高产酯化酶红曲菌的筛选、鉴定及酶学性质研究
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摘要
该研究从研究室保藏的10株红曲霉菌株中筛选高产酯化酶菌株,通过分子生物学技术对其进行鉴定,并对其最适培养基进行选择,最后对其所产的酯化酶酶学特性进行初步研究。结果表明,筛选获得一株高产酯化酶菌株X1,并被鉴定为血红红曲霉(Monascus sanguineus)。其最适产酶培养基为可溶性淀粉70 g/L,大豆蛋白胨20 g/L,NaNO_3 2 g/L,KH_2PO_4 1 g/L,MgSO_4·7H_2O 2 g/L,初始pH值4.5。采用最优培养基,在30℃、180 r/min条件下发酵4 d,酯化酶活力为315.19 U/m L。该酯化酶的最适反应温度为40℃,在25～35℃范围内稳定性较好;最适pH值为5.0,在pH为6.0时稳定性较高;金属离子Ca~(2+)可提高该酯化酶活性,Mg~(2+)、Fe~(2+)、Na~+和Ag~+则有不同程度抑制作用,且Ag~+抑制作用最明显。
A high-yield esterase Monascus was screened from 10 Monascus strains in the laboratory and identified by molecular biology technology,the optimal medium was screened, and the enzymatic properties of esterase produced by Monascus were preliminary studied. The results showed that a high-yield esterase Monascus strain X1 was screened and was identified as Monascus sanguineous. The optimum medium for esterase production was soluble starch 70 g/L, soybean peptone 20 g/L, NaNO_3 2 g/L, KH_2PO_4 1 g/L, MgSO_4·7H_2O 2 g/L and initial pH 4.5. The strain was fermented for 4 d at 30 ℃ and 180 r/min in the optimal medium, the esterase activity was 315.19 U/ml. The optimum reaction temperature of the esterase was 40 ℃,and the stability was better at 25-35 ℃. The optimum reaction pH value was 5.0, and the stability was better at 6.0. The metal ions Ca~(2+) increased the activity of the esterase, while Mg~(2+), Fe~(2+), Na~+ and Ag~+ had different degrees of inhibition, and the inhibitory effect of Ag~+ was the most obvious.
A high-yield esterase Monascus was screened from 10 Monascus strains in the laboratory and identified by molecular biology technology,the optimal medium was screened, and the enzymatic properties of esterase produced by Monascus were preliminary studied. The results showed that a high-yield esterase Monascus strain X1 was screened and was identified as Monascus sanguineous. The optimum medium for esterase production was soluble starch 70 g/L, soybean peptone 20 g/L, NaNO_3 2 g/L, KH_2PO_4 1 g/L, MgSO_4·7H_2O 2 g/L and initial pH 4.5. The strain was fermented for 4 d at 30 ℃ and 180 r/min in the optimal medium, the esterase activity was 315.19 U/ml. The optimum reaction temperature of the esterase was 40 ℃,and the stability was better at 25-35 ℃. The optimum reaction pH value was 5.0, and the stability was better at 6.0. The metal ions Ca~(2+) increased the activity of the esterase, while Mg~(2+), Fe~(2+), Na~+ and Ag~+ had different degrees of inhibition, and the inhibitory effect of Ag~+ was the most obvious.
引文
[1]CHEN W P,HE Y,ZHOU Y X,et al.Edible filamentous fungi from the species Monascus:Early traditional fermentations,modern molecular biology,and future genomics[J].Compr Rev Food Sci Food Safety,2015,14(5):555-567.
[2]胡娜,吴鑫颖,李付丽,等.紫色红曲霉FBKL3.0018产酯化酶的酶学性质研究[J].中国酿造,2017,36(5):123-127.
[3]谢恩举,徐世江.红曲酯化酶制剂在浓香型粮食酒生产中的应用[J].中国酿造.2000,19(1):27.
[4]罗小叶,胥思霞,邱树毅,等.高酯化大曲催化增香技术在浓香型白酒生产上的应用[J].中国酿造,2018,37(10):141-144.
[5]刘新宇.红曲霉的分离纯化及在清香型白酒中的应用[D].临汾:山西师范大学,2014.
[6]杜春迎.浓香型大曲中高产酯化酶菌株的筛选及产酶条件优化究[D].哈尔滨:黑龙江大学,2012.
[7]张磊,张华玲,刘绪,等.红曲霉产酯化酶条件的研究[J].酿酒科技,2013(9):68-70.
[8]赵吉兴,李耀,李凯.红曲霉固体发酵生产酿造增香发酵剂的研究与应用[J].中国酿造.2013,32(3):114-117.
[9]刘丹,杨帆,薛意斌,等.红曲霉Monascus sanguineus X1处理黄水的培养基优化及酯化液制备.食品科学技术学报,http://kns.cnki.net/kcms/detail/10.115/TS.20190130.1537.002.html.
[10]CHEN S,LIU K Y,ZHENG J,et al.Study on enzymatic esterification conditions of yellow water optimized by response surface methodology[J].Sci Technol Food Ind,2012,33(12):205-209.
[11]CHENG G.Liquor industry status and prospects for Monascus[J].Mod Food,2016,12(6):15-17.
[12]艾金忠,马美荣,聂建光,等.高酯化酶活性红曲霉及生香酵母在二锅头白酒生产中的应用[J].酿酒,2013,40(4):33-34.
