原核来源L-天冬氨酸-α-脱羧酶的活性较低,是限制生物法合成β-丙氨酸的主要因素,该研究将目光由原核来源转向真核来源,意在寻找具有更高催化活性的酶。该文通过基因合成获得埃及伊蚊来源半胱亚磺酸脱羧酶,并添加sumo标签实现了其在大肠杆菌中的高效表达,酶学性质表征实验结果表明,重组酶可催化L-天冬氨酸生成β-丙氨酸,比酶活力为(5.00±0.074) U/mg,添加sumo标签后比酶活力为(5.40±0.129) U/mg;最适反应条件为40 ℃,pH 6.0;带标签酶的稳定性要明显优于野生型,在50 ℃处理30 min后酶活力仍保留40%,在pH 5.0~8.0酶活力都维持在80%左右。利用大肠杆菌自身的天冬氨酸裂解酶与重组半胱亚磺酸脱羧酶相偶联,实现了从富马酸到天冬氨酸的一步转化,产物的摩尔转化率达到93.03%。该研究中半胱亚磺酸脱羧酶对于L-天冬氨酸具有较高的催化活性,为β-丙氨酸的工业化生产提供理论支持和技术参考。
Prokaryotic source L-aspartate-α-decarboxylase has low activity, which is the main factor restricting the biosynthesis of β-alanine. In this study, we shifted our attention from prokaryotic sources to eukaryotic sources, and aimed to find higher catalytically active enzymes. We obtained cysteine sulfinic decarboxylase from Aedes aegypti through gene synthesis, and added the sumo tag to achieve high-efficiency expression in Escherichia coli. The results of enzymatic characterization experiments showed that the recombinant enzyme could catalyze L-aspartic acid to produce β-alanine, the specific enzyme activity was (5.60±0.074) U/mg, after adding the sumo tag, the specific enzyme activity was (5.80±0.129) U/mg. The optimal reaction conditions were 40 ℃ and pH 6.0. The stability of the tagged enzyme was significantly better than that of the wild type. The enzyme activity still retained 40% after treatment at 50 ℃ for 30 min, and the enzyme activity remained at about 80% between pH 5.0-8.0. The cysteine sulfinic decarboxylase was coupled with L-aspartic acid lyase of E. coli to realize the one-step conversion from fumaric acid to aspartic acid, and the molar conversion rate of the product reached 93.03%. In this study, cysteine sulfinic decarboxylase had high catalytic activity for L-aspartic acid, which provided technical reference and theoretical support for the industrial production of β-alanine.
[1] ZHAO X L, LI Q, MENG Q, et al.Identification and expression of cysteine sulfinate decarboxylase, possible regulation of taurine biosynthesis in Crassostrea gigas in response to low salinity[J].Scientific Reports, 2017, 7:5505.
[2] LI J H, LING Y Q, FAN J J, et al.Expression of cysteine sulfinate decarboxylase (CSD) in male reproductive organs of mice[J].Histochemistry & Cell Biology, 2006, 125(6):607-613.
[3] LIU P Y, DING H Z, CHRISTENSEN B M., et al.Cysteine sulfinic acid decarboxylase activity of Aedes aegypti aspartate 1-decarboxylase:The structural basis of its substrate selectivity[J].Insect Biochemistry & Molecular Biology, 2012, 42(6):396-403.
[4] LIU Z M, ZHENG W H, YE W Q, et al.Characterization of cysteine sulfinic acid decarboxylase from Tribolium castaneum and its application in the production of β-alanine[J].Applied Microbiology and Biotechnology, 2019, 103(23-24):9 443-9 453.
[5] LIU P Y, TORRENS-SPENCE M P, DING H Z, et al.Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases[J].Amino Acids, 2013, 44(2):391-404.
[6] PIAO X Y, WANG L, LIN B X, et al.Metabolic engineering of Escherichia coli for production of L-aspartate and its derivative β-alanine with high stoichiometric yield[J].Metabolic Engineering, 2019, 54:244-254.
[7] JOSEPH J M, EIMEAR D, PAUL A S, et al.The effect of carnosine or β-alanine supplementation on markers of glycaemic control and insulin resistance in human and animal studies:A protocol for a systematic review and meta-analysis[J].Systematic Reviews, 2020, 9(1):282.
[8] GREEN J R B,LOBO A J,HOLDSWORTH C D, et al.Balsalazide is more effective and better tolerated than mesalamine in the treatment of acute ulcerative colitis[J].Gastroenterology, 1998, 114(1):15-22.
[9] NOFRE C, TINTI J M, OUAR F.Sweeteners derived from glycine and β-alanine, process for sweetening various products and compositions containing such sweeteners, European:EP0195730[P].1986-03-13.
[10] 齐博. β-丙氨酸对肉仔鸡生产性能、肌肉品质和肌源活性肽的影响[D].北京:中国农业科学院,2017.
QI B.The effect of β-alanine on broiler performance, muscle quality and muscle-derived active peptides[D].Beijing:Chinese Academy of Agricultural Sciences, 2017.
[11] LEE J W, NA D, PARK J M, et al.Systems metabolic engineering of microorganisms for natural and non-natural chemicals[J].Nature Chemical Biology, 2012, 8(6):536-546.
[12] El-WASSEF A.β-alanine as lead antidote[J].Oriental Journal of Chemistry, 1988, 4 (1):102-103.
[13] LEE B I, SUH S W.Crystal structure of the schiff base intermediate prior to decarboxylation in the catalytic cycle of aspartate α-decarboxylase[J].Journal of Molecular Biology, 2004, 340(1):1-7.
[14] RAMJEE M K, GENSCHEL U, ABELL C, et al.Escherichia coli L-aspartate-α-decarboxylase:Preprotein processing and observation of reaction intermediates by electrospray mass spectrometry[J].Biochemical Journal, 1997, 323(3):661-669.
[15] 莫芹,李由然,石贵阳.磷酸吡哆醛依赖型天冬氨酸α-脱羧酶的研究[J].发酵科技通讯,2019,48(2):63-68;78.
MO Q, LI Y R, SHI G Y.Study on pyridoxal phosphate-dependent aspartate α-decarboxylase[J].Bulletin of Fermentation Science and Technology, 2019, 48(2):63-68;78.
[16] 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 via β-alanine[J].Metabolic Engineering, 2015, 27:57-64.
[17] 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.
GAO Y.The construction and process optimization of a catalytic system for the conversion of fumaric acid to β-alanine by one-kettle and two-enzyme method[D].Wuxi:Jiangnan University, 2017.
[18] 王超,叶文琪,薛岚,等.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[J].食品与发酵工业,2019,45(11):7-13.
WANG C, YE W Q, XUE L, et al.Modification of aspartate α-decarboxylase from Tribolium castaneum andits application in producing β-alanine[J].Food and Fermentation Industries, 2019, 45(11):7-13.
[19] WANG L J, KONG X D, ZHANG H Y, et al.Enhancement of the activity of L-aspartase from Escherichia coli W by directed evolution[J].Biochem Biophys Res Commun, 2000, 276(1):346-349.
[20] ZHANG Y F, WANG Q, HESS H.Increasing enzyme cascade throughput by pH-engineering the microenvironment of individual enzymes[J].ACS Catalysis, 2017, 7(3):2 047-2 051.
