Screening, identification and enzymatic properties of α-cyclodextrin glycosyltransferase producing strains
TAO Dawei1, NING Xibin1,2,3,4*
1(College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China) 2(Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China) 3(Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306, China) 4(National R&D Branch Center for Freshwater Aquatic Products Processing Technology (Shanghai), Shanghai 201306, China)
Abstract: A high-yield α-cyclodextrin glycosyltransferase strain was screened from planting fields on a farm in Xinchang town, Pudong New Area, Shanghai, and its enzymatic properties were studied to improve the thermal stability and pH stability of this enzyme. According to the morphological characteristics, physiological and biochemical identification and 16S rRNA sequence alignment, the strain was identified as Paenibacillus macerans, and was named P. macerans TLLY7. Enzymatic properties indicated that 50 ℃ and pH 6.0 were the most suitable reaction conditions. It showed good stability under the conditions of 40-45 ℃ and pH 6.0-9.0. Ca2+, Mn2+ and Zn2+ had positive effects on the enzyme, while Fe2+, Cu2+ and Co2+ had negative effects. Ca2+ had the strongest positive effect on enzyme activity with an increase of 25%, Cu2+ had the strongest negative effect with an inhibition rate of 33%. The effect of K+ and Mg2+ on the enzyme activity was not obvious. Preliminary studies on the enzymatic properties of α-cyclodextrin glucosyltransferase produced by strain TLLY7 indicate that it has the value of further research and potential application.
TERADA Y, YANASE M, TAKATA H, et al.Cyclodextrins are not the major cyclic alpha-1,4-glucans produced by the initial action of cyclodextrin glucanotransferase on amylose[J].J Biol Chem, 1997, 272(25):15 729-15 733.
[2]
SINGH M, SHARMA R, BANERJEE U C.Biotechnological applications of cyclodextrins[J].Biotechnology Advances, 2002, 20(5):341-359.
[3]
王蕾. 环糊精葡萄糖基转移酶的产物特异性分子改造及发酵制备研究[D].无锡:江南大学, 2018.WANG L.Product specificity engineering and fermentation of cyclodextrin glycosyltransferase[D].Wuxi:Jiangnan University, 2018.
[4]
陈龙然. 产α-环糊精葡萄糖基转移酶的菌株分离、产酶条件优化与酶学特性研究[D].杭州:浙江大学, 2005.CHEN L R.Breeding and optimized fermentation of an α-glucanotransferase producing strain and its enzymatic characteristics[D].Hangzhou:Zhejiang University, 2005.
[5]
雷新辉. α-环糊精葡萄糖基转移酶高产菌株的选育及产酶条件的研究[D].西安:陕西科技大学, 2013.LEI X H.Research on α-cyclodextrin glycosyltransferase high-yield strain breeding and enzyme production conditions[D].XÍan:Shaanxi University of Science & Technology, 2013.
[6]
VEEN B A, ALEBEEK G J, UITDEHAAG J C, et al.The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms[J].Eur J Biochem, 2000, 267(3):658-665.
[7]
金征宇, 柏玉香, 王金鹏.环糊精系列综述之一 环糊精葡萄糖基转移酶的筛选及其定向改造[J].食品与生物技术学报, 2012, 31(2):113-123.JIN Z Y, BAI Y X, WANG J P.Screen and modification of cyclodextrin glycosyltransferase[J].Journal of Food Science and Biotechnology, 2012, 31(2):113-123.
[8]
MARTINS R F, HATTI-KAUL R.Bacillus agaradhaerens LS-3C cyclodextrin glycosyltransferase:Activity and stability features[J].Enzyme and microbial technology, 2003,33(6):819-827.
[9]
ATANASOVA N, PETROVA P, IVANOVA V, et al.Isolation of novel alkaliphilic Bacillus strains for cyclodextrin glucanotransferase production[J].Applied Biochemistry and Biotechnology, 2008,149(2):155-167.
[10]
VIVIAN M, ANTONIO J G, PAULO R A, et al.Cyclodextrin glycosyltransferase production by new Bacillus sp.strains isolated from brazilian soil[J].Brazilian Journal of Microbiology:Publication of the Brazilian Society for Microbiology, 2008, 39(4):682-688.
[11]
FREITAS T L D, MONTI R,CONTIERO J, et al.Production of CGTase by a Bacillus alkalophilic CGII strain isolated from wastewater of a manioc flour industry[J].Brazilian Journal of Microbiology, 2004,35(3):255-260.
[12]
COELHOS L D A,MAGALHÃES V C,MARBACH P A S, et al.A new alkalophilic isolate of Bacillus as a producer of cyclodextrin glycosyltransferase using cassava flour[J].Brazilian Journal of Microbiology, 2016, 47(1):120-128.
