Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacterium <i>Pseudoalteromonas</i> sp. NJ-21 -- Journal of Oceanology and Limnology,2018,36(4):1304-1314

	Cite this paper:
	XIE Maisheng, LI Jiang, HE Peiqing, LIN Xuezheng. Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacterium Pseudoalteromonas sp. NJ-21[J]. Journal of Oceanology and Limnology, 2018, 36(4): 1304-1314

Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacterium Pseudoalteromonas sp. NJ-21

XIE Maisheng¹, LI Jiang^1,2, HE Peiqing^1,2, LIN Xuezheng^1,2

1 Key Laboratory of Marine Bioactive Substances, The First Institute of Oceanography, State Oceanic Administration(SOA), Qingdao 266061, China;
2 Key Laboratory of Marine Natural Products of Qingdao, Qingdao 266061, China

Abstract:

In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6 (PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity (258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃for 30 min. Al163 activity was stimulated by Cd²⁺, Ca²⁺, Fe³⁺, and Mn²⁺, but inhibited by Cu²⁺, Si²⁺, Fe²⁺, and Ni²⁺. Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, polyM, and polyG, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.

Key words: Pseudoalteromonas sp.|alginate lyase|PL-6 family

Received: 2017-03-15 Revised:

Tools

PDF (640 KB) Free

Print this page

Add to favorites

Email this article to others

Authors

Articles by XIE Maisheng

Articles by LI Jiang

Articles by HE Peiqing

Articles by LIN Xuezheng

References:
Akiyama H, Endo T, Nakakita R, Murata K, Yonemoto Y, Okayama K. 1992. Effect of depolymerized alginates on the growth of bifidobacteria. Bioscience, Biotechnology, and Biochemistry, 56(2):355-356.
An Q D, Zhang G L, Wu H T, Zhang Z C, Zheng G S, Luan L, Murata Y, Li X. 2009. Alginate-deriving oligosaccharide production by alginase from newly isolated flavobacterium sp. LXA and its potential application in protection against pathogens. Journal of Applied Microbiology, 106(1):161-170.
Ariyo B, Tamerler C, Bucke C, Keshavarz T. 1998. Enhanced penicillin production by oligosaccharides from batch cultures of Penicillium chrysogenum in stirred-tank reactors. FEMS Microbiology Letters, 166(1):165-170.
Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2):248-254.
Cantarel B L, Coutinho P M, Rancurel C, Bernard T, Lombard V, Henrissat B. 2009. The Carbohydrate-Active Enzymes database (CAZy):an expert resource for glycogenomics.Nucleic Acids Research, 37(Database issue):D233-D238.
Carrasco M, Rozas J M, Barahona S, Alcaíno J, Cifuentes V, Baeza M. 2012. Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-antarctic region. BMC Microbiology, 12:251.
Chung K H, Park J S, Hwang H S, Kim J C, Lee K Y. 2007.Detection and kinetics of mucosal pathogenic bacteria binding with polysaccharides. Journal of Microbiology and Biotechnology, 17(7):1 191-1 197.
Enquist-Newman M, Faust A M E, Bravo D D, Santos C N S, Raisner R M, Hanel A, Sarvabhowman P, Le C, Regitsky D D, Cooper S R, Peereboom L, Clark A, Martinez Y, Goldsmith J, Cho M Y, Donohoue P D, Luo L, Lamberson B, Tamrakar P, Kim E J, Villari J L, Gill A, Tripathi S A, Karamchedu P, Paredes C J, Rajgarhia V, Kotlar H K, Bailey R B, Miller D J, Ohler N L, Swimmer C, Yoshikuni Y. 2014. Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature, 505(7482):239-243.
Garron M L, Cygler M. 2010. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology, 20(12):1 547-1 573.
Hashimoto W, Miyake O, Ochiai A, Murata K. 2005. Molecular identification of sphingomonas sp. A1 Alginate lyase (A1-IV') as a member of novel polysaccharide lyase family 15 and implications in alginate lyase evolution. Journal of Bioscience and Bioengineering, 99(1):48-54.
Haug A, Larsen B, Smidsrød O. 1967. Studies on the sequence of uronic acid residues in alginic acid. Acta Chemica Scandinavica, 21:691-704.
Hu X K, Jiang X L, Hwang H, Liu S L, Guan H S. 2004.Promotive effects of alginate-derived oligosaccharide on maize seed germination. Journal of Applied Phycology, 16(1):73-76.
Inoue A, Takadono K, Nishiyama R, Tajima K, Kobayashi T, Ojima T. 2014. Characterization of an alginate lyase, FlAlyA, from Flavobacterium sp. strain UMI-01 and its expression in Escherichia coli. Marine Drugs, 12(8):4 693-4 712.
Iwamoto M, Kurachi M, Nakashima T, Kim D, Yamaguchi K, Oda T, Iwamoto Y, Muramatsu T. 2005. Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells. FEBS Letters, 579(20):4 423-4 429.
Iwamoto Y, Araki R, Iriyama K I, Oda T, Fukuda H, Hayashida S, Muramatsu T. 2001. Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain No.272 and its action on saturated oligomeric substrates.Bioscience, Biotechnology, and Biochemistry, 65(1):133-142.
