Heterologous Expression and Characterization of Endo-type Alginate Lyase from Yeosuana marina sp. JLT21

doi:10.13560/j.cnki.biotech.bull.1985.2021-0539

Abstract

Abstract:

Alginate oligosaccharides（AO）have rich biological activity,preparing AO using enzymatic approach is of important,practical and applicable value. To mine highly active and stable enzymes for preparing AO,a gene from an alginate lyase YAM-1 of Yeosuana marina sp. JLT21 was expressed in Escherichia coli,and the enzyme was purified and characterized. As one of polysaccharide lyase family 7（PL7）members,YMA-1 consisted of 306 amino acids. The recombinant YMA-1 showed the optimal activity of 1.3×10⁴ U/mg at pH 9.0 and 55℃,and Cu²⁺ efficiently stimulated its activity. YMA-1 also demonstrated a highly catalytic activity towards sodium alginate,poly G and poly M,and their specific activities were（5 201.21±86.46）,（6 399.73±253.12）and（3 751.68±116.25）U/mg,respectively under 37℃ and pH 9.0. The end-products from the enzymolysis of sodium alginate were mainly unsaturated trisaccharides and tetrasaccharides. Duo to its broad substrate spectrum,high activity and thermostability,YMA-1 as endo-type alginate lyase in PL7 family has potential applications in green and efficient production of alginate oligosaccharides.

Key words: endo-type alginate lyase, recombinant expression, enzymatic properties, Yeosuana marina sp. JLT21

CHANG Qing, SHU Yue-rong, WANG Wen-tao, JIANG Hao, YAN Quan-de, QIAN Zheng, GAO Xue-chun, WU Jin-hong, ZHANG Yong. Heterologous Expression and Characterization of Endo-type Alginate Lyase from Yeosuana marina sp. JLT21[J]. Biotechnology Bulletin, 2022, 38(2): 123-131.

Figures/Tables 6

Fig.1 Phylogenetic tree of alginate lyase YMA-1 and amino acid sequence alignment analysis of PL7 family enzyme A：Bootstraps are the confidence values obtained from 1,000 replications（%）. Species of alginolytic bacteria and the accession number from GenBank are listed on the right. Scale bar indicates approximately 20% sequence difference. B：FlAlyA from Flavobacterium sp. UMI-01（BAP05660）,A1-II’ from Sphingomonas sp. A1（BAD16656）,AlyA from Klebsiella pneumoniae（AAA25049）,AlyA5 from Zobellia galactanivorans DsijT（CAZ98266.1）,AlyA1 from Z. galactanivorans DsiJT（CAZ95239）,AlgAT5 from Defluviitalea phaphyphila（WP_058486006.1）,PA1167 from Pseudomonas aeruginosa PAO1（AAG04556）,and AlyC3 from Psychromonas sp.（QOP59290.1）

Fig.2 Recombinant expression,purification and analysis of alginate lyase YMA-1 A：Detecting the purity of recombinase YMA-1 via SDS-PAGE. M：Protein marker. 1：Crude enzyme. 2：Flow-through with 20 mmol/L imidazole buffer;3：Flow-through with 30 mM imidazole buffer. 4：Flow-through with 50 mmol/L imidazole buffer. 5：Flow-through with 80 mmol/L imidazole buffer. 6：Purified recombinant enzyme YMA-1. B：Gel filtration chromatography analysis of recombinant enzyme YMA-1

Fig.3 Activities of alginate lyase YMA-1 under different pH,temperature and metal ions A：Determination of YMA-1 activity at pH 5.0 to 10.0. YMA-1 catalysis was optimal at pH 9.0,and its specific activity of the enzyme was 4.7×103 U/mg. B：Examination of YMA-1 activity at 10℃ to 60℃. YMA-1 showed the optimal activity of 1.3×104 U/mg at 55℃. C：Measurement of YMA-1 thermostability Tm value was determined by differential scanning calorimetry（DSC）. D：Effects of various metal ions on the activity of YMA-1

Fig.4 Substrate preference of alginate lyase YMA-1 A：Substrate specificity of alginate lyases. Alginate lyases can be classified into Poly M lyase and Poly G lyase and bi-functional lyase according to chemical structures of substrates acting with alginate lyase. B：Preference of YMA-1 on 3 substrates in different structures,it presents high activity on 3 alginic acid substrates in different structure,confirming it is a bi-functional alginate lyase

Fig.5 Michaelis-Menten kinetics of alginate lyase YMA-1

Fig.6 LC-MS analysis of the oligosaccharide products from degrading sodium alginates by YMA-1 catalysis A：HPLC analysis of products of YMA-1 catalysis. Four different end-products were detected,and the peak times were 3.92,4.04,4.13 and 4.23 min,respectively. B：ESI-MST analysis of products of YMA-1 catalysis. The end-products were unsaturated disaccharides（ΔDP2）（[M-H]-=351.0）,unsaturated trisaccharides（ΔDP3）（[M-H]-=527.09）,unsaturated tetrasaccharide（ΔDP4）（[M-H]-=703.12）and unsaturated pentasaccharide（ΔDP5）（[M-H]-=879.15）,respectively

