Elucidating the degradation pattern of a new cold-tolerant pectate lyase used for efficient preparation of pectin oligosaccharides

Abstract

The cold-active pectate lyases have drawn increasing attention in food and biotechnological applications due to their ability to retain high catalytic efficiency under lower temperatures, which could be helpful for energy saving, cost reduction and flavor preservation. Herein, a new cold-tolerant pectate lyase (ErPelPL1) gene from Echinicola rosea was cloned and heterologously expressed in Escherichia coli. Interestingly, ErPelPL1 retained high catalytic activity even at a low temperature (4 °C). ErPelPL1 exhibited optimal activity at 35 ℃, pH 8.0 with 1 mM of Ca²⁺. It showed high specific activity towards polygalacturonic acid (34.7 U/mg) and sodium polygalacturonate (59.3 U/mg). The combined thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC) and electrospray ionization mass spectrometry (ESI-MS) results indicated that ErPelPL1 endolytically degraded pectic substances into the oligosaccharides with degrees of depolymerization (Dps) of 1–6. In conclusion, this study mainly conducted biochemical characterization and product analysis of a cold-tolerant pectate lyase. Therefore, it provides a promising enzyme candidate for food and biotechnological applications.

Keywords

Pectate lyase / Echinicola rosea / Cold-adapted / Product analysis

References

[1] Bekli S, Aktas B, Gencer D, Aslim B. Biochemical and molecular characterizations of a novel pH- and temperature-stable pectate lyase from Bacillus amyloliquefaciens S6 for industrial application. Mol Biotechnol, 2019, 61(9): 1-13.

[2] Bhatia RK, Ullah S, Zahirhoque M, Ahmad I, Yang YH, Bhatt AK, Bhatia SK. Psychrophiles: a source of cold-adapted enzymes for energy efficient biotechnological industrial processes. J Environ Chem Eng, 2020

[3] Carrasco M, Rozas JM, Alcaíno J, Cifuentes V, Baeza M. Pectinase secreted by psychrotolerant fungi: identification, molecular characterization and heterologous expression of a cold-active polygalacturonase from Tetracladium sp. Microb Cell Factories, 2019, 18(1): 45.

[4] Chen J, Liang RH, Liu W, Li T, Liu CM, Wu SS, Wang ZJ. Pectic-oligosaccharides prepared by dynamic high-pressure microfluidization and their in vitro fermentation properties. Carbohyd Polym, 2013, 91(1): 175-182.

[5] Gómez B, Yáñez R, Parajó J, Alonso JL. Production of pectin-derived oligosaccharides from lemon peels by extraction, enzymatic hydrolysis and membrane filtration. J Chem Technol Biot, 2014, 91(1): 234-247.

[6] Gummadi SN, Panda T. Purification and biochemical properties of microbial pectinases—a review. Process Biochem, 2003, 38(7): 987-996.

[7] Henrissat B, Heffron SE, Yoder MD, Lietzke SE, Jurnak F. Functional implications of structure-based sequence alignment of proteins in the extra-cellular pectate lyase superfamily. Plant Physiol, 1995, 107(3): 963-976.

[8] Hu F, Zhu B, Li Q, Yin H, Ming DM. Elucidation of a unique pattern and the role of carbohydrate binding module of an alginate lyase. Mar Drugs, 2019, 18(1): 32.

[9] Jayani RS, Saxena S, Gupta R. Microbial pectinolytic enzymes: a review. Process Biochem, 2005, 40(9): 2931-2944.

[10] Kamijo J, Sakai K, Suzuki H, Suzuki K, Kunitake E, Shimizu M, Kato M. Identification and characterization of a thermostable pectate lyase from Aspergillus luchuensis var. saitoi. Food Chem, 2019, 276: 503-510.

[11] Kang HJ, Jo C, Kwon JH, Son JH, An BJ, Byun MW. Antioxidant and cancer cell proliferation inhibition effect of citrus pectin-oligosaccharide prepared by irradiation. J Med Food, 2006, 9(3): 313-320.

[12] Kashyap DR, Vohra PK, Chopra S, Tewari R. Applications of pectinases in the commercial sector: a review. Bioresour Technol, 2001, 77(3): 215-227.

[13] Klug-Santner BG, Schnitzhofer W, Vršanská M, Weber JR, Agrawal PB, Nierstrasz VA, Guebitz GM. Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2. J Biotechnol, 2006, 121(3): 390-401.

[14] Kohli P, Gupt R. Alkaline pectinases: a review. Biocatal Agric Biotechnol, 2015, 4(3): 279-285.

[15] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259): 680-685.

[16] Li YP, Li WH, Zhang GL, Lü XQ, Hwang H, Aker WG, Guan H, Wang P. Purification and characterization of polysaccharides degradases produced by Alteromonas sp. A321. Int J Biol Macromol, 2016, 86: 96-104.

[17] Li Q, Hu F, Wang YM, Zhu BW, Ni F, Yao Z. Elucidation of degradation pattern and immobilization of a novel alginate lyase for preparation of alginate oligosaccharides. Int J Biol Macromol, 2020, 146: 579-587.

[18] Lietzke SE, Scavetta RD, Yoder MD, Jurnak F. The refined three-dimensional structure of pectate lyase E from Erwinia chrysanthemi at 2.2 Å resolution. Plant Physiol, 1996, 111(1): 73-92.

[19] Liu MQ, Huo WK, Dai X, Dang YH. Preparation of low-molecular-weight citrus pectin by recombinant Bacillus subtilis pectate lyase and promotion of growth of Bifidobacterium longum. Catal Commun, 2018, 107: 39-42.

[20] Margesin R, Schinner F. Characterization of a metalloprotease from psychrophilic Xanthomonas maltophilia. FEMS Microbiol Lett, 1991, 79(2–3): 257-261.

[21] Miller LG. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Biochem, 1959, 31(3): 426-428.

[22] Ogawa A, Sawada K, Saito K, Hakamada Y, Sumitomo N, Hatada Y, Kobayashi T, Ito S. A new high-alkaline and high-molecular-weight pectate lyase from a Bacillus isolate: enzymatic properties and cloning of the gene for the enzyme. Biosci Biotech Bioch, 2000, 64(6): 1133-1141.

[23] Olano-Martin E, Gibson GR, Rastall RA. Comparison of the in vitro bifidogenic properties of pectins and pectic oligosaccharides. J Appl Microbiol, 2002, 93(3): 505-511.

[24] Ramya LN, Pulicherla KK. