Recombinant Expression and Characterization of A Novel Cold-Adapted <i>β</i>-Galactosidase from <i>Paenibacillus polymyxa</i>

XIANG Zhixuan; GUAN Leying; LI Jing; YAN Qiaojuan; JIANG Zhengqiang

doi:10.13386/j.issn1002-0306.2023010185

Volume 44 Issue 22

Nov. 2023

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Science and Technology of Food Industry > 2023 > 44(22): 125-133. > DOI: 10.13386/j.issn1002-0306.2023010185

XIANG Zhixuan, GUAN Leying, LI Jing, et al. Recombinant Expression and Characterization of A Novel Cold-Adapted β-Galactosidase from Paenibacillus polymyxa[J]. Science and Technology of Food Industry, 2023, 44(22): 125−133. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023010185.

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Recombinant Expression and Characterization of A Novel Cold-Adapted β-Galactosidase from Paenibacillus polymyxa

1.
Key Laboratory of China National Light Industry and Food Bioengineering, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
2.
College of Engineering, China Agricultural University, Beijing 100083, China
3.
Food Laboratory of Zhongyuan, Luohe 462300, China

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Received Date: February 01, 2023
Available Online: September 17, 2023

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Abstract

Abstract

Objective: To analyze a novel cold-adapted β-galactosidase, which provided the basis for producing low lactose dairy products or synthesizing galactooligosaccharides (GOS) at low temperatures. Methods: The β-galactosidase (PpBgal42A) gene was cloned from the genome of Paenibacillus polymyxa. The recombinant plasmid (pET-28a-PpBgal42A) was constructed and successfully expressed in E.coli BL21(DE3). After purification by affinity chromatography, the enzymatic properties of recombinant β-galactosidase were studied. To evaluate the transglycosylation ability of PpBgal42A, the concentration of each component was determined by high performance liquid chromatography with the conversion rate of GOS as evaluation index. Results: PpBgal42A shared the highest identity of 59.9% with the GH family 42 β-galactosidase from Alicyclobacillus acidocaldarius. After purification, the specific activity of PpBgal42A was 163.7 U/mg and the molecular weight was about 79 kDa. Its optimal pH and temperature were pH7.5 and 35 ℃, respectively. It was stable in the range of pH7.0~9.5 and 4~35 ℃. It was rapidly inactivated at 50 ℃. The half-life of PpBgal42A at 35 ℃ was 1777 min. PpBgal42A synthesized 31.6% of GOS using 350 g/L lactose as the substrate at 30 ℃ for 6 h. Conclusion: PpBgal42A is a novel cold-adapted enzyme. It can hydrolyze lactose and synthesize galactooligosaccharides, which has potential application value.
- Paenibacillus polymyxa,
- β-galactosidase,
- enzymatic characterization,
- cold adaption,
- galactooligosaccharides