Iwamoto Y, Xu X, Tamura T, Oda T, Muramatsu T. 2003.Enzymatically depolymerized alginate oligomers that cause cytotoxic cytokine production in human mononuclear cells. Bioscience Biotechnology & Biochemistry, 67(2):258-263.
Kawada A, Hiura N, Tajima S, Takahara H. 1999. Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells. Archives of Dermatological Research, 291(10):542-547.
Kim H S, Lee C G, Lee E Y. 2011. Alginate lyase:structure, property, and application. Biotechnology and Bioprocess Engineering, 16(5):843-851.
Kobayashi T, Uchimura K, Miyazaki M, Nogi Y, Horikoshi K. 2009. A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp. Extremophiles, 13(1):121-129.
Laurienzo P. 2010. Marine polysaccharides in pharmaceutical applications:an overview. Marine Drugs, 8(9):2 435-2 465.
Lee S I, Choi S H, Lee E Y, Kim H S. 2012. Molecular cloning, purification, and characterization of a novel polyMGspecific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2. Applied Microbiology and Biotechnology, 95(6):1 643-1 653.
Li S Y, Wang L N, Han F, Gong Q H, Yu W G. 2016. Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp.Kz7. Journal of Biochemistry, 159(1):77-86.
Maki H, Mori A, Fujiyama K, Kinoshita S, Yoshida T. 1993.Cloning, sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp. OS-ALG-9. Journal of General Microbiology, 139(5):987-993.
Miller G L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3):426-428.
Ogura K, Yamasaki M, Mikami B, Hashimoto W, Murata K. 2008. Substrate recognition by family 7 alginate lyase from sphingomonas sp. A1. Journal of Molecular Biology, 380(2):373-385.
Qin Q L, Xie B B, Yu Y, Shu Y L, Rong J C, Zhang Y J, Zhao D L, Chen X L, Zhang X Y, Chen B, Zhou B C, Zhang Y Z. 2014. Comparative genomics of the marine bacterial genus Glaciecola reveals the high degree of genomic diversity and genomic characteristic for cold adaptation.Environmental Microbiology, 16(6):1 642-1 653.
Takeda H, Yoneyama F, Kawai S, Hashimoto W, Murata K. 2011. Bioethanol production from marine biomass alginate by metabolically engineered bacteria. Energy & Environmental Science, 4(7):2 575-2 581.
Tomoda Y, Umemura K, Adachi T. 1994. Promotion of barley root elongation under hypoxic conditions by alginate lyase-lysate (A.L.L.). Bioscience Biotechnology & Biochemistry, 58(1):202-203.
Uchimura K, Miyazaki M, Nogi Y, Kobayashi T, Horikoshi K. 2010. Cloning and sequencing of alginate lyase genes from deep-sea strains of vibrio and Agarivorans and characterization of a new vibrio enzyme. Marine Biotechnology, 12(5):526-533.
Wargacki A J, Leonard E, Win M N, Regitsky D D, Santos C N S, Kim P B, Cooper S R, Raisner R M, Herman A, Sivitz A B, Lakshmanaswamy A, Kashiyama Y, Baker D, Yoshikuni Y. 2012. An engineered microbial platform for direct biofuel production from brown macroalgae.Science, 335(6066):308-313.
Wong T Y, Preston L A, Schiller N L. 2000. Alginate lyase:review of major sources and enzyme characteristics,structure-function analysis, biological roles, and applications. Annual Review of Microbiology, 54:289-340.
Xu F, Dong F, Wang P, Cao H Y, Li C Y, Li P Y, Pang X H, Zhang Y Z, Chen X L. 2017. Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGC from polysaccharide lyase family 6. Journal of Biological Chemistry, 292(11):4 457-4 468.
Xu X, Iwamoto Y, Kitamura Y, Oda T, Muramatsu T. 2003.Root growth-promoting activity of unsaturated oligomeric uronates from alginate on carrot and rice plants.Bioscience, Biotechnology, and Biochemistry, 67(9):2 022-2 025.
Yamamoto Y, Kurachi M, Yamaguchi K, Oda T. 2007.Stimulation of multiple cytokine production in mice by alginate oligosaccharides following intraperitoneal administration. Carbohydrate Research, 342(8):1 133-1 137.
Yamasaki M, Ogura K, Hashimoto W, Mikami B, Murata K. 2005. A structural basis for depolymerization of alginate by polysaccharide lyase family-7. Journal of Molecular Biology, 352(1):11-21.
Zhang R X, Zhou J H, Jia Z P, Zhang Y X, Gu G M. 2004.Hypoglycemic effect of Rehmannia glutinosa oligosaccharide in hyperglycemic and alloxan-induced diabetic rats and its mechanism. Journal of Ethnopharmacology, 90(1):39-43.
Zhu B W, Tan H D, Qin Y Q, Xu Q S, Du Y G, Yin H. 2015.Characterization of a new endo-type alginate lyase from Vibrio sp. W13. International Journal of Biological Macromolecules, 75:330-337.
Zhu B W, Yin H. 2015. Alginate lyase:review of major sources and classification, properties, structure-function analysis and applications. Bioengineered, 6(3):125-131.
Zong W R, Cheong K L, Wu D T, Li J, Zhao J, Li S P. 2014.Preparation and purification of raffinose family oligosaccharides from Rehmannia glutinosa Libosch. by fast protein liquid chromatography coupled with refractive index detection. Separation and Purification Technology, 138:98-103.