南极假丝酵母脂肪酶B(Candida antarctic lipase B,CALB)对水溶性和非水溶性底物均有良好的催化效率,广泛应用于各种工业领域,例如,生物柴油、对映体酮洛芬、甘油碳酸酯等的合成。为提高CALB热稳定性,将CALB与48条不同来源的脂肪酶序列进行比对获得共识序列,并初步确定了可显著提升CALB保守性的14个突变位点。随后,通过定点突变获得热稳定性显著提升的组合突变体G114A/A284 N(CALBm)。结果显示,与CALB相比,CALBm在50 ℃下的半衰期(t1/2)提高了74.7%,达到14.5 min,但比酶活(16.1 U/mg)和kcat/Km[5 932.3 mmol/(L·min)]分别下降了36.3%和23%。分子动力学模拟表明,增加的疏水相互作用(G114 vs M83,G114 vs V84,G114 vs I121)和氢键(A284 vs G41,A284 vs A281)提升了CALBm蛋白分子的整体刚性和稳定性。分子对接分析显示,CALBm与底物分子之间作用力的减少可能是其活性降低的主要原因。研究结果将有助于提升CALB高温条件下的应用效果。
Candida antarctica lipase B (CALB) is an enzyme with excellent catalytic efficiency for both water-soluble and non-water-soluble substrates. As a result, it is widely used in various industries such as the synthesis of biodiesel, enantiomeric ketoprofen, and glycerol carbonate, etc. To improve the thermal stability, CALB was aligned with 48 lipase sequences from diverse sources to generate a consensus sequence, and 14 mutations were initially detected that considerably strengthened the conservation of CALB. Subsequently, the combined mutant G114A/A284N (CALBm) with significantly improved thermal stability was determined by site-directed mutation analysis. The results showed that the half-life(t1/2) of CALBm at 50 ℃ was increased by 74.7% compared with CALB, reaching 14.5 min; however, the specific activity (16.1 U/mg) and kcat/Km [5 932.3 mmol/(L·min)] decreased by 36.3% and 23%, respectively. According to molecular dynamics simulation, the improved hydrophobic interaction (G114 vs M83, G114 vs V84, G114 vs I121) and hydrogen bonding (A284 vs G41, A284 vs A281) enhanced the overall rigidity and stability of CALBm. Meanwhile, molecular docking analysis showed that the decrease in the interaction between CALBm and substrate molecules was the main reason for the reduced activity. These results will benefit the application of CALB at high temperatures.
[1] GOTOR-FERNÁNDEZ V, BUSTO E, GOTOR V.Candida antarctica lipase B:An ideal biocatalyst for the preparation of nitrogenated organic compounds[J].Advanced Synthesis & Catalysis, 2006, 348(7-8):797-812.
[2] NAMAL SENANAYAKE S P J, SHAHIDI F.Incorporation of docosahexaenoic acid (DHA) into evening primrose (Oenothera biennis L.) oil via lipase-catalyzed transesterification[J].Food Chemistry, 2004, 85(4):489-496.
[3] 李站胜, 颜晨麟, 江宁, 等.脂肪酶处理白酒丢糟提升复糟酒的品质[J].现代食品科技, 2019, 35(5):191-197;123.
LI Z S, YAN C L, JIANG N, et al.Lipase treatment of distiller's grains wine to promote its quality[J].Modern Food Science and Technology, 2019, 35(5):191-197;123.
[4] PARK H J, PARK K, KIM Y H, et al.Computational approach for designing thermostable Candida antarctica lipase B by molecular dynamics simulation[J].Journal of Biotechnology, 2014, 192(Part A):66-70.
[5] DU W, XU Y Y, LIU D H, et al.Comparative study on lipase-catalyzed transformation of soybean oil for biodiesel production with different acyl acceptors[J].Journal of Molecular Catalysis B:Enzymatic, 2004, 30(3-4):125-129.
[6] BARBOSA O, ORTIZ C, TORRES R, et al.Effect of the immobilization protocol on the properties of lipase B from Candida antarctica in organic media:Enantiospecifc production of atenolol acetate[J].Journal of Molecular Catalysis B:Enzymatic, 2011, 71(3-4):124-132.
[7] TAMAYO J J, LADERO M, SANTOS V E, et al.Esterification of benzoic acid and glycerol to α-monobenzoate glycerol in solventless media using an industrial free Candida antarctica lipase B[J].Process Biochemistry, 2012, 47(2):243-250.
