Abstract: To develop high-quality glutamate decarboxylase (GAD), the affinity of cellulose-binding domain (CBD) and the self-cleavage of DnaB (an intein) were used for the construction of CBD-DnaB-GAD fusion enzyme using Enterococcus faecium as gadB donor, and the purification of GAD and its enzymatic properties were also investigated. The results showed that recombinant Escherichia coli GDMCC60446 carrying pRPOCDN-EfagadB could efficiently express CBD-DnaB-GAD, and the optimal solution for self-cleavage of DnaB was 0.2 mol/L phosphate buffer saline (pH 6.5, containing 0.5 mol/L NaCl and 1 mmol/L EDTA). The GAD, prepared by one-step purification and self-cleavage, showed a single band on SDS-PAGE electrophoresis with a molecular weight of 55.68 kDa, and consists of only one subunit. The purified GAD showed maximum activity at pH 5.0 and 55 ℃, and was stable at the range of pH 4.8 to 5.8 and -25 to 55 ℃. Its activity was hardly affected by 5 mmol/L of NaCl, CaCl2 or ethylene glycol, but was inactivated by 5 mmol/L of KCl, EDTA-2Na, ZnSO4, CuSO4, MnSO4, MgSO4, FeCl2, FeCl3, AlCl3, AgNO3 or Pb(CH3COO)2 at different levels. The purified GAD only catalyzed the decarboxylation of L-glutamic acid with apparent Km and Vmax at 10.51 mmol/L and 3.41 μmol/(mL·min) respectively, and the reaction activities were not inhibited by the substrate and product. The purity and enzymatic properties of GAD were excellent, and the preparation process was simple. Due to the high purity and excellent enzymatic properties of GAD, as well as the simple preparation process, this technology has potential to be applied to industrial production.
 VILLEGAS J M, BROWN L, DE GIORI G S, et al.Optimization of batch culture conditions for GABA production by Lactobacillus brevis CRL 1942, isolated from quinoa sourdough[J].LWT-Food Science and Technology, 2016, 67:22-26.  杨成丽, 马子玉, 胡晓丽, 等.化学－酶催化法制备D-谷氨酸与γ-氨基丁酸[J].化学与生物工程, 2013, 30(11):55-56. YANG C L, MA Z Y, HU X L, et al.Production of D-glutamic acid and γ-aminobutyric acid using chem-enzymic method[J].Chemistry and Bioengineering, 2013, 30(11):55-56.  ZHU H, SADIQ F A, LI Y, et al.Application of ion-exchange resin as solid acid for buffer-free production of γ-aminobutyric acid using Enterococcus faecium cells[J].LWT-Food Science and Technology, 2018, 98:341-348.  杨胜远, 李云.双向单因素与田口法优化屎肠球菌产谷氨酸脱羧酶培养基[J].食品科学, 2018, 39(4):90-98. YANG S Y, LI Y.Optimization of fermentation medium for glutamate decarboxylase production by Enterococcus faecium by bidirectional One-Factor-at-a-Time and Taguchi methods[J].Food Science, 2018, 39(4):90-98.  YANG S Y, LIU S M, JIANG M, et al.Enhancing effect of macroporous adsorption resin on gamma-aminobutyric acid production by Enterococcus faecium in whole-cell biotransformation system[J].Amino Acids, 2020, 52(5):771-780.  DIVYASHRI G, PRAPULLA S G.An insight into kinetics and thermodynamics of gamma-aminobutyric acid production by Enterococcus faecium CFR3003 IN batch fermentation[J].Annals of Microbiology, 2015, 65:1 109-1 118.  YANG S Y, LIN Q, LU Z X, et al.Characterization of a novel glutamate decarboxylase from Streptococcus salivarius ssp.thermophilus Y2[J].Journal of Chemical Technology & Biotechnology, 2008, 83(6):855-861.  CHEN K J, WU Y T, LEE C K.Cellulose binding domain fusion enhanced soluble expression of fructosyl peptide oxidase and its simultaneous purification and immobilization[J].International Journal of Biological Macromolecules, 2019, 133:980-986.  