[13]葛龙,赵艳,章亭洲.固态发酵技术的特点与应用[J].饲料与畜牧,2010(8):54-56.
[14]林琳,王昌禄,李贞景,等.mok E基因过表达对红曲霉Monacolin K产量、菌丝及孢子形态的影响[J].食品科学,2018,39(8):45-49.
[15]潘名志.红曲霉产酯化酶特性及其酶学性质的研究[D].贵阳:贵州大学,2009.
[16]MANTER D K,VIVANCO J M.Use of the ITS primers,ITS1F and ITS4,to characterize fungal abundance and diversity in mixed-template samples by qPCR and length heterogeneity analysis[J].J Microbiol Meth,2007,71(1):7-14.
[17]KUMAR S,TAMURA K,NEI M.MEGA:Molecular evolutionary genetics analysis software for microcomputers[J].Bioinformatics,1994,10(2):189-191.
[18]CSOTA J,VENDRUSCOLO F.Production of red pigments by Monascusruber CCT 3802 using lactose as a substrate[J].Biocatal Agr Biotech,2017,11:50-55.
[19]HILARES R T,DE-SOUZA R A,MARCELINO P F,et al.Sugarcane bagasse hydrolysate as a potential feedstock for red pigment production by Monascus ruber[J].Food Chem,2017,doi:https://doi.org/10.1016/j.food chem.2017.11.111.
[20]VAREJAO N,DE-ANDRADE R A,ALMEIDA R V,et al.Structural mechanism for the temperature-dependent activation of the hyperthermophilicPf2001 esterase[J].Structure,2018,26(2):doi:10.1016/j.str.2017.12.004.
[21]CHEN L,DANG G,DENG X,et al.Characterization of a novel exported esterase Rv3036c from Mycobacterium tuberculosis[J].Protein Expres Purif,2014,104:50-56.
[2]胡娜,吴鑫颖,李付丽,等.紫色红曲霉FBKL3.0018产酯化酶的酶学性质研究[J].中国酿造,2017,36(5):123-127.
[3]谢恩举,徐世江.红曲酯化酶制剂在浓香型粮食酒生产中的应用[J].中国酿造.2000,19(1):27.
[4]罗小叶,胥思霞,邱树毅,等.高酯化大曲催化增香技术在浓香型白酒生产上的应用[J].中国酿造,2018,37(10):141-144.
[5]刘新宇.红曲霉的分离纯化及在清香型白酒中的应用[D].临汾:山西师范大学,2014.
[6]杜春迎.浓香型大曲中高产酯化酶菌株的筛选及产酶条件优化究[D].哈尔滨:黑龙江大学,2012.
[7]张磊,张华玲,刘绪,等.红曲霉产酯化酶条件的研究[J].酿酒科技,2013(9):68-70.
[8]赵吉兴,李耀,李凯.红曲霉固体发酵生产酿造增香发酵剂的研究与应用[J].中国酿造.2013,32(3):114-117.
[9]刘丹,杨帆,薛意斌,等.红曲霉Monascus sanguineus X1处理黄水的培养基优化及酯化液制备.食品科学技术学报,http://kns.cnki.net/kcms/detail/10.115/TS.20190130.1537.002.html.
[10]CHEN S,LIU K Y,ZHENG J,et al.Study on enzymatic esterification conditions of yellow water optimized by response surface methodology[J].Sci Technol Food Ind,2012,33(12):205-209.
[11]CHENG G.Liquor industry status and prospects for Monascus[J].Mod Food,2016,12(6):15-17.
[12]艾金忠,马美荣,聂建光,等.高酯化酶活性红曲霉及生香酵母在二锅头白酒生产中的应用[J].酿酒,2013,40(4):33-34.
[13]葛龙,赵艳,章亭洲.固态发酵技术的特点与应用[J].饲料与畜牧,2010(8):54-56.
[14]林琳,王昌禄,李贞景,等.mok E基因过表达对红曲霉Monacolin K产量、菌丝及孢子形态的影响[J].食品科学,2018,39(8):45-49.
[15]潘名志.红曲霉产酯化酶特性及其酶学性质的研究[D].贵阳:贵州大学,2009.
[16]MANTER D K,VIVANCO J M.Use of the ITS primers,ITS1F and ITS4,to characterize fungal abundance and diversity in mixed-template samples by qPCR and length heterogeneity analysis[J].J Microbiol Meth,2007,71(1):7-14.
[17]KUMAR S,TAMURA K,NEI M.MEGA:Molecular evolutionary genetics analysis software for microcomputers[J].Bioinformatics,1994,10(2):189-191.
[18]CSOTA J,VENDRUSCOLO F.Production of red pigments by Monascusruber CCT 3802 using lactose as a substrate[J].Biocatal Agr Biotech,2017,11:50-55.
[19]HILARES R T,DE-SOUZA R A,MARCELINO P F,et al.Sugarcane bagasse hydrolysate as a potential feedstock for red pigment production by Monascus ruber[J].Food Chem,2017,doi:https://doi.org/10.1016/j.food chem.2017.11.111.
[20]VAREJAO N,DE-ANDRADE R A,ALMEIDA R V,et al.Structural mechanism for the temperature-dependent activation of the hyperthermophilicPf2001 esterase[J].Structure,2018,26(2):doi:10.1016/j.str.2017.12.004.
[21]CHEN L,DANG G,DENG X,et al.Characterization of a novel exported esterase Rv3036c from Mycobacterium tuberculosis[J].Protein Expres Purif,2014,104:50-56.