赤拟谷盗来源天冬氨酸α-脱羧酶分子改造及催化合成β-丙氨酸工艺的建立
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摘要
L-天冬氨酸α-脱羧酶活性较低,稳定性较差,使得其在工业应用中受到限制。该研究旨在提高L-天冬氨酸α-脱羧酶的催化性能,促进生物法生产β-丙氨酸的工业化进程。依据嗜热蛋白酶的氨基酸内在进化趋势,对赤拟谷盗来源L-天冬氨酸α-脱羧酶进行分子改造,以期提高稳定性。实验共构建21个突变体,获得催化性能优良的突变体K221R,该突变体的比酶活较野生型提高20. 3%;野生型经50℃处理30 min,残余酶活接近0,而突变体K221R的残余酶活为43%。建立了基因工程菌全细胞催化天冬氨酸生成β-丙氨酸的工艺,K221R菌株的产量达到134. 72 g/L,摩尔转化率为94. 52%,是迄今为止的最高产量。该研究构建的基因工程菌具有工业应用潜力,同时也为生物法制备β-丙氨酸提供理论与技术基础。
Low catalytic ability and poor stability limit industrial applications of L-aspartate α-decarboxylase.This study was therefore conducted to improve the catalytic activity of L-aspartate α-decarboxylase to promote biological production of β-alanine in industries. Based on evolutionary information of thermophilic bacteria,L-aspartate α-decarboxylase from Tribolium castaneum was molecularly modified to improve its enzyme stability. The mutant strain K221 R was screened,as it had improved thermal stability and enzymatic activity. Compared with the wild type,the specific enzyme activity of K221 R increased 20. 3%. Moreover,after incubating the enzyme at 50 ℃ for 30 min,the residual activity of the wild type was 0,while K221 R remained 43% activity. Furthermore,up to 134. 72 g/L β-alanine was produced using K221 R-expression whole cells,which was the highest production level achieved up-to-date,with 94. 52% molar conversion rate. In conclusion,the engineered strain containing K221 variant has great potential for industrial production of β-alanine.
Low catalytic ability and poor stability limit industrial applications of L-aspartate α-decarboxylase.This study was therefore conducted to improve the catalytic activity of L-aspartate α-decarboxylase to promote biological production of β-alanine in industries. Based on evolutionary information of thermophilic bacteria,L-aspartate α-decarboxylase from Tribolium castaneum was molecularly modified to improve its enzyme stability. The mutant strain K221 R was screened,as it had improved thermal stability and enzymatic activity. Compared with the wild type,the specific enzyme activity of K221 R increased 20. 3%. Moreover,after incubating the enzyme at 50 ℃ for 30 min,the residual activity of the wild type was 0,while K221 R remained 43% activity. Furthermore,up to 134. 72 g/L β-alanine was produced using K221 R-expression whole cells,which was the highest production level achieved up-to-date,with 94. 52% molar conversion rate. In conclusion,the engineered strain containing K221 variant has great potential for industrial production of β-alanine.
引文
[1]罗积杏,薛建萍,沈寅初.β-氨基丙酸的合成与应用[J].氨基酸和生物资源,2005,27(1):52-55.
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[13]陈夏林,李由然,顾正华,等.两种L-天冬氨酸α-脱羧酶的表达与酶学性质分析[J].微生物学通报,2017(10):2 337-2 344.
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[16]高宇.一釜双酶法转化富马酸制备β-丙氨酸催化体系的构建及工艺优化[D].无锡:江南大学,2017.
[17] LIU P,TORRENS-SPENCE M P,DING H,et al. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acidα-decarboxylases[J]. Amino Acids,2013,44(2):391-404.
[18] ARAKANE Y,LOMAKIN J,BEEMAN R W,et al. Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum[J]. Journal of Biological Chemistry,2009,284(24):16 584.
[19] MOUSSIAN B. Recent advances in understanding mechanisms of insect cuticle differentiation[J]. Insect Biochemistry&Molecular Biology,2010,40(5):363-375.
[20] KRAMER K J,MORGAN T D,HOPKINS T L,et al. Catecholamines andβ-alanine in the red flour beetle,Tribolium castaneum:roles in cuticle sclerotization and melanization[J]. Insect Biochemistry,1984,14(3):293.