[13]
张佳瑜, 吴丹, 李兆丰, 等.来源于软化芽孢杆菌的环糊精葡萄糖基转移酶在毕赤酵母和枯草杆菌中的表达[J].生物工程学报, 2009, 25(12):1 948-1 954.ZHANG J Y, WU D, LI Z F, et al.Expression of Paenibacillus macerans cyclodextrin glycosyltransferase in Pichia pastoris and Bacillus subtilis[J].Chinese Journal of Biotechnology, 2009, 25(12):1 948-1 954.
[14]
LEJEUNE A, SAKAGUCHI K, IMANAKA T.A spectrophotometric assay for the cyclization activity of cyclomaltohexaose (α-cyclodextrin) glucanotransferase[J].Analytical Biochemistry, 1989,181(1):6-11.
[15]
李兆丰. 软化类芽孢杆菌α-环糊精葡萄糖基转移酶在大肠杆菌中的表达及其产物特异性分析[D].无锡:江南大学, 2009.LI Z F.Expression of α-cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli[D].Wuxi:Jiangnan University, 2009.
[16]
GARRITY G, BELL J, LILBURN T.Bergey's manual of systematic Bacteriology[M].(Ninth Edition) New York:Springer, 2004.
[17]
东秀珠, 蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社, 2001.DONG X Z, CAI M Y.Identification Manual of Common Bacterial Systems[M].Beijing:Science Press, 2001.
[18]
陈成, 宁喜斌.产β-内酰胺酶菌株的筛选、鉴定及酶学性质初步研究[J].食品与发酵工业, 2019, 45(24):77-83.CHEN C, NING X B.Screening, identification and enzymatic properties of β-lactamase producing strains[J].Food and Fermentation Industries, 2019, 45(24):77-83.
[19]
张晓磊. 一株α-环糊精葡萄糖基转移酶产生菌的选育、鉴定、产酶条件优化和酶学性质的研究[D].上海:上海海洋大学, 2015.ZHANG X L.Breeding, identification and optimized fermentation of an α-cyclomaltodextrin glucanotransferase producing strain and its enzymatic characterization[D].Shanghai:Shanghai Ocean University, 2015.
[20]
陈龙军, 林陈强, 张慧, 等.嗜热芽胞杆菌α-环糊精葡萄糖基转移酶在枯草芽胞杆菌中的表达[J].福建农业学报, 2019,34(5):600-605.CHEN L J, LIN C Q, ZHANG H, et al.Expression of α-cyclodextrin glycosyltransferase gene of Gebacillius sp.CHB1 in Bacillus subtilis[J].Fujian Journal of Agricultural Sciences, 2019, 34(5):600-605.
[21]
叶学军. 地芽孢杆菌CHB1产CGTase的分离纯化及基因的克隆与表达[D].福州:福州大学, 2014.YE X J.Purification, characterization, gene cloning and expression of cyclodextrin glycosyltransferase from Genbacillus caldoxylosilyticus CHB1[D].Fuzhou:Fuzhou University, 2014.
[22]
刘德海, 权淑静, 杨文玲, 等.高产α-半乳糖苷酶菌株的筛选鉴定及发酵培养基的优化[J].中国酿造, 2019, 38(12):80-86.LIU D H, QUAN S J, YANG W L, et al.Screening, identification and fermentation medium optimization of high α-galactosidase-producing strain[J].China Brewing, 2019, 38(12):80-86.
[23]
王文娟, 刘志东, 宁喜斌.南极磷虾产低温淀粉酶菌株的筛选、鉴定及酶学性质研究[J].食品与发酵工业, 2019, 45(19):38-44.WANG W J, LIU Z D, NING X B.Screening, identification and enzymatic properties of low-temperature amylase producing strain in Antarctic kril[J].Food and Fermentation Industries, 2019, 45(19):38-44.
[24]
张昌志. 罗汉果内生菌中产环糊精葡萄糖基转移酶菌株的筛选及酶法改性罗汉果苦味苷的研究[D].桂林:广西师范大学, 2018.ZHANG C Z.Screening of cyclodextrin glucosyltransferase producing strains from endophytic of Siraitia grosvenorii and study on enzymatic modification of Siraitia grosvenorii bitter glycoside[D].Guilin:Guangxi Normal University, 2018.
[25]
曹冬梅. 产β-环糊精葡萄糖基转移酶高温菌株HY15发酵条件的优化及酶动力学研究[D].长春:吉林农业大学, 2011.CAO D M.Research on kinetics and fermentation conditions of β-cyclodextrin glycosyltransferase production from thermophiles HY15[D].Changchun:Jilin Agricultural University, 2011.
[26]
LI Z, LI B, GU Z, et al.Extracellular expression and biochemical characterization of α-cyclodextrin glycosyltransferase from Paenibacillus macerans[J].Carbohydrate Research, 2010, 345(7):886-892.
[27]
NOOR Y M, SAMSULRIZAL N H, JEMA'ON N A, et al.A comparative genomic analysis of the alkalitolerant soil bacterium Bacillus lehensis G1[J].Gene, 2014, 545(2):253-261.
2021, Vol. 47 
