耐热β-半乳糖苷酶(Pyrococcus furiosus DSM 3638)活性位点N415S点突变对其酶活性的影响

doi:10.6040/j.issn.1671-9352.0.2016.399

Abstract

Abstract: β-galactosidase has both activities to hydrolyze lactose into galactose /glucose and to polymerize a couple of galactoses into oligo-galactose. These two β-galactosidase activities have application potential in manufactory industries. By exploiting site mutation technique, a mutant N415S(Asn was replaced by Ser)of thermo-stable β-galactosidase(Pyrococcus furiosus DSM 3638)was obtained. The mutant cDNA was cloned into yeast expression vector, and a yeast expression cell strain was selected to express the mutant enzyme protein. By fermentation, we obtained a product of this mutant enzyme. We further characterized the mutant enzyme, and found that the optimum pH is 5.5, the optimum temperature is 95 ℃. The enzyme activity of this mutant to hydrolyze lactose is much better than that of the wild type(WT); while the enzyme activity to polymerize galactose into oligomer is better that that of the wild type, but not so good as expected for industry application. This mutant will be useful for milk treatment to largely reduce the content of lactose.

Key words: β-galactosidase, galactooligosaccharides, point mutation, enzyme activity

CLC Number:

Q939.97

YIN Lei, WANG Yuan-yuan, XIN Zhi-hao, WANG Xiang-dong. The effect of point mutation N415S in Pyrococcus furiosus DSM 3638 (a thermo-stable β-galactosidase)on its enzymatic activities[J].JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2016, 51(11): 74-78.

