Objective: To obtain industrial standard lipase with high activity and stability, Butelase 1 ligase was used to connect the N- and C-termini of Thermomyces lanuginosus lipase (TLL), and then the protein characteristics, including heat stability, protease resistance, and enzymatic kinetics were analyzed. Methods: His6 affinity column was used to purify recombinant Butelase 1 (rButelase 1) and TLL (rTLL) from mammalian cells and E. co- li; rButelase 1 to was utilized connect the N- and C-termini of rTLL to generate circular rTLL (cTLL); the enzymatic activity, the heat stability, the protease resistance, the resistance to heat-induced precipitation, and the enzymatic kinetics were analyzed to determine the difference of rTLL and cTLL. Results: Recombinant rButelase 1 and rTLL expressed in mammalian cells and E. co-li were purified; cTLL was obtained, and the comparable enzymatic activity was found in both cTLL and rTLL. After heat treatment at 70 ℃ for 180 min, most of cTLL remained soluble and 95% of its activity, while rTLL was almost completely precipitated and lost most of its activity. cTLL has characteristics of stable proteins, such as uniform particle size and molecular weight distribution. Conclusion: Butelase 1 can effectively circularize lipase from T. lanuginosus. cTLL maintained its enzymatic activity. Meanwhile, its protein characteristics, including thermal stability and protease resistance, has also been improved.
Wen, SONG; Xia-xia, WU; Hai-wei, LOU; and Hui-min, FANG
"Study on the stability of lipase of terminally cyclized Thermomyces lanuginosus,"
Food and Machinery: Vol. 38:
8, Article 7.
Available at: https://www.ifoodmm.cn/journal/vol38/iss8/7
 WALTON N J,MAYER M J,NARBAD A.Molecules of interest:Vanillin[J].Cheminform,2003,34(38):505-515.
 MAKKAR H P S,BECKER K.Isolation of tannins from leaves of some trees and shrubs and their properties[J].Journal of Agricultural & Food Chemistry,1994,42(3):731-734.
 黄艳凤.微生物转化香兰素的初步研究[D].天津:天津科技大学,2004:21-22.HUANG Y F.Primary studies on biotransformation of vanillin[D].Tianjin:Tianjin University of Science and Technology,2004:21-22.
 FURUYA T,KUROIWA M,KINO K.Biotechnological production of vanillin using immobilized enzymes[J].Journal of Biotechnology,2017,243:25-28.
 薛岳.乙醛酸法合成香兰素工艺研究[D].上海:华东理工大学,2011:22-25.XUE Y.Synthesisof vanillin by glyoxylic acid method[D].Shanghai:East China University of Science and Technology,2011:22-25.
 SINGH A,MUKHOPADHYAY K,SACHAN S G.Biotransforma-tion of eugenol to vanillin by a novel strain Bacillus safensis SMS1003[J].Biocatalysis and Biotransformation,2018,37(4):1-13.
 HAN Z C,LONG L K,DING S J.Expression and characterization of carotenoid cleavage oxygenases from Herbaspirillum seropedicae and Rhodobacteraceae bacterium capable of biotransforming isoeugenol and 4-vinylguaiacol to vanillin[J].Frontiers in Microbiology,2019,10:1 869-1 869.
 许美玲.生物法合成香兰素和香草酸的研究[D].北京:北京化工大学,2007:18-25.XU M L.Producyion of vanillin and vanillic acid by microbial conversion[D].Beijing:Beijing University of Chemical Technology,2007:18-25.
 高锐,杨威,宋鹏飞,等.微生物制备烟用香料的研究进展[J].安徽农业科学,2017,45(2):92-96.GAO R,YANG W,SONG P F,et al.Research progress on production of tobacco flavor by microbial fermentation[J].Journal of Anhui Agricultural Sciences,2017,45(2):92-96.
 党玥,陈雪峰,刘欢,等.香兰素生物合成的研究进展[J].微生物学通报,2020,47(11):3 678-3 688.DANG Y,CHEN X F,LIU H,et al.Research progress on vanillin biosynthesis[J].Microbiology China,2020,47(11):3 678-3 688.
 SHIMONI E,RAVID U,SHOHAM Y.Isolation of a Bacillus sp capable of transforming isoeugenol to vanillin[J].Journal of Biotechnology,2000,78(1):1-9.
 OVERHAGE J,PRIEFERT H,STEINBUCHEL A.Biochemical and genetic analyses of ferulic acid catabolism in Pseudomonas sp.strain HR199[J].Applied and Environmental Microbiology,1999,65(11):4 837-4 847.
