Cloning,Recombinant Expression and Enzymatic Properties of α-Amylase Gene from Laceyella sp.

doi:10.13560/j.cnki.biotech.bull.1985.2019-1053

Abstract

Abstract: Cloning and heterologous expression of amylase gene in the amylase-producing strain screened from soil were conducted and their enzymatic properties were analyzed. An amylase-producing strain was isolated and numbered MF-8-1,which was from the soil sample near the Anyang Flour Mill by using the solid starch screening medium. Then the amylase gene AmyL was cloned by corresponding primers with homology cloning. Further the recombinant plasmid of pET-22b-AmyL was constructed by ligating Amy and expression vector pET-22b（+）,and transformed into expression host strain Escherichia coli BL21（DE3）. Finally,the recombinant enzyme was isolated and purified and its enzymatic properties were determined. As results,AmyL was expressed successfully in E. coli BL21（DE3）,and the molecular weight of recombinant enzyme AmyL was 55 kD. Recombinant enzyme presented maximum activity at pH 6.0 and 45℃. It had better temperature stability when the temperature was lower than 60℃. The enzyme was highly stable at pH 6.0-10.0. The kinetics studies of recombinant enzyme showed that the specific activity,K_m and V_max of the enzyme were 185.39 U/mg,0.95 mg/mL and 343.12 μmol/（min ·mg）,respectively. AmyL is characterized by stable enzyme activity under medium temperature and alkalinity,thus it has potential application value in detergent,textile,paper making and other industries.

Key words: Laceyella sp., α-amylase, gene cloning, heterologous expression, enzymatic properties

ZHAO Hai-yan, SONG Chen-bin, LIU Zheng-ya, MA Xing-rong, SHANG Hui-hui, LI An-hua, GUAN Xian-jun, WANG Jian-she. Cloning,Recombinant Expression and Enzymatic Properties of α-Amylase Gene from Laceyella sp.[J]. Biotechnology Bulletin, 2020, 36(8): 23-33.