[21] DAI F,LIANG Q,CAO C,et al. Aspartate decarboxylase is required for a normal pupa pigmentation pattern in the silkworm,bombyx mori[J]. Scientific Reports,2015,5:10 885.
[22] LIU P,DING H,CHRISTENSEN B M,et al. Cysteine sulfinic acid decarboxylase activity of Aedes aegypti aspartate L-decarboxylase:the structural basis of its substrate selectivity[J]. Insect Biochemistry&Molecular Biology,2012,42(6):396-403.
[23] RICHARDSON G,DING H,ROCHELEAU T,et al. An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes[J]. Molecular Biology Reports,2010,37(7):3 199-3 205.
[24] BORODINA I,KILDEGAARD K R,JENSEN N B,et al. Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae viaβ-alanine[J]. Metabolic Engineering,2015,27:57-64.
[25] BRADFORD M M. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72(S1-2):248-254.
[26] ARGOS P,ROSSMANN M G,GRAU U M,et al. Thermal stability and protein structure[J]. Evolution of Protein Structure&Function,1980,18(25):159-169.
[27] ZHANG X J,BAASE W A,MATTHEWS B W. Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent,additive effects on both structure and stability[J]. Protein Science,2010,1(6):761-776.
[28] MRABET N T,BROECK A V D,BRANDE I V D,et al. Arginine residues as stabilizing elements in proteins[J]. Biochemistry,1992,31(8):2 239.
[29]叶双双,周丽,周哲敏.定点突变提高苯丙氨酸羟化酶的热稳定性[J].生物工程学报,2016,32(9):1 243-1 254.
[30] JAKOB F,MARTINEZ R,MANDAWE J,et al. Surface charge engineering of a Bacillus gibsonii subtilisin protease[J]. Applied Microbiology&Biotechnology,2013,97(15):6 793-6 802.
[31]黄楠,朱龙宝,周丽,等.鱼腥藻苯丙氨酸脱氨酶的基因克隆、表达及最适反应p H改造[J].微生物学通报,2015,42(7):1 208-1 215.
[32]王哲.重组大肠杆菌产腈水合酶发酵优化及烟酰胺生产工艺的建立[D].无锡:江南大学,2017.
[2]任怡,王彦青,舒宏,等.β-氨基丙酸的合成工艺研究[J].辽宁化工,2006,35(4):187-188.
[3]楼坚.生物转化法生产β-丙氨酸的研究[D].杭州:浙江工业大学,2006.
[4] SHEN Yan,ZHAO Lianzhen,LI Youran,el al. Synthesis ofβ-alanine from L-aspartate using L-aspartate-α-decarboxylase from Corynebacterium glutamicum[J]. Biotechnology Letters,2014,36(8):1 681-1 686.
[5]高丽娟,裘娟萍. L-天冬氨酸脱羧酶研究进展[J].工业微生物,2007,37(5):54-59.
[6] SONG C W,LEE J,KO Y S,et al. Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid[J]. Metabolic Engineering,2015,30(3):121-129.
[7] WILLIAMSON J M,BROWN G M. Purification and properties of L-aspartateα-decarboxylase,an enzyme that catalyzes the formation ofβ-alanine in Escherichia coli[J]. Journal of Biological Chemistry,1979,254(16):8 074-8 082.
[8] LEE B I,SUH S W. Crystal structure of the schiff base intermediate prior to decarboxylation in the catalytic cycle of aspartateα-decarboxylase[J]. Journal of Molecular Biology,2004,340(1):1-7.
[9] GOPALAN G,CHOPRA S,RANGANATHAN A,et al.Crystal structure of uncleaved L-aspartate-α-decarboxylase from Mycobacterium tuberculosis[J]. Proteins-structure Function&Bioinformatics,2010,65(4):796-802.
[10] CUIW,SHI Z,FANG Y,et al. Significance of Arg3,Arg54,and Tyr58 of L-aspartateα-decarboxylase from Corynebacterium glutamicum,in the process of self-cleavage[J]. Biotechnology Letters,2014,36(1):121-126.