References

[1] REUTER S, RUSBORG NYGAAARD A, ZIMMERMANN W. β-Galactooligosaccharide synthesis with β-galactosidases from Sulfolobus solfataricus,Aspergillus oryzae,and Escherichia coli[J]. Enzyme and Microbial Techilology, 1999, 25(6):509-516.
[2] 徐晨,陈历俊,石惟沈,等. 低聚半乳糖的研究进展及应用[J]. 中国食品添加剂,2011(1):205-209. XU Chen, CHEN Lijun, SHI Weishen, et al. Advanced in the research and application of Galactooligosaccharide[J]. China Food Additives, 2011(1):205-209.
[3] 张莉,李庆章,田雷.β-半乳糖苷酶研究进展[J].东北农业大学学报,2009,40(7):128-131. ZHANG Li, LI Qingzhang, TIAN Lei. Research advancement of β-galactosidase[J]. Journal of Northeast Agricultural University, 2009, 40(7):128-131.
[4] LEBBINK J H, KAPER Thijs, BRON Peter, et al. Improving low-temperature catalysis in the hyperthermostable pyrococcus furiosus β-Glucosidase celb by directed evolution[J]. Biochemistry, 2000, 39(13):3656-3665.
[5] KAPER Thijs, LEBBINK J H, POUWELS Jeroen, et al. Comparative structural analysis and substrate specificity engineering of the hyperthermostable β-glucosidase celb from Pyrococcus furiosus[J]. Biochemistry, 2000, 39(17):4963-4970.
[6] PARK Sunghoon, ALLI Inteaz, PARK Kwanhwa, et al. Mutation studies in the active site of β-glucosidase from Pyrococcus furiosus DSM 3638[J]. Protein & Peptide Letters, 2013, 20(1):107-114.
[7] 李正义,肖敏,卢丽丽,等. 转糖基β-半乳糖苷酶生产含低聚半乳糖的低乳糖牛奶[J]. 食品科学,2007,28(5):241-244. LI Zhengyi, XIAO Min, LU Lili, et al. Production of low-lactose milk containing galactooligosaccharides with β-Galactosidase-catalyzed transgalactosylation from Enterobacter sp.B5[J]. Food Science, 2007, 28(5):241-244.
[8] LI Bin, WANG Zemin, LI Shiwu, et al. Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus[J]. BMC Biotechnology, 2013, 13(1):1-10.
[9] HUNG M N, LEE B H. Purification and characterization of a recombinant β-glucosidase with transgalactosylation activity from Bifidobacterium infantis HL96[J]. Appl Microbiol Biotechnol, 2002, 58(4):439-445.
[10] LIU G X, KONG J, LU W W, et al. β-Galactosidase with transgalactosylation activity from Lactobacillus fermentum K4[J]. Journal of Dairy Science, 2011, 94(12):5811-5820.
[11] 魏东芝,王筱兰,陈少欣,等.固定化嗜热脂肪芽孢杆菌连续合成半乳糖寡糖的研究[J].生物工程学报:2003,16(3):392-395. WEI Dongzhi, WANG Xiaolan, CHEN Shaoxin, et al. Study on continuous synthesis of galacto-oligosaccharide by immobilized bacillus stearothermophilus[J]. Chinese Journal of Biotechnology, 2003, 16(3):392-395.
[12] ZARATE S, LOPEZ-LEIVA M H. Oligosaccharide formation during enzymatic lactose hydrolysis: a literature review[J]. Journal of Food Protection, 1990, 53(3):262-268.
[13] 徐海波.α-半乳糖苷酶的酶学性质分析及基因的定点突变研究[D].武汉:华中农业大学,2013. XU Haibo. Characterization and site-directed mutagenesis of α-galactosidase[D].Wuhan: Huazhong Agricultural University, 2013.
[14] 张志刚.耐热β-半乳糖苷酶的结构分析及同源建模[J].现代食品科技,2013,29(4):706-709. ZHANG Zhigang. Structure analysis and homology modeling of thermostable β-galactosidase[J]. Modern Food Science and Technology, 2013, 29(4):706-709.
[15] PARK A R, OH D K. Effects of galactose and glucose on the hydrolysis reaction of a thermostable β-galactosidase from Caldicellulosiruptor saccharolyticus[J]. Applied Microbiology & Biotechnology, 2010, 85(5):1427-1435.
[16] 陈少欣,魏东芝,胡振华,等.嗜热脂肪芽孢杆菌β-半乳糖苷酶合成低聚半乳糖动力学模型[J]. 生物工程学报,2000,16(6),735-739. CHEN Shaoxin, WEI Dongzhi, HU Zhenhua, et al. Kinetic model of galacto-oligosaccharide synthesis by β-galactosidase from bacillus stearothermophilus[J]. Chinese Journal of Biotechnology, 2000, 16(6):735-739.
[17] NGUYEN T T, NGUYEN H M, GEIGER Barbara, et al.Heterologous expression of a recombinant lactobacillal β-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system[J]. Microbial Cell Factories, 2015, 14(1):1-11.
[18] HARJU M. Lactose hydrolysis[J]. Bulletin of the International Dairy Federation, 1987(212):50-54.
[19] SANTOS A, LADERO M, GARCIA-OCHOA F. Kinetic modeling of lactose hydrolysis by aβ-galactosidase from Kluyveromices fragilis[J]. Enzyme & Microbial Technology, 1998, 22(7):558-567.
[20] JOHNSON A O, SEMENYA J G, BUCHOWSKI M S, et al. Correlation of lactose maldigestion, lactose intolerance, and milk intolerance[J]. American Journal of Clinical Nutrition, 1993, 57(3):399-401.
[21] SYLWIA W, JÓZEF S. Thermostable β-glucosidase with a broad substrate specifity suitable for processing of lactose-containing products[J]. Food Chemistry, 2004, 85(2):181-187.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

The effect of point mutation N415S in Pyrococcus furiosus DSM 3638 (a thermo-stable β-galactosidase)on its enzymatic activities

PDF (PC)

Abstract

Cite this article

share this article

References

Related Articles 3

Metrics

Comments

Recommended 0

[1]	LU Wen-wei,KONG Wen-tao,SUN Zhi-lan,KONG Jian*,JI Ming-jie . Cloning and expression of the beta-galactosidase gene of Paenibacillus sp. K1 in E.coli [J]. J4, 2008, 43(7): 69-73 .
[2]	LU Wen-wei,KONG Wen-tao,KONG Jian,JI Ming-jie* . Transgalactosylation activity of beta-galactosidase produced by Lactobacillus fermentum K4 [J]. J4, 2008, 43(7): 83-87 .
[3]	WANG Hong-mei,XIAO Min*,LI Zheng-yi,LI Yu-mei,QIAN Xin-min, . Screening and identification of βgalactosidaseproducing microorganism and enzymatic synthesis of galactooligosaccharides using its transgalactosylation [J]. J4, 2006, 41(1): 133-139 .