References

[1] 赵国华, 张蕴玉, 雷琳, 等. 淀粉对微生物的结合作用及应用研究进展[J]. 中国食品学报, 2019, 3:1-12.
Zhao GH, Zhang YY, Lei L, et al.Research advances in the binding mechanism of starch to microorganisms and its applications[J]. Journal of Chinese Institute of Food Science and Technology, 2019, 3 :1-12.
[2] Sajilata MG, Singhal RS, Kulkarni PR.Resistant starch-a review[J]. Comprehensive Reviews in Food Science and Food Safety, 2006, 5(1):1-17.
[3] Gupta R, Gigras P, Mohapatra H, et al.Microbial α-amylases:a biotechnological perspective[J]. Process Biochemistry, 2003, 38(11):1599-1616.
[4] 中华人民共和国农业部. 中国农业统计资料[M]. 北京:中国农业出版社, 2018.
Ministry of Agriculture, PRC. China agriculture statistical report[J]. Beijing:China Agriculture Press, 2018.
[5] 左丽君, 张爱忠, 姜宁. 直链支链淀粉比对动物肠道内消化、肠道菌群和肠道组织形态影响的研究进展[J]. 饲料博览, 2018(1):1-3.
Zuo LJ, Zhang AZ, Jiang N.Research progress on the effect of the proportion of amylose and amylopectin on starch digestion, intestinal microflora and intestinal tissue morphology in the intestines of animals[J]. Feed Review, 2018(1):1-3.
[6] 杨力权, 杨国保, 陈贵元等. 一株高温酸性淀粉酶产生菌的分离、筛选、鉴定及其酶学性质研究[J]. 云南大学学报:自然科学版, 2019, 41(5):1047-1054.
Yang LQ, Yang BG, Chen GY, et al.Isolation, screening and identification of a thermostable acidic amylase-producing strain and its enzymatic properties[J]. Journal of Yunnan University: Natural Sciences Edition, 2019, 41(5):1047-1054.
[7] 王培力. 高温α-淀粉酶耐热嗜酸的分子改良及其高效表达的研究[D]. 北京:中国农业科学院, 2016.
Wang PL.Improvement of the thermal and acidic stability and efficient expression of the alpha amylase BLA[D]. Beijing:Chinese Academy of Agriculture Science, 2016.
[8] Shah IJ, Gami PN, Shukla RM, et al.Optimization for α-amylase production by Aspergillus oryzae using submerge fermentation technology[J]. Basic Research Journal Microbiology, 2014, 1:1-10.
[9] Wang X, Kan G, Shi C, et al.Purification and characterization of a novel wild-type α-amylase from Antarctic sea ice bacterium Pseudoalteromonas sp. M175[J]. Protein Expression and Purification, 2019, 164:105444.
[10] Gopinath SCB, Aubu P, Arshad MKM, et al.Biotechnological processes in microbial amylase production[J]. BioMed Research International, 2017:1272193.
[11] Struyf N, Verspreet J, Verstrepen KJ, et al.Investigating the impact of α-amylase, α-glucosidase and glucoamylase action on yeast-mediated bread dough fermentation and bread sugar levels[J]. Journal of Cereal Science, 2017, 75:35-44.
[12] Amel EO, Gagaoua M, Bourekoua H, et al.Improving bread quality with the application of a newly purified thermostable α-amylase from Rhizopus Oryzae FSIS4[J]. Foods, 2017, 6(1):1.
[13] 朱蓓蓓, 周杰, 朱娣, 等. 超声波辅助退浆节能强化技术[J]. 印染, 2016, 42(21):19-22.
Zhu BB, Zhou J, Zhu D, et al.Ultrasonic assisted desizing technology for energy saving[J]. Dyeing and Finishing, 2016, 42(21):19-22.
[14] Morais RR, Pascoat AM, Pereira-Júnior MA, et al.Bioethanol production from Solanum lycocarpum starch:A sustainable non-food energy source for biofuels[J]. Renewable Energy, 2019:140.
[15] 李文钊, 臧传刚, 李义, 等. α-淀粉酶的研究与应用进展[J]. 当代化工, 2017, 46(11):2292-2299.
Li WZ, Zang CG, Li Y, et al.Research and application progress of α-Amylase[J]. Contemporary Chemical Industry, 2017, 46(11):2292-2299.
[16] 张余慧, 程晓芳, 袁丹丹, 等. 浅谈淀粉酶在动物生产中的应用研究进展[J]. 广东饲料, 2018, 27(3):33-35.
Zhang YH, Cheng XF, Yuan DD, et al.Application of amylase in animal towel production[J]. Guangdong Feed, 2018, 27(3):33-35.
[17] Sindhu R, Binod P, Madhavan A, et al.Molecular improvements in microbial α-amylases for enhanced stability and catalytic efficiency[J]. Bioresource Technology, 2017, 245:1740-1748.
[18] Dey TB, Kumar A, Banerjee R, et al.Improvement of microbial α-amylase stability:strategic approaches[J]. Process Biochemistry, 2016, 51(10):1380-1390.
[19] Shukla RJ, Singh SP.Characteristics and thermodynamics of α-amylase from thermophilic actinobacterium, Laceyella sacchari TSI-2[J]. Process Biochemistry, 2015, 50(12):2128-2136.
[20] 姜钊. 高温放线菌科系统演化与菌丝分化的多组学研究[D]. 昆明:云南大学, 2018.
