用于β-半乳糖苷酶高效发酵制备的廉价工业培养基配方开发
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摘要
以乳清粉为主要成分开发了一套高产β-半乳糖苷酶且廉价的工业培养基配方.首先,考察了乳清粉替代原培养基中碳源(乳糖)和部分氮源的可行性,优化了乳清粉最佳浓度和氮源(酵母膏、蛋白胨)的最佳组合,筛选了利于β-半乳糖苷酶分泌合成的无机盐种类,获得了一套较理想的工业培养基配方:乳清粉60 g/L,酵母膏9g/L,蛋白胨3 g/L,MnCl20.03 g/L,ZnCl_2 0.058 g/L,pH 8.0,应用于乳酸克鲁维酵母发酵制备β-半乳糖苷酶,酶活达到33.4 U/mL(摇瓶培养)和77.47 U/mL(发酵罐培养),单位酶活力达到13.74 U/mg生物量,显著优于传统发酵培养基;且乳清粉廉价易得,特别适合作为工业培养基主成分应用于β-半乳糖苷酶的规模制备.
In this study, cheap industrial media using whey powder as the main substrate was developed successfully for high-production of β-galactosidase by Kluyveromyces lactis. Firstly, the feasibility that whey powder replacing of the carbonyl(lactose) and the partial nitrogen source in the traditional media was investigated. Then the concentration of whey powder and the nitrogen source(yeast extract, peptone)were optimized, and inorganic salts that were beneficial to the synthesis of β-galactosidase were screened.Finally, cheap industrial media was obtained as follows: whey powder 60 g/L, yeast extract 9 g/L, peptone 3 g/L, MnCl20.03 g/L, ZnCl20.058 g/L and pH 8.0. Under the optimized conditions, the β-galactosidase activity was reached up to 33.40 U/mL(in shaken flasks) and 77.47 U/mL(in fermenter), and the special activity reached 13.74 U/mg(biomass). These results were significantly better than that of the traditional media. Since whey powder as a by-product of dairy industry is a cheap waste source, the optimized industrial media using whey powder as the main substrate is particularly suitable for large-scale production of β-galactosidase.
In this study, cheap industrial media using whey powder as the main substrate was developed successfully for high-production of β-galactosidase by Kluyveromyces lactis. Firstly, the feasibility that whey powder replacing of the carbonyl(lactose) and the partial nitrogen source in the traditional media was investigated. Then the concentration of whey powder and the nitrogen source(yeast extract, peptone)were optimized, and inorganic salts that were beneficial to the synthesis of β-galactosidase were screened.Finally, cheap industrial media was obtained as follows: whey powder 60 g/L, yeast extract 9 g/L, peptone 3 g/L, MnCl20.03 g/L, ZnCl20.058 g/L and pH 8.0. Under the optimized conditions, the β-galactosidase activity was reached up to 33.40 U/mL(in shaken flasks) and 77.47 U/mL(in fermenter), and the special activity reached 13.74 U/mg(biomass). These results were significantly better than that of the traditional media. Since whey powder as a by-product of dairy industry is a cheap waste source, the optimized industrial media using whey powder as the main substrate is particularly suitable for large-scale production of β-galactosidase.
引文
1张敏文,顾取良,张博,等.乳糖酶研究进展[J].微生物学杂志,2011,31(3):81-86.
2 Lifran E V,Hourigan J A,Sleigh R W,et al.New wheys for lactose[J].Food Australia,2000,52(4):120-125.
3 Panesar P S,Panesar R,Singh R S,et al.Microbial production,immobilization and applications ofβ-D-galactosidase[J].Journal of Chemical Technology&Biotechnology,2006,81(4):530-543.
4 Husain Q.Beta-galactosidases and their potential applications:A review[J].Critical Reviews in Biotechnology,2010,30(1):41-62.
5 Siso M I G.The biotechnological utilization of cheese whey:A review[J].Bioresource Technology,1996,57(1):1-11.
6 Ptanzeres A R,Carvalho F,Rivas Javier.Cheese whey management:A review[J].Journal of Environmental Management,2012,110:48-68.
7 Ornelas A P,Silceira W B,Sampaio F C,et al.The activity ofβ-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate[J].Journal of Applied Microbiology,2008,104:1 008-1 013.
8 Xu J L,Zhao J,Wang L F,et al.Enhancedβ-galactosidase production from whey powder by a mutant of the psychrotolerant yeast guehomyces pullulans 171 for hydrolysis of lactose[J].Applied Biochemistry and Biotechnology,2012,166(3):599-611.
9张帅帅,王梦凡,刑肖肖,等.乳酸克鲁维酵母透性化细胞β-半乳糖苷酶催化合成低聚半乳糖[J].南开大学学报:自然科学版,2013,46(5):99-105.
10 Gupte A M,NAIR J S.β-galactosidase production and ethanol fermentation from whey using Kluyveromyces marxianus NCIM 3551[J].Journal of Scientific&Industrial Research,2010,69(11):855-859.
11 Pinheiro R,Belo I,Mota M.Growth andβ-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure[J].Letters in Applied Microbiology,2003,37(6):438-442.
12 Inchaurrondo V,Flores M,Voget C.Growth andβ-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis[J].Journal of Industrial Microbiology and Biotechnology,1998,20(5):291-298.
13 You S P,Zhang J Y,Yin Q D,et al.Development of a novel integrated process for co-production ofβ-galactosidase and ethanol using lactose as substrate[J].Bioresource Technology,2017,230:15-23.