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References

[1]	KOLEV P, ROCHA-MENDOZA D, RUIZ-RAMÍREZ S, et al. Screening and characterization of β-galactosidase activity in lactic acid bacteria for the valorization of acid whey[J]. JDS Communications,2022,3(1):1−6. doi: 10.3168/jdsc.2021-0145
[2]	LU L L, GUO L C, WANG K, et al. β-Galactosidases:A great tool for synthesizing galactose-containing carbohydrates[J]. Biotechnology Advances,2020,39:107465. doi: 10.1016/j.biotechadv.2019.107465
[3]	GAO X, WU J, WU D. Rational design of the β-galactosidase from Aspergillus oryzae to improve galactooligosaccharide production[J]. Food Chemistry,2019,286:362−367. doi: 10.1016/j.foodchem.2019.01.212
[4]	ARSOV A, IVANOV I, TSIGORIYNA L, et al. In vitro production of galactooligosaccharides by a novel β-galactosidase of Lactobacillus bulgaricus[J]. International Journal of Molecular Sciences,2022,23(22):14308. doi: 10.3390/ijms232214308
[5]	YIN H F, BULTERMA J B, DIJKHUIZEN L, et al. Reaction kinetics and galactooligosaccharide product profiles of the β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae[J]. Food Chemistry,2017,225:230−238. doi: 10.1016/j.foodchem.2017.01.030
[6]	HUNG M N, LEE B H. Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96[J]. Applied Microbiology and Biotechnology,2002,58(4):439−445. doi: 10.1007/s00253-001-0911-6
[7]	LIU Y, CHEN Z, JIANG Z Q, et al. Biochemical characterization of a novel β-galactosidase from Paenibacillus barengoltzii suitable for lactose hydrolysis and galactooligosaccharides synthesis[J]. International Journal of Biological Macromolecules,2017,104:1055−1063. doi: 10.1016/j.ijbiomac.2017.06.073
[8]	SUN X J, DUAN X G, WU D, et al. Characterization of Sulfolobus solfataricus β-galactosidase mutant F441Y expressed in Pichia pastoris[J]. Journal of the Science of Food and Agriculture,2014,94(7):1359−1365. doi: 10.1002/jsfa.6419
[9]	RUTKIEWICZ M, BUJACZ A, BUJACZ G. Structural features of cold-adapted dimeric GH2 β-D-galactosidase from Arthro bacter sp. 32cB[J]. BBA - Proteins and Proteomics,2019,1867(9):776−786. doi: 10.1016/j.bbapap.2019.06.001
[10]	FAN Y T, HUA X, ZHANG Y Z, et al. Cloning, expression and structural stability of a cold-adapted β-galactosidase from Rahnella sp. R3[J]. Protein Expression and Purification,2015,115:158−164. doi: 10.1016/j.pep.2015.07.001
[11]	MANGIAGALLI M, LOTTI M. Cold-active β-galactosidases:insight into cold adaption mechanisms and biotechnological exploitation[J]. Marine Drugs,2021,19(1):43. doi: 10.3390/md19010043
[12]	NAKAGAWA T, FUJIMOTO Y, IKEHATA R, et al. Purification and molecular characterization of cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2[J]. Applied Microbiology and Biotechnology,2006,72(4):720−725. doi: 10.1007/s00253-006-0339-0
[13]	PAWLAK-SZUKALSKA A, WANARSKA M, TOMASZ A, et al. A novel cold-active β-d-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp. 32cB-gene cloning, purification and characterization[J]. Process Biochemistry,2014,49(12):2122−2133. doi: 10.1016/j.procbio.2014.09.018
[14]	TIMMUSK S, COPOLOVICI D, COPOLOVICI L, et al. Paenibacillus polymyxa biofilm polysaccharides antagonise Fusarium graminearum[J]. Scientific Reports,2019,9(1):1−11. doi: 10.1038/s41598-018-37186-2
[15]	SAAD M A, ABDRABOU H S, ELKHTAB E, et al. Occurrence of toxic biogenic amines in various types of soft and hard cheeses and their control by Bacillus polymyxa D05-1[J]. Fermentation,2022,8(7):327. doi: 10.3390/fermentation8070327
[16]	YUAN Y, ZHANG X Y, ZHANG H, et al. Degradative GH5 β-1, 3-1, 4-glucanase PpBglu5A for glucan in Paenibacillus polymyxa KF-1[J]. Process Biochemistry,2020,98:183−192. doi: 10.1016/j.procbio.2020.08.008
[17]	HORI K, KAWABATA Y, NAKAZAWA Y, et al. A novel β-1, 4-mannanase isolated from Paenibacillus polymyxa KT551[J]. Food Science and Technology Research,2014,20(6):1261−1265. doi: 10.3136/fstr.20.1261
[18]	张春园, 张鸣明, 胡先望, 等. 多粘类芽孢杆菌新普鲁兰酶基因克隆及其在大肠杆菌中的表达[J]. 食品工业科技,2020,41(15):119−123,128 doi: 10.13386/j.issn1002-0306.2020.15.019 ZHANG C Y, ZHANG M M, HU X W, et al. Cloning of neopullulanase gene from Paenibacillus polymyxa and its expression in Escherichia coli[J]. Science and Technology of Food Industry,2020,41(15):119−123,128. doi: 10.13386/j.issn1002-0306.2020.15.019