Jiang Z.The systematics of family thermoactinomycetaceae and the multi-omics research for its hyphae differentiation[J]. Kunming:Yunnan University, 2018.
[21] Yoon JH, Kim IG, Shin YK, et al.Proposal of the genus Thermoactinomyces sesu strict and three new genera, Laceyella, Thermoflavimicrobium and Seinonella, on the basis of phenotypic, phylogenetic and chemotaxonomic analyses[J]. International Journal of Systematic and Evolutionary Microbiology, 2005, 55:395-400.
[22] Li D, Huang W, Wang CX, et al.The complete genome sequence of the thermophilic bacterium Laceyella sacchari FBKL4. 010 reveals the basis for tetramethylpyrazine biosynthesis in Moutai-flavor Daqu[J]. Microbiologyopen, 2019:e922.
[23] 李豆南, 黄魏, 王晓丹, 等. 酱香型大曲中高温放线菌的筛选及风味成分分析[J]. 食品科学, 2018, 39(6):171-176.
Li DN, Huang W, Wang XD, et al.Identification and flavor profile of a thermoactinomycetaceae strain separated from moutai-flavor Daqu[J]. Food Science, 2018, 39(6):171-176.
[24] Ming H, Ji WL, Li S, et al.Laceyella thermophila sp. nov. , a thermophilic bacterium isolated from a hot spring[J]. International Journal of Systematic and Evolutionary Microbiology, 2017, 67(8):2953-2958.
[25] Zhu WJ, Li Y, Jia HH, et al.Expression, purification and characterization of a thermostable leucine dehydrogenase from the halophilic thermophile Laceyella sacchari[J]. Biotechnology Letters, 2016, 38(5):855-861.
[26] Lomthong T, Hanphakphoom S, Kongsaeree P, et al.Enhancement of poly(L-lactide)-degrading enzyme production by Laceyella sacchari LP175 using agricultural crops as substrates and its degradation of poly(L-lactide)polymer[J]. Polymer Degradation and Stability, 2017, 143:64-73.
[27] 施庆珊, 梁文涛, 疏秀林, 等. 一株高温放线菌及其在造纸污泥堆肥过程中的应用[J]. 农业环境科学学报, 2008(1):368-371.
Shi QS, Liang WT, Shu XL, et al.A Thermophylic actinomycete strain and its application on composting of pulp and paper mill sludges[J]. Journal of Agro-Environment Science, 2008(1):368-371.
[28] 朱国东, 陈波, 张兰兰, 等. 高温放线菌莱斯氏属RHA1菌株产低分子量α-淀粉酶的初步研究[J]. 生物技术通报, 2011(10):199-205.
Zhu GD, Chen B, Zhang LL, et al.Purification and characterization of a novel low molecular weight α-amylase from thermophilic actinomycete strain Laceyella sp.RHA1[J]. Biotechnology Bulletin, 2011(10):199-205.
[29] 孟庆龙. 热稳定5-氨基乙酰丙酸合成酶的筛选、表达、酶学性质研究及其应用[D]. 天津:天津大学, 2016.
Meng QL.Screening, expression, characterization and application of thermostable 5-aminolevulinlic acid synthase[D]. Tianjin:Tianjin University, 2016.
[30] Xiao Y, Zeng GM, Yang ZH, et al.Changes in the acrinomycetal communities during continuous thermophilic composing as revealed by denaturing gradient gel electrophoresis and quantitative PCR[J]. Bioresource Technology, 2011, 102(2):1383-1388.
[31] Singh V, Pandey VC, Agrawal S.Potential of Laceyella sacchari strain B42 crude xylanase in biobleaching of kraft pulp[J]. African Journal of Biotechnology, 2013, 12:570-579.
[32] Dolashki A, Voelter W, Gushterova A, et al.Isolation and characterization of novel tyrosinase from Laceyella sacchari[J], Protein Pept Lett, 2012, 19(5):538-543.
[33] Jørgensen CM, Madsen SM, Vrang A, et al.Recombinant expression of Laceyella sacchari thermitase in Lactococcus lactis[J]. Protein Expression and Purification, 2013, 92(2):148-155.
[34] Kobayashi T, Uchimura K, Kubota T, et al.Biochemical and genetic characterization of beta-1, 3 glucanase from a deep subseafloor Laceyella putida[J]. Appl Microbiol Biotechnol, 2016, 100(1):203-214.
[35] Lomthong T, Lertwattanasakul N, Kitpreechavanich V.Production of raw starch degrading enzyme by the thermophilic filamentous bacterium Laceyella sacchari LP175 and its application for ethanol production from dried cassava chips[J]. Starch-Starke, 2016:68.
[36] Ahmed KA, Mohamed I, Noha FO.Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3[J]. International Journal of Biological Macromolecules, 2019, 132:1274-1281.
[37] El-Sayed AKA, Abou-Dobara MI, El-Fallal AA, et al.Gene sequence, modelling and enzymatic characterization of α-amylase AmyLa from the Thermophile Laceyella sp. DS3[J]. Starch-Stärke, 2016:69.
[38] Shukla RJ, Singh SP.Structural and catalytic properties of immobilized α-amylase from Laceyella sacchari TSI-2[J]. International Journal of Biological Macromolecules, 2016, 85:208-216.
[39] Kumar S, Stecher G, Tamura K.MEGA7:Molecular evolutionary genetics analysis version 7. 0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7):1870-1874.
[40] Saitou N, Nei M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, 4(4):406-425.
[41] Tuzlakoglu MO, Akbulut N, Issever SO, et al.Ligase-independent cloning of amylase gene from a local Bacillus subtilis isolate and biochemical characterization of the purified enzyme[J]. New Biotechnology, 2013, 171(2):263-278.
[42] Zakalskiy AE, Zakalska OM, Rzhepetskyy YA, et al.Overexpression of(His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography[J]. Protein Expression and Purification, 2012, 81(1):63-68.
[43] Miller GL.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical Biochemistry, 1959, 31(3):426-428.
[44] Lineweaver H, Burk D.The determination of enzyme dissociation constants[J]. Journal of America Chemistry Society, 1934, 56(3):658-666.
[45] Takenaka S, Miyatake A, Tanaka K, et al.Characterization of the native form and the carboxy-terminally truncated halotolerant form of α-amylases from Bacillus subtilis strain FP-133[J]. Journal of Basic Microbiology, 2015, 55(6):780-789.
[46] Vrsalović PA, Findrik BZ, Durđa VR.Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis:effect of calcium, pH and temperature[J]. Bioprocess and Biosystems Engineering, 2013, 36(1):117-126.
[47] Priyadarshini S, Pradhan SK, Ray P.Production, characterization and application of thermostable, alkaline alpha-amylase(AA11)from Bacillus cereus strain SP-CH11 isolated from Chilika Lake[J]. International Journal of Biological Macromolecules, 2020, 145:804-812.
[48] Gutiérrez-García AK, Alvarez-Guzmán CL, Leon-Rodriguez Ade.Autodisplay of alpha amylase from Bacillus megaterium in E. coli for the bioconversion of starch into hydrogen, ethanol and succinic acid[J]. Enzyme and Microbial Technology, 2020, 134:109477.
[49] 何江红, 王荣钰, 舒小芳, 等. 川西藏区牦牛酸醡肉中产蛋白酶和淀粉酶菌株的分离鉴定及其生理特性分析[J]. 中国调味品, 2018, 43(12):60-65.
He JH, Wang RY, Shu XF, et al.Isolation and identification of protease and amylase producing strains in Sour Yak Meat from western Sichuan Tibetan areas and analysis of their physiological characteristics[J]. China Condiment, 2018, 43(12):60-65.
[50] El-Sayed AK1, Abou Dobara MI, El-Fallal AA, et al. Purification, sequencing, and biochemical characterization of a novel calcium-independent α-amylase AmyTVE from Thermoactinomyces vulgaris[J]. Applied Biochemistry and Biotechnology, 2013, 170(3):483-497.
[51] Wang YC, Zhao N, Ma JW, et al.High-level expression of a novel α-amylase from Thermomyces dupontiiin Pichia pastoris and its application in maltose syrup production[J]. International Journal of Biological Macromolecules, 2019, 127:683-692.
[52] Hofemeister B, Konig S, Hoang V, et al.The gene amyE(TV1)codes for a nonglucogenic α-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis[J]. Applied and Environmental Microbiology, 1994, 60:3381-3389.
[53] 韩蕴琪. 蛋白质半理性设计提高Candida Rugosa Lipase1的热稳定性[D]. 长春:吉林大学, 2011.
Han YQ.Increasing the thermostability of CRL1 by protein semi-rational design[J]. Changchun:Jilin University, 2011.
[54] Suvd D, Fujimoto Z, Takase K, et al.Crystal structure of Bacillus stearothermophilus alpha-amylase:possible factors determining the thermostability[J]. The Journal of Biochemistry, 2001, 129(3):461-468.
[55] 高艳云. 分子改造提高酸性α-淀粉酶热稳定性[D]. 上海:华东理工大学, 2016.
Gao YY.Improving thermostability of acidic α-amylase through molecular modification[D]. Shanghai: East China University of Science and Technology, 2016.
[56] Mabrouk SB, Aghajari N, Ali MB, et al.Enhancement of the thermostability of the maltogenic amylase MAUS149by Gly312Ala and Lys436Arg substitutions[J]. Bioresource Technology, 2011, 102:1740-1746.