 DIGNUM M,KERLER J,VERPOORTE R.Vanilla production:Technological,chemical,and biosynthetic[J].Food Reviews International,2001,17(2):199-219.
 FALCONNIER B,LAPIERRE C,LESAGE-MEESSEN L,et al.Vanillin as a product of ferulic acid biotransformation by the white-rot fungus Pycnoporus cinnabarinus I-937:Identification of metabolic pathways[J].Journal of Biotechnolog,1994,37(2):123-132.
 LESAGE-MEESSEN L,DELATTRE M,HAON M,et al.A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus[J].Journal of Biotechnology,1996,50(3):107-113.
 TOMS A,WOOD J M.Degradation of trans-ferulic acid by Pseudomonas acidovorans[J].Biochemistry,1970,9(2):337-343.
 LUZIATELLI F,BRUNETTI L,FICCA A G,et al.Maximizing the efficiency of vanillin production by biocatalyst enhancement and process optimization[J].Frontiers in Bioengineering and Biotechnology,2019,7:279.
 严晓娟,宋勇强,牛犇,等.一株转化阿魏酸产香兰素菌株的筛选与鉴定[J].中国酿造,2015,34(10):47-52.YAN X J,SONG Y Q,NIU B,et al.Screening and identification of a strain transforming ferulic acid to vanillin[J].China Brewing,2015,34(10):47-52.
 郑璇,黄瑾,林洁,等.气相色谱—质谱法测定奶粉中香兰素含量的方法建立[J].食品与机械,2016,32(11):35-38.ZHENG X,HUANG J,LIN J,et al.Determination of vanillin and ethyl-vanillin in milk powder by gas chromatography-mass spectrometry[J].Food & Machinery,2016,32(11):35-38.
 王洁.一种芽孢杆菌J4-1发酵生产香兰素的研究[D].武汉:湖北大学,2012.WANG J.Studying one kind of Bacillus J4-1 fermentation production of the vanillin[D].Wuhan:Huibei University,2012.
 王家宝,黄美凤,杨笛,等.脂肪酶替代双乙酰酒石酸单双甘油酯对压面面团流变学和面包烘焙特性的影响[J].食品与机械,2020,36(9):38-42.WANG J B,HUANG M F,YANG D,et al.A comparative study of lipase and diacetyl tartaric esters of mono-glycerides(DATEM)on rheological properties of dough by pressing process and baking properties of bread[J].Food & Machinery,2020,36(9):38-42.
 LOKHA Y,ARANA-PEA S,RIOS N S,et al.Modulating the properties of the lipase from Thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions[J].Enzyme and Microbial Technology,2020,133:109461.
 BAUER T L,BUCHHOLZ P C F,PLEISS J.The modular structure of α/β-hydrolases[J].The FEBS Journal,2020,287(5):1 035-1 053.
 HAMDAN S H,MAIANGWA J,ALI M S M,et al.Thermostable lipases and their dynamics of improved enzymatic properties[J].Applied Microbiology and Biotechnology,2021,105(19):7 069-7 094.
 PURKAYASTHA A,KANG T J.Stabilization of proteins by covalent cyclization[J].Biotechnology and Bioprocess Engineering,2019,24(5):702-712.
 HEMU X Y,ZHANG X H,BI X B,et al.Butelase 1-mediated ligation of peptides and proteins[J].Enzyme-Mediated Ligation Methods,2019,2 012:83-109.
 CAO Y,BI X.Butelase-1 as the prototypical peptide asparaginyl ligase and its applications:A review[J].International Journal of Peptide Research and Therapeutics,2022,28(1):1-16.
 CAO Y,NGUYEN G K T,TAM J P,et al.Butelase-mediated synthesis of protein thioesters and its application for tandem chemoenzymatic ligation[J].Chem Commun(Camb),2015,51(97):17 289-17 292.
 季亚美,王立梅,齐斌.常压室温等离子体诱变选育高产脂肪酶解脂耶氏酵母[J].食品与机械,2020,36(11):10-15.JI Y M,WANG L M,QI B.Breeding of Yarrowial ipolytica with high lipase production by atmospheric room temperature plasma mutagenesis[J].Food & Machinery,2020,36(11):10-15.
 HEMU X,ZHANG X,NGUYEN G K T,et al.Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization[J].RSC Advances,2021,11(37):23 105-23 112.
 NAUMANN T A,SAVINOV S N,BENKOVIC S J.Engineering an affinity tag for genetically encoded cyclic peptides[J].Biotechnol Bioeng,2005,92(7):820-830.