Glutaminase is an important food enzyme. In order to improve the thermal stability of glutaminase, Discovery studio 2017 was used to virtually mutate forty-nine amino acids in Bacillus subtilis glutaminase (YbgJ) that have adverse interactions. Key amino acid loci E3, E55, and D213 that affect the thermal stability of YbgJ were identified and saturated mutated. Compared with wild type enzyme, half-lives (t1/2, 55 ℃) of the mutants E3C, E55F, and D213T increased by 58%, 69%, and 41%, respectively. The t1/2 of the compounded mutants E3C/E55F/D213T, E3C/E55F, E3C/D213T, and E55F/D213 were 1.73, 1.44, 1.22, and 0.97 times higher than that of the wild type enzyme, respectively. Among them, the specific activity of E3C/E55F raised by 23%, reaching 693 U/mg. Structural analysis indicated that E3C/E55F/D213T, E3C/E55F, E3C/D213T, and E55F/D213T had thirty, twenty-three, eleven, and eight more hydrogen bonds, respectively, than that in wild type enzyme. Meanwhile, their adverse interactions decreased by 5, 4, 4, and 3, respectively. The results revealed that substituting key amino acids in the enzyme molecule had great influences on the thermal stability of YbgJ. Decreases in intramolecular adverse interactions and increases in hydrogen bonds may account for enhanced thermal stability of YbgJ.
CHEN Xiao
,
LI Jianghua
,
LIU Song
,
DU Guocheng
. Improved thermal stability of glutaminase through saturated mutations[J]. Food and Fermentation Industries, 2019
, 45(5)
: 1
-7
.
DOI: 10.13995/j.cnki.11-1802/ts.018768
[1] 康维民,肖念新.枯草菌产生的谷氨酰胺酶在酱油酿造中的作用[J].中国酿造, 1997, 16(4):36-37.
[2] 黎兵华,丁义涛,余德才.谷氨酰胺酶与肿瘤的关系研究进展[J].转化医学电子杂志, 2017, 4(12):1-6.
[3] 浦荷芳.硝基还原假单胞菌谷氨酰胺酶基因的克隆、表达及其在L-茶氨酸酶法合成中的应用[D].南京:南京师范大学,2012.
[4] BINOD P, SINDHU R, MADHAVAN A et al.Recent developments in L-glutaminase production and applications-an overview[J].Bioresour Technol, 2017, 245(Pt B):1 766-1 774.
[5] WAKAYAMA M, YAMAGATA T, KAMEMURA A et al.Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation[J].Journal of Industrial Microbiology and Biotechnology, 2005, 32(9):383-390.
[6] 汪正华,朱蓓霖,赵云等.蛋白质谷氨酰胺酶基因的合成表达及性质研究[J].中国生物工程杂志,2012, 32(11):55-60.
[7] 周尚庭,李沛,郭辉.谷氨酰胺酶和酵母抽提物对无添加酱油的品质提升研究[J].中国调味品,2016, 41(5):45-50.
[8] DURA M A, FLORES M, TOLDRA F. Effects of curing agents and the stability of a glutaminase from Debaryomyces spp.[J].Food Chemistry, 2004, 86(3):385-389.
[9] MATSUURA S I, CHIBA M, TSUNODA T et al.Enzyme Immobilization in Mesoporous silica for enhancement of Thermostability[J].Journal of Nanoscience and Nanotechnology, 2018, 18(1):104-109.
[10] ZHANG Junhui, JIANG Yuyan, LIN Ying et al.Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids[J].Plos One, 2013, 8(7):e67 892.
[11] LIPPOW S M,WITTRUP K D,TIDOR B.Computational design of antibody-affinity improvement beyond in vivo maturation[J].Nature Biotechnology, 2007, 25(10):1 171-1 176.
[12] LI G, FANG X, SU F, et al.Enhancing the Thermostability of Rhizomucor miehei lipase with a limited screening library by rational design point mutations and disulfide bonds[J].Applied and Environmental Microbiology, 2018, 84(2):9-17.
[13] KOIDE A, JORDAN MR,HORNOR SR et al.Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface[J].Biochemistry, 2001, 40:10 326-10 333.
[14] ZHENG P, CAO Y, BU T et al.Single molecule force spectroscopy reveals that electrostatic interactions affect the mechanical stability of proteins[J]. Biophysical Journal, 2011, 100(6):1 534-1 541.
[15] PANDIAN S R K, DEEPAK V, SIVASUBRAMANIAM S D et al.Optimization and purification of anticancer enzyme L-glutaminase from Alcaligenes faecalis KLU102[J].Biologia, 2014, 69(12):1 644-1 651.
[16] IMADA A, IGARASI S, NAKAHAMA K et al.Asparaginase and glutaminase activities of micro-organisms[J].Journal of General Microbiology, 1973, 76(1):85-99.
[17] FIELDS R.The rapid determination of amino groups with TNBS[J].Methods in Enzymology, 1972, 25:464-468.
[18] JR K J, NYBERG K, SALI D et al. Contribution of hydrophobic interactions to protein stability[J].Journal of Molecular Biology,2011,408(3), 514-528.
[19] TANNER J J,AND R M H,KRAUSE K L.Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus d-glyceraldehyde-3-phosphate dehydrogenase at 2.5 resolution [J].Biochemistry, 1996, 35(8):2 597.
[20] BEN M S, AYADI D Z, BEN H H et al.Thermostability improvement of maltogenic amylase MAUS149 by error prone PCR[J].Journal of Biotechnology, 2013, 168(4):601-606.
