyridine (50mmol·L-1) was hydrolyzed into nicotinicacid within30min, whereas only less than5%of nicotinamide was detected.
Therefore, cloning and heterologous expression of fungal nitrilase gene was carried outusing recombinant DNA technology due to the catalytic potential of this strain for nitrilecompounds. Partial cDNA sequence was amplified from the total RNA of wild Gibberellaintermedia through reverse transcription-PCR. A coding gene of fungal nitrilase wassubsequently cloned through further PCR from cDNA. The open reading frame consisted of963bp and potentially encoded a protein of320amino acid residues with a theoreticalmolecular mass of35.94kDa. The fungal nitrilase from G. intermedia CA3-1showed97%identity with the putative nitrilase from G. moniliformis (ABF83489). The identities of G.intermedia CA3-1nitrilase gene to the other reported nitrilases were lower than40%. Theencoding gene was transformed into E. coli Rosetta-gami (DE3) and recombinant strain E.coli Rosetta-gami (DE3)/pET28a(+)-Nit was successfully constructed. The recombinant straindisplayed good nitrile converting activity and specific activity of resting cells could reach upto0.5U·mg-1with3-cyanopyridine as the substrate. The half-lives of resting cells at30°C,40°C, and50°C were24.75h,2.55h, and1.34h, respectively.
Purification and characterization of recombinant nitrilase was studied. The recombinantprotein was purified to electrophoretic homogeneity and the actual molecular mass was about 37.0kDa. The purified enzyme exhibited optimal activity at45°C and pH7.8. This nitrilasewas specific towards aliphatic and aromatic nitriles, especially3-and4-cyanopyridine. Thekinetic parameters Vmaxand Kmfor3-cyanopyridine were determined to be0.81μmol·min-1·mg-1and12.11mmol·L-1through Hanes-Woolf plot, respectively. On the otherhand, the catalytic triad (Glu-45, Lys-127, and Cys-162) was proposed and confirmed byoverlap extension PCR.
G. intermedia nitrilase was modified at the gene level through gene engineering approachesin order to improve its enzymatic properties in catalytic application. Site saturationmutagenesis of128-Ile and161-Asn near the active site was conducted to improve thespecific activity and reducing the amide formation of G. intermedia CA3-1nitrilase. The twomutant libraries were constructed and several positive mutants were obtained. Two mostsignificant mutants I128V and N161Q were selected for further study. And double mutationswere subsequently introduced to construct mutant I128L-N161Q, which supported higherspecific activity and lower amide formation. The theremostability of all three mutants wasobviously improved compared with wild-type nitrilase, especially at30°C and40°C. However,double mutation did not further improve thermostability on the basis of single mutation.I128V and N161Q still exhibited wide reaction pH range, while I128L-N161Q becamerelatively narrow. The reaction temperature of I128V and N161Q was increased to50°Csimultaneously and I128L-N161Q remained the same. Mutations led to obvious changes ofsubstrate spectrum, especially for4-cyanopyridine, which resulted in an increase of relativeactivity by more that40%. Also, the catalytic efficiency of mutants was improved.
The application studies of G. intermedia nitrilase mutant were performed to fully explore itsapplication potential. Complex immobilization of resting cells harboring nitrilase was utilizedto evaluate the application potential of immobilized nitrilase. Chitosan and PVA were selectedfor entrapment experiment, and acceptable residual activity and mechanical strength wereobserved. The immobilization conditions were8%of PVA and4%of chitosan, and theentrapped beads were maintained in saturated borate solution containing6%of sodiumtripolyphosphate. Interestingly, the thermostability of immobilized cells at30°C and40°Cwas improved by over100%. Also, immobilization led to obvious improvement of storagestability at4°C and-20°C. Conversion of3-cyanopyridine with different concentrationsdemonstrated that100mM3-cyanopyridine in the reaction mixture was the optimumconcentration of substrate. Fed-batch reaction using immobilized cells as the catalyst wascarried out for bioconversion of3-cyanopyridine. Finally,208g·L-1nicotinic acid wasobtained after18feedings (100mmol·L-1) within525min. Repetitive batch reaction modecould further improve nicotinic acid production through22feedings.