14 Guimaraes P M R,Teixeira J A,Dominingues L.Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey[J].Biotechnology Advances,2010,28(3):375-384.
15 Nor Z M,Tamer M I,Scharer J M,et al.Automated fed-batch culture of Kluyveromyces fragilis based on a novel method for online estimation of cell specific growth rate[J].Biochemical Engineering Journal,2001,9(3):221-231.
16 Lukondeh T,Ashbolt N J,Rogers P L.Fed-batch fermentation for production of Kluyveromyces marxianus FII510700 cultivated on a lactose-based medium[J].Journal of Industrial Microbiology&Biotechnology,2005,32(7):284-288.
17 Somawat H M,Agrawat A,Dutta S M.Production ofβ-galactosidase from Kluyveromyces fragilis grown on whey[J].Folia Microbiologica,1981,26(5):370-376.
18 Hsu C A,Yu R C,Lee S L,et al.Cultural condition affecting the growth and production of beta-galactosidase by Bifidobacterium longum CCRC 15708 in a jar fermenter[J].International Journal of Food Microbiology,2007,116(1):186-189.
19 Zheng Jia,Zhao Wei,Guo Ning,et al.Development of an industrial medium and a novel fed-batch strategy for high-level expression of recombinantβ-mananase by Pichia pastoris[J].Bioresource Technology,2012,118(14):257-264.
20 Sun H S,You S P,Wang M F,et al.Recyclable strategy for the production of high-purity galacto-oligosaccharides by Kluyveromyces lactis[J].Journal of Agricultural&Food Chemistry,2016,64(28):5 679-5 685.
2 Lifran E V,Hourigan J A,Sleigh R W,et al.New wheys for lactose[J].Food Australia,2000,52(4):120-125.
3 Panesar P S,Panesar R,Singh R S,et al.Microbial production,immobilization and applications ofβ-D-galactosidase[J].Journal of Chemical Technology&Biotechnology,2006,81(4):530-543.
4 Husain Q.Beta-galactosidases and their potential applications:A review[J].Critical Reviews in Biotechnology,2010,30(1):41-62.
5 Siso M I G.The biotechnological utilization of cheese whey:A review[J].Bioresource Technology,1996,57(1):1-11.
6 Ptanzeres A R,Carvalho F,Rivas Javier.Cheese whey management:A review[J].Journal of Environmental Management,2012,110:48-68.
7 Ornelas A P,Silceira W B,Sampaio F C,et al.The activity ofβ-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate[J].Journal of Applied Microbiology,2008,104:1 008-1 013.
8 Xu J L,Zhao J,Wang L F,et al.Enhancedβ-galactosidase production from whey powder by a mutant of the psychrotolerant yeast guehomyces pullulans 171 for hydrolysis of lactose[J].Applied Biochemistry and Biotechnology,2012,166(3):599-611.
9张帅帅,王梦凡,刑肖肖,等.乳酸克鲁维酵母透性化细胞β-半乳糖苷酶催化合成低聚半乳糖[J].南开大学学报:自然科学版,2013,46(5):99-105.
10 Gupte A M,NAIR J S.β-galactosidase production and ethanol fermentation from whey using Kluyveromyces marxianus NCIM 3551[J].Journal of Scientific&Industrial Research,2010,69(11):855-859.
11 Pinheiro R,Belo I,Mota M.Growth andβ-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure[J].Letters in Applied Microbiology,2003,37(6):438-442.
12 Inchaurrondo V,Flores M,Voget C.Growth andβ-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis[J].Journal of Industrial Microbiology and Biotechnology,1998,20(5):291-298.
13 You S P,Zhang J Y,Yin Q D,et al.Development of a novel integrated process for co-production ofβ-galactosidase and ethanol using lactose as substrate[J].Bioresource Technology,2017,230:15-23.
14 Guimaraes P M R,Teixeira J A,Dominingues L.Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey[J].Biotechnology Advances,2010,28(3):375-384.
15 Nor Z M,Tamer M I,Scharer J M,et al.Automated fed-batch culture of Kluyveromyces fragilis based on a novel method for online estimation of cell specific growth rate[J].Biochemical Engineering Journal,2001,9(3):221-231.
16 Lukondeh T,Ashbolt N J,Rogers P L.Fed-batch fermentation for production of Kluyveromyces marxianus FII510700 cultivated on a lactose-based medium[J].Journal of Industrial Microbiology&Biotechnology,2005,32(7):284-288.
17 Somawat H M,Agrawat A,Dutta S M.Production ofβ-galactosidase from Kluyveromyces fragilis grown on whey[J].Folia Microbiologica,1981,26(5):370-376.
18 Hsu C A,Yu R C,Lee S L,et al.Cultural condition affecting the growth and production of beta-galactosidase by Bifidobacterium longum CCRC 15708 in a jar fermenter[J].International Journal of Food Microbiology,2007,116(1):186-189.
19 Zheng Jia,Zhao Wei,Guo Ning,et al.Development of an industrial medium and a novel fed-batch strategy for high-level expression of recombinantβ-mananase by Pichia pastoris[J].Bioresource Technology,2012,118(14):257-264.
20 Sun H S,You S P,Wang M F,et al.Recyclable strategy for the production of high-purity galacto-oligosaccharides by Kluyveromyces lactis[J].Journal of Agricultural&Food Chemistry,2016,64(28):5 679-5 685.