References 20

[1]	Kloareg B, Demarty M, Mabeau S. Ion-exchange properties of isolated cell walls of brown algae:the interstitial solution[J]. J Exp Bot, 1987, 38(10):1652-1662. doi: 10.1093/jxb/38.10.1652 URL
[2]	Sellimi S, Younes I, Ayed HB, et al. Structural, physicochemical and antioxidant properties of sodium alginate isolated from a Tunisian brown seaweed[J]. Int J Biol Macromol, 2015, 72:1358-1367. doi: 10.1016/j.ijbiomac.2014.10.016 URL
[3]	Tang JC, Taniguchi H, Chu H, et al. Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes[J]. Lett Appl Microbiol, 2009, 48(1):38-43. doi: 10.1111/j.1472-765X.2008.02481.x pmid: 19018967
[4]	Wong TY, Preston LA, Schiller NL. Alginate lyase:review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications[J]. Annu Rev Microbiol, 2000, 54(1):289-340. doi: 10.1146/micro.2000.54.issue-1 URL
[5]	Jain S, Ohman DE. Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa[J]. Infect Immun, 2005, 73(10):6429-6436. doi: 10.1128/IAI.73.10.6429-6436.2005 URL
[6]	Cheng D, Jiang C, Xu J, et al. Characteristics and applications of alginate lyases:a review[J]. Int J Biol Macromol, 2020, 164:1304-1320. doi: 10.1016/j.ijbiomac.2020.07.199 URL
[7]	Zhu B, Yin H. Alginate lyase:Review of major sources and classification, properties, structure-function analysis and applications[J]. Bioengineered, 2015, 6(3):125-131. doi: 10.1080/21655979.2015.1030543 URL
[8]	Xu F, Wang P, Zhang YZ, et al. Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases[J]. Appl Environ Microbiol, 2018, 84(3):e02040-17.
[9]	Lombard V, Bernard T, Rancurel C, et al. A hierarchical classification of polysaccharide lyases for glycogenomics[J]. Biochem J, 2010, 432(3):437-444. doi: 10.1042/BJ20101185 URL
[10]	Wang B, Ji SQ, Lu M, et al. Biochemical and structural characterization of alginate lyases:an update[J]. Curr Biotechnol, 2015, 4(3):223-239. doi: 10.2174/2211550104666150723231423 URL
[11]	Nakata S, Murata K, Hashimoto W, et al. Uncovering the reactive nature of 4-deoxy- l - erythro -5-hexoseulose uronate for the utilization of alginate, a promising marine biopolymer[J]. Sci Rep, 2019, 9:17147. doi: 10.1038/s41598-019-53597-1 pmid: 31748627
[12]	Kim HS, Lee CG, Lee EY. Alginate lyase:Structure, property, and application[J]. Biotechnol Bioprocess Eng, 2011, 16(5):843-851. doi: 10.1007/s12257-011-0352-8 URL
[13]	Wang Y, Han F, Hu B, et al. In vivo prebiotic properties of alginate oligosaccharides prepared through enzymatic hydrolysis of alginate[J]. Nutr Res, 2006, 26(11):597-603. doi: 10.1016/j.nutres.2006.09.015 URL
[14]	Islan GA, Bosio VE, Castro GR. Alginate lyase and ciprofloxacin co-immobilization on biopolymeric microspheres for cystic fibrosis treatment[J]. Macromol Biosci, 2013, 13(9):1238-1248. doi: 10.1002/mabi.201300134 URL
[15]	Tang L, Wang Y, Gao S, et al. Biochemical characteristics and molecular mechanism of an exo-type alginate lyase V_xAly7D and its use for the preparation of unsaturated monosaccharides[J]. Biotechnol Biofuels, 2020, 13:99. doi: 10.1186/s13068-020-01738-4 URL
[16]	Lu D, Zhang Q, Wang S, et al. Biochemical characteristics and synergistic effect of two novel alginate lyases from Photobacterium sp. FC615[J]. Biotechnol Biofuels, 2019, 12:260. doi: 10.1186/s13068-019-1600-y URL
[17]	Yamasaki M, Moriwaki S, Miyake O, et al. Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7:a novel alginate lyase with a beta-sandwich fold[J]. J Biol Chem, 2004, 279(30):31863-31872. doi: 10.1074/jbc.M402466200 URL
[18]	Kobayashi T, Uchimura K, Miyazaki M, et al. A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp[J]. Extremophiles, 2009, 13(1):121-129. doi: 10.1007/s00792-008-0201-7 pmid: 19002649
[19]	Inoue A, Anraku M, Nakagawa S, et al. Discovery of a novel alginate lyase from Nitratiruptor sp. SB155-2 thriving at deep-sea hydrothermal vents and identification of the residues responsible for its heat stability[J]. J Biol Chem, 2016, 291(30):15551-15563. doi: 10.1074/jbc.M115.713230 URL
[20]	Li S, Wang L, Chen X, et al. Cloning, expression, and biochemical characterization of two new oligoalginate lyases with synergistic degradation capability[J]. Mar Biotechnol:NY, 2018, 20(1):75-86. doi: 10.1007/s10126-017-9788-y URL