本研究首先分别以3-氰基吡啶和甘氨腈为唯一氮源从环境样品中进行产腈水解酶真菌的筛选，通过施加选择性压力的两轮富集培养以及简单快速的苯酚-次氯酸钠法初筛及精确的HPLC方法最终筛选，最后从初筛得到的81株腈转化酶产生菌中获得一株具有最高3-氰基吡啶转化活力以及较高热稳定性的真菌CA3-1用于后续研究。采用分子生物学手段对该真菌菌株进行鉴定和系统进化分析，确认该菌株为Gibberellaintermedia CA3-1。该菌株现保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.4903。对G. intermedia CA3-1的腈化合物催化特征进行了初步考察。结果证明该真菌拥有较广泛的底物谱和良好的热稳定性，能转化利用各类腈化合物尤其3-氰基吡啶和脂肪族腈化合物表现出极高的特异性；在30°C，40°C和50°C下的半衰期分别为231.1h,72.9h和6.4h；在5%(v/v)的丙醇中仍能保留较高酶活；50mmol·L-13-氰基吡啶能在30min内被转化完全，同时产生约＜5%的烟酰胺。
鉴于筛选获得的G. intermedia CA3-1对腈类化合物的生物转化表现出较大潜力，利用基因重组技术对该菌株中所包含的真菌腈水解酶基因进行克隆和异源表达。采用反转录PCR的方法，从G. intermedia CA3-1野生菌的RNA通过反转录PCR扩增cDNA序列，并进一步PCR获得真菌腈水解酶的编码基因。开放读码框由963bp碱基所组成，共编码320个氨基酸，理论分子量为35.94kDa。该真菌腈水解酶基因与G. moniliformis(ABF83489)的假定腈水解酶基因具有97%的同源性，与其他各类已报道的细菌及真菌腈水解酶同源性均低于40%。将目的基因转入表达宿主Escherichia coli Rosetta-gami(DE3)，成功构建产真菌腈水解酶的重组菌株E. coli Rosetta-gami (DE3)/pET28a(+)-Nit，重组菌表现出较好的腈化合物水解活力，以3-氰基吡啶为底物时游离菌体的比酶活能达到0.5U·mg-1，游离细胞在30°C、40°C和50°C下的半衰期t1/2分别为24.75h，2.55h和1.34h。
对重组酶进行分离纯化和酶学表征。通过Ni-NTA琼脂糖凝胶柱对重组酶进行分离纯化，得到了电泳纯的腈水解酶蛋白，实际分子量约为37.0kDa；并对纯化酶的酶学性质进行了研究。其最适反应条件为温度45°C和pH7.8；重组真菌腈水解酶能水解脂肪族和芳香族腈化合物，尤其对3-氰基吡啶和4-氰基吡啶特异性较高；以3-氰基吡啶为底物的动力学参数Vmax和Km分别为0.81μmol·min-1·mg-1和12.11mmol·L-1。采用重叠延伸PCR技术确立Glu-45，Lys-127和Cys-162为G. intermedia CA3-1腈水解酶的催化活性中心。
为提高G. intermedia腈水解酶的应用性能，通过基因工程手段对该酶进行分子改造。采用点饱和突变的方式对G. intermedia CA3-1腈水解酶活性中心附近的128位异亮氨酸及161位天冬酰胺残基进行饱和突变改造，构建了这两个位点的饱和突变库，筛选获得多株比酶活提高、酰胺生成量降低的突变株；选取其中酶活提高和酰胺降低幅度较为显著的2株突变株I128V和N161Q，进行进一步的组合突变，成功构建了组合突变株I128L-N161Q，组合突变株的酶活进一步提高、酰胺生成相应减少；研究了3株突变株游离细胞的热稳定性，结果表明，单点突变株的稳定性均有明显提高，尤其是在30°C和40°C情况下，然而，组合突变并未导致稳定性的进一步提高，突变导致酶在酸性pH条件下的酶活降低，碱性pH条件下的酶活则有一定提升，突变酶I128L和N161Q依然拥有广泛的作用pH范围，而I128L-N161Q作用范围相比野生型则相对变窄；I128L和N161Q的最适反应温度有所提高均为50°C，突变酶I128L-N161Q的最适反应温度仍不变；突变酶的底物谱发生了一定改变，尤其是对4-氰基吡啶的活性大幅提高，达到40%以上；突变导致酶的催化效率也有不同程度提高。
为充分挖掘其应用潜能，进行了G. intermedia腈水解酶突变体的应用研究。采用复合固定化的方式对腈水解酶游离细胞进行固定化以考察其应用潜力。选取了壳聚糖和聚乙烯醇这两种材料进行包埋固定能获得较佳的酶活回收和机械强度，固定条件为在8%的PVA浓度、4%的壳聚糖浓度下进行固定，固化操作在6%的三聚磷酸钠的饱和硼酸溶液中进行。复合固定化细胞在30°C和40°C下的热稳定性相比游离细胞提高了2倍以上；固定化还导致催化剂在4°C和-20°C的贮藏稳定性显著提高。转化不同浓度3-氰基吡啶的结果表明，以100mmol·L-13-氰基吡啶为底物时表现出最高酶活力。采用底物流加模式，通过固定化细胞进行生物转化，经18批次的100mmol·L-1底物流加，最终在525min-1转化时间内累积产物浓度达到208g·L。通过分批转化模式能进一步促进烟酸产量，利用批次提升至22次。
Nitrilases are an important biocatalyst in the nitrilase superfamily, which can directlyconvert a wide range of nitrile compounds into corresponding carboxylic acids. Thus theenzyme is well applied in organic acid biosynthesis, surface modification of polyacrylonitrileand bioremediation of high toxic nitrile wastes. However, its application might be hindered byseveral limitations, including less variety, low catalytic activity, poor operational stability, andso on. On the other hand, conventional studies mainly focused on the bacterial nitrilase andthe potential of fungal nitrilase has been far from being fully explored. These factors becamethe barrier to the utilization of nitrilase in industrial biocatalysis. In this study, fungal nitrilaseis selected as the research object and a series of relevant works are carried out as follows.