[8] KAPOOR M, GUPTA M N.Obtaining monoglycerides by esterification of glycerol with palmitic acid using some high activity preparations of Candida antarctica lipase B[J].Process Biochemistry, 2012, 47(3):503-508.
[9] PETERSON M E, DANIEL R M, DANSON M J, et al.The dependence of enzyme activity on temperature:Determination and validation of parameters[J].The Biochemical Journal, 2007, 402(2):331-337.
[10] KÖSE Ö, TÜTER M, AKSOY H A.Immobilized Candida antarctica lipase-catalyzed alcoholysis of cotton seed oil in a solvent-free medium[J].Bioresource Technology, 2002, 83(2):125-129.
[11] KIM S C, KIM Y H, LEE H, et al.Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification[J].Journal of Molecular Catalysis B:Enzymatic, 2007, 49(1-4):75-78.
[12] LIU Q, XUN G H, FENG Y.The state-of-the-art strategies of protein engineering for enzyme stabilization[J].Biotechnology Advances, 2019, 37(4):530-537.
[13] RATH A, DAVIDSON A R.The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis[J].Protein Science, 2000, 9(12):2457-2469.
[14] TOKURIKI N, TAWFIK D S.Stability effects of mutations and protein evolvability[J].Current Opinion in Structural Biology, 2009, 19(5):596-604.
[15] STEIPE B, SCHILLER B, PLÜCKTHUN A, et al.Sequence statistics reliably predict stabilizing mutations in a protein domain[J].Journal of Molecular Biology, 1994, 240(3):188-192.
[16] STEVENS A J, BROWN Z Z, SHAH N H, et al.Design of a split intein with exceptional protein splicing activity[J].Journal of the American Chemical Society, 2016, 138(7):2162-2165.
[17] PAATERO A, ROSTI K, SHKUMATOV A V, et al.Crystal structure of an engineered LRRTM2 synaptic adhesion molecule and a model for neurexin binding[J].Biochemistry, 2016, 55(6):914-926.
[18] 张玲敏. 南极假丝酵母脂肪酶B在黑曲霉中的分泌表达及硅藻土固定化应用研究[D].广州:华南理工大学, 2019.
ZHANG L M.Secretory expression of Candida antarctica lipase B in Aspergillus niger and its application in diatomite immobilization[D].Guangzhou:South China University Of Technology, 2019.
[19] HAYAT S M G, FARAHANI N, GOLICHENARI B, et al.Recombinant protein expression in Escherichia coli (E.coli):What we need to know[J].Current Pharmaceutical Design, 2018, 24(6):718-725.
[20] STERNKE M, TRIPP K W, BARRICK D.The use of consensus sequence information to engineer stability and activity in proteins[J].Methods in Enzymology, 2020, 643:149-179.
[21] ANBAR M, GUL O, LAMED R, et al.Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis[J].Applied and Environmental Microbiology, 2012, 78(9):3458-3464.
[22] LEHMANN M, LOCH C, MIDDENDORF A, et al.The consensus concept for thermostability engineering of proteins further proof of concept[J].Protein Engineering, 2002, 15(5):403-411.
[23] KIM H S, LE Q A T, KIM Y H.Development of thermostable lipase B from Candida antarctica (CalB) through in silico design employing B-factor and RosettaDesign[J].Enzyme and Microbial Technology, 2010, 47(1-2):1-5.
[24] LE Q A T, JOO J C, YOO Y J, et al.Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge[J].Biotechnology and Bioengineering, 2012, 109(4):867-876.
[25] 温露文, 徐岩, 喻晓蔚.理性设计提高酯合成催化反应脂肪酶的热稳定性[J].微生物学通报, 2020, 47(7):2106-2118.
WEN L W, XU Y, YU X W.Rational design to improve lipase thermostability for ester synthesis[J].Microbiology, 2020, 47(7):2106-2118.
[26] XIE Y, AN J, YANG G Y, et al.Enhanced enzyme kinetic stability by increasing rigidity within the active site[J].Journal of Biological Chemistry, 2014, 289(11):7994-8006.
[27] SU B M, WU D Y, XU X Q, et al.Design of a PL18 alginate lyase with flexible loops and broader entrance to enhance the activity and thermostability[J].Enzyme and Microbial Technology, 2021, 151:109916.
[28] SIDDIQUI K S.Defying the activity-stability trade-off in enzymes:taking advantage of entropy to enhance activity and thermostability[J].Critical Reviews in Biotechnology, 2017, 37(3):309-322.