[11]邓思颖,张君丽,蔡真,等.枯草芽胞杆菌L-天冬氨酸α-脱羧酶的酶学性质[J].生物工程学报,2015,31(8):1 184-1 193.
[12]石增秀,崔文璟,周丽,等.谷氨酸棒杆菌L-天冬氨酸α-脱羧酶基因的克隆及重组酶性质研究[J].生物技术通报,2013(4):110-115.
[13]陈夏林,李由然,顾正华,等.两种L-天冬氨酸α-脱羧酶的表达与酶学性质分析[J].微生物学通报,2017(10):2 337-2 344.
[14] KWON A R,LEE B I,HAN B W,et al. Crystallization and preliminary X-ray crystallographic analysis of aspartate L-decarboxylase from Helicobacter pylori[J]. Acta Crystallographica,2002,58(5):861-863.
[15] SCHMITZBERGER F,KILKENNY M L,LOBLEY C M C,et al. Structural constraints on protein self-processing in L-aspartate-α-decarboxylase[J]. Embo Journal,2014,22(23):6 193-6 204.
[16]高宇.一釜双酶法转化富马酸制备β-丙氨酸催化体系的构建及工艺优化[D].无锡:江南大学,2017.
[17] LIU P,TORRENS-SPENCE M P,DING H,et al. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acidα-decarboxylases[J]. Amino Acids,2013,44(2):391-404.
[18] ARAKANE Y,LOMAKIN J,BEEMAN R W,et al. Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum[J]. Journal of Biological Chemistry,2009,284(24):16 584.
[19] MOUSSIAN B. Recent advances in understanding mechanisms of insect cuticle differentiation[J]. Insect Biochemistry&Molecular Biology,2010,40(5):363-375.
[20] KRAMER K J,MORGAN T D,HOPKINS T L,et al. Catecholamines andβ-alanine in the red flour beetle,Tribolium castaneum:roles in cuticle sclerotization and melanization[J]. Insect Biochemistry,1984,14(3):293.
[21] DAI F,LIANG Q,CAO C,et al. Aspartate decarboxylase is required for a normal pupa pigmentation pattern in the silkworm,bombyx mori[J]. Scientific Reports,2015,5:10 885.
[22] LIU P,DING H,CHRISTENSEN B M,et al. Cysteine sulfinic acid decarboxylase activity of Aedes aegypti aspartate L-decarboxylase:the structural basis of its substrate selectivity[J]. Insect Biochemistry&Molecular Biology,2012,42(6):396-403.
[23] RICHARDSON G,DING H,ROCHELEAU T,et al. An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes[J]. Molecular Biology Reports,2010,37(7):3 199-3 205.
[24] BORODINA I,KILDEGAARD K R,JENSEN N B,et al. Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae viaβ-alanine[J]. Metabolic Engineering,2015,27:57-64.
[25] BRADFORD M M. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72(S1-2):248-254.
[26] ARGOS P,ROSSMANN M G,GRAU U M,et al. Thermal stability and protein structure[J]. Evolution of Protein Structure&Function,1980,18(25):159-169.
[27] ZHANG X J,BAASE W A,MATTHEWS B W. Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent,additive effects on both structure and stability[J]. Protein Science,2010,1(6):761-776.
[28] MRABET N T,BROECK A V D,BRANDE I V D,et al. Arginine residues as stabilizing elements in proteins[J]. Biochemistry,1992,31(8):2 239.
[29]叶双双,周丽,周哲敏.定点突变提高苯丙氨酸羟化酶的热稳定性[J].生物工程学报,2016,32(9):1 243-1 254.
[30] JAKOB F,MARTINEZ R,MANDAWE J,et al. Surface charge engineering of a Bacillus gibsonii subtilisin protease[J]. Applied Microbiology&Biotechnology,2013,97(15):6 793-6 802.
[31]黄楠,朱龙宝,周丽,等.鱼腥藻苯丙氨酸脱氨酶的基因克隆、表达及最适反应p H改造[J].微生物学通报,2015,42(7):1 208-1 215.
[32]王哲.重组大肠杆菌产腈水合酶发酵优化及烟酰胺生产工艺的建立[D].无锡:江南大学,2017.
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