Isolation of nitrilase-producing fungus from environmental samples was firstly performedwith3-cyanopyridine and glycinonitrile as the sole nitrogen source in the present study. Afungus CA3-1displaying high3-cyanopyridine hydrolyzing activity and good thermostabilitywas isolated from81strains through two rounds of enrichment culture, subsequent primaryscreening with phenol-hypochlorite method and final screening with HPLC method. Thisfungal strain was designated as Gibberella intermedia CA3-1using molecular identification,and phylogenetic analysis of this strain was also performed. The strain was deposited in theChina General Microbiological Culture Collection Center and the accession number isCGMCC4903. The catalytic properties of G. intermedia resting cells were determined.Results showed that this fungus showed a wide substrate spectrum with high specificity forheterocyclic and aliphatic nitriles. It also displayed good thermostability and the half-lives at30°C,40°C, and50°C were231.1h,72.9h, and6.4h, respectively. It remained extremelyactive in5%(v/v) propanol.3-Cyanopyridine (50mmol·L-1) was hydrolyzed into nicotinicacid within30min, whereas only less than5%of nicotinamide was detected.
Therefore, cloning and heterologous expression of fungal nitrilase gene was carried outusing recombinant DNA technology due to the catalytic potential of this strain for nitrilecompounds. Partial cDNA sequence was amplified from the total RNA of wild Gibberellaintermedia through reverse transcription-PCR. A coding gene of fungal nitrilase wassubsequently cloned through further PCR from cDNA. The open reading frame consisted of963bp and potentially encoded a protein of320amino acid residues with a theoreticalmolecular mass of35.94kDa. The fungal nitrilase from G. intermedia CA3-1showed97%identity with the putative nitrilase from G. moniliformis (ABF83489). The identities of G.intermedia CA3-1nitrilase gene to the other reported nitrilases were lower than40%. Theencoding gene was transformed into E. coli Rosetta-gami (DE3) and recombinant strain E.coli Rosetta-gami (DE3)/pET28a(+)-Nit was successfully constructed. The recombinant straindisplayed good nitrile converting activity and specific activity of resting cells could reach upto0.5U·mg-1with3-cyanopyridine as the substrate. The half-lives of resting cells at30°C,40°C, and50°C were24.75h,2.55h, and1.34h, respectively.
Purification and characterization of recombinant nitrilase was studied. The recombinantprotein was purified to electrophoretic homogeneity and the actual molecular mass was about 37.0kDa. The purified enzyme exhibited optimal activity at45°C and pH7.8. This nitrilasewas specific towards aliphatic and aromatic nitriles, especially3-and4-cyanopyridine. Thekinetic parameters Vmaxand Kmfor3-cyanopyridine were determined to be0.81μmol·min-1·mg-1and12.11mmol·L-1through Hanes-Woolf plot, respectively. On the otherhand, the catalytic triad (Glu-45, Lys-127, and Cys-162) was proposed and confirmed byoverlap extension PCR.
G. intermedia nitrilase was modified at the gene level through gene engineering approachesin order to improve its enzymatic properties in catalytic application. Site saturationmutagenesis of128-Ile and161-Asn near the active site was conducted to improve thespecific activity and reducing the amide formation of G. intermedia CA3-1nitrilase. The twomutant libraries were constructed and several positive mutants were obtained. Two mostsignificant mutants I128V and N161Q were selected for further study. And double mutationswere subsequently introduced to construct mutant I128L-N161Q, which supported higherspecific activity and lower amide formation. The theremostability of all three mutants wasobviously improved compared with wild-type nitrilase, especially at30°C and40°C. However,double mutation did not further improve thermostability on the basis of single mutation.I128V and N161Q still exhibited wide reaction pH range, while I128L-N161Q becamerelatively narrow. The reaction temperature of I128V and N161Q was increased to50°Csimultaneously and I128L-N161Q remained the same. Mutations led to obvious changes ofsubstrate spectrum, especially for4-cyanopyridine, which resulted in an increase of relativeactivity by more that40%. Also, the catalytic efficiency of mutants was improved.
The application studies of G. intermedia nitrilase mutant were performed to fully explore itsapplication potential. Complex immobilization of resting cells harboring nitrilase was utilizedto evaluate the application potential of immobilized nitrilase. Chitosan and PVA were selectedfor entrapment experiment, and acceptable residual activity and mechanical strength wereobserved. The immobilization conditions were8%of PVA and4%of chitosan, and theentrapped beads were maintained in saturated borate solution containing6%of sodiumtripolyphosphate. Interestingly, the thermostability of immobilized cells at30°C and40°Cwas improved by over100%. Also, immobilization led to obvious improvement of storagestability at4°C and-20°C. Conversion of3-cyanopyridine with different concentrationsdemonstrated that100mM3-cyanopyridine in the reaction mixture was the optimumconcentration of substrate. Fed-batch reaction using immobilized cells as the catalyst wascarried out for bioconversion of3-cyanopyridine. Finally,208g·L-1nicotinic acid wasobtained after18feedings (100mmol·L-1) within525min. Repetitive batch reaction modecould further improve nicotinic acid production through22feedings.
引文
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