JONES R W, PEREZ F G.A small cellulose-binding-domain protein (CBM1) in Phytophthora is highly variable in the non-binding amino terminus[J].Current Microbiology, 2017, 74:1 287-1 293.  MNDEZ-LTER J A, GIL-MUOZ J, NIETO-DOMNGUEZ M, et al.A novel, highly efficient β-glucosidase with a cellulose-binding domain:characterization and properties of native and recombinant proteins[J].Biotechnology for Biofuels, 2017, 10:256-271.  BULAKHOV A G, GUSAKOV A V, ROZHKOVA A M, et al.Properties of chimeric polysaccharide monooxygenase with an attached cellulose binding module and its use in the hydrolysis of cellulose-containing materials in the composition of cellulase complexes[J].Catalysis in Industry, 2018, 10(2):152-158.  RICHINS R D, MULCHANDANI A, CHEN W.Expression, immobilization, and enzymatic characterization of cellulose-binding domain-organophosphorus hydrolase fusion enzymes[J].Biotechnology and Bioengineering, 2015, 69(6):591-596.  LIN Q, LI D, QIN H.Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2[J].Electronic Journal of Biotechnology, 2017, 27:8-13  MATHYS S, EVANS T C, CHUTE I C, et al.Characterization of self-splicing mini-intein and its conversion into autocatalytic N-and C-terminal cleavage elements:Facile production of protein building blocks for protein ligation[J].Gene, 1999, 231(1/2):1-13.  HONG J, WANG Y, YE X, et al.Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage[J].Journal of Chromatography A, 2008, 1194:150-154.  ZHAO Z L, LU W, DUN B Q, et al.Purification of green fluorescent protein using a two-intein system[J].Applied Microbiology and Biotechnology, 2008, 77(5):1 175-1 180.  杨胜远, 林谦, 赖丽萍, 等.屎肠球菌纤维素结合域谷氨酸脱羧酶构建及其酶学性质[J].食品与发酵工业, 2019, 45(21):22-30. YANG S Y, LIN Q, LAI L P, et al.Construction and characteristics of recombinant cellulose-binding domain-glutamate decarboxylase of Enterococcus faecium[J].Food and Fermentation Industries, 2019, 45(21):22-30.  杨胜远, 林谦, 张晓宁, 等.大肠杆菌高效表达屎肠球菌纤维素结合域谷氨酸脱羧酶的条件优化[J].食品与机械, 2019, 35(7):31-38. YANG S Y, LIN Q, ZHANG X N, et al.Optimization of conditions for efficient expression of recombinant cellulose-binding domain-glutamate decarboxylase of Enterococcus faecium in Escherichia coli[J].Food and Machinery, 2019, 35(7):31-38.  HONG J, YE X, WANG Y, et al.Bioseparation of recombinant cellulose-binding module-proteins by affinity adsorption on an ultra-high-capacity cellulosic adsorbent[J].Analytica Chimica Acta, 2008, 621(2):193-199.  STRAUSBAUCH P H, FISCHER E H.Chemical and physical properties of Escherichia coli glutamate decarboxylase[J].Biochemistry, 1970, 9(2):226-232.  SEO M J, NAM Y D, LEE S Y, et al.Expression and characterization of a glutamate decarboxylase from Lactobacillus brevis 877G producing γ-aminobutyric acid[J].Bioscience, Biotechnology, and Biochemistry, 2013, 77(4):853-856.  KOMATSUZAKI N, NAKAMURA T, KIMURA T, et al.Characterization of glutamate decarboxylase from a high γ-aminobutyric acid (GABA)-producer, Lactobacillus paracasei[J].Bioscience, Biotechnology, and Biochemistry, 2008, 72(2):278-285.  SA H D, PARK J Y, JEONG S J, et al.Characterization of glutamate decarboxylase (GAD) from Lactobacillus sakei A156 isolated from Jeot-gal[J].Journal of Microbiology & Biotechnology, 2015, 25(5):696-703.  LIU Q, CHENG H, MA X, et al.Expression, characterization and mutagenesis of a novel glutamate decarboxylase from Bacillus megaterium[J].Biotechnology Letters, 2016, 38:1 107-1 113.  DE BIASE D, TRAMONTI A, BOSSA F, et al.The response to stationary-phase stress conditions in Escherichia coli:Role and regulation of the glutamic acid decarboxylase system[J].Molecular Microbiology, 1999, 32(6):1 198-1 211.