[19]	LAEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature,1970,227(5259):680−685. doi: 10.1038/227680a0
[20]	KATROLIA P, YAN Q J, JIA H Y, et al. Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris[J]. Journal of Molecular Catalysis. B:Enzymatic, 2011, 69(3):112-119.
[21]	LOWRY O H, ROSEBROUGH N J, FARR A L, et al. Protein measurement with the folin phenol reagent[J]. The Journal of Biological Chemistry,1951,193(1):265−275. doi: 10.1016/S0021-9258(19)52451-6
[22]	LIU Y, YAN Q J, GUAN L Y, et al. Biochemical characterization of a novel protease-resistant α-galactosidase from Paecilomyces thermophila suitable for raffinose family oligosaccharides degradation[J]. Process Biochemistry,2020,94:370−379. doi: 10.1016/j.procbio.2020.04.013
[23]	QIN Z M, LI S F, HUANG X, et al. Improving galactooligosaccharide synthesis efficiency of β-galactosidase Bgal1-3 by reshaping the active site with an intelligent hydrophobic amino acid scanning[J]. Journal of Agriculture and Food Chemistry,2019,67(40):11158−11166. doi: 10.1021/acs.jafc.9b04774
[24]	KOLEV P, ROCHA-MENDOZA D, RUIZ-RAMÍREZ S, et al. Screening and characterization of β-galactosidase activity in lactic acid bacteria for the valorization of acid whey[J]. JDS Communications,2021,3(1):1−6.
[25]	ZERVA A, LIMNAIOS A, KRITIKOU A S, et al. A novel thermophile β-galactosidase from Thermothielavioides terrestris producing galactooligosaccharides from acid whey[J]. New Biotechnology,2021,63:45−53. doi: 10.1016/j.nbt.2021.03.002
[26]	PEPRAH A F, WANG T T, KOSIBA A A, et al. Integration of elastin-like polypeptide fusion system into the expression and purification of Lactobacillus sp. B164 β-galactosidase for lactose hydrolysis[J]. Bioresource Technology,2020,311:123513. doi: 10.1016/j.biortech.2020.123513
[27]	LIN Q B, WANG S D, WANG M L, et al. A novel glycoside hydrolase family 42 enzyme with bifunctional β-galactosidase and α-L-arabinopyranosidase activities and its synergistic effects with cognate glycoside hydrolases in plant polysaccharides degradation[J]. International Journal of Biological Macromolecules,2019,140:129−139. doi: 10.1016/j.ijbiomac.2019.08.037
[28]	ABURTO C, CASTILLO C, CORNEJO F, et al. β-Galactosidase from Exiguobacterium acetylicum:cloning, expression, purification and characterization[J]. Bioresource Technology,2019,277:211−215. doi: 10.1016/j.biortech.2019.01.005
[29]	KATROLIA P, ZHANG M, YAN Q J, et al. Characterisation of a thermostable family 42 β-galactosidase (BgalC) family from Thermotoga maritima showing efficient lactose hydrolysis[J]. Food Chemistry,2011,125(2):614−621. doi: 10.1016/j.foodchem.2010.08.075
[30]	SUN J J, YAO C Y, WANG W, et al. Cloning, expression and characterization of a novel cold-adapted β-galactosidase from the deep-sea bacterium Alteromonas sp. ML52[J]. Marine Drugs,2018,16(12):469. doi: 10.3390/md16120469
[31]	WANG K, LI G, YU S Q, et al. A novel metagenome-derived β-galactosidase:gene cloning, overexpression, purification and characterization[J]. Applied Microbiology and Biotechnology,2010,88(1):155−165. doi: 10.1007/s00253-010-2744-7
[32]	WANG L J, MOU Y Z, GUAN B, et al. Genome sequence of the psychrophilic Cryobacterium sp. LW097 and characterization of its four novel cold-adapted β-galactosidases[J]. International Journal of Biological Macromolecules,2020,163:2068−2083. doi: 10.1016/j.ijbiomac.2020.09.100
[33]	CARNEIRO L A B C, LI Y, DUPREE P, et al. Characterization of a β-galactosidase from Bacillus subtilis with transgalactosylation activity[J]. International Journal of Biological Macromolecules,2018,120:279−287. doi: 10.1016/j.ijbiomac.2018.07.116
[34]	JUAJUN O, NGUYEN T H, MAISCHBERGER T, et al. Cloning, purification, and characterization of β-galactosidase from Bacillus licheniformis DSM 13[J]. Applied Microbiology and Biotechnology,2011,89(3):645−654. doi: 10.1007/s00253-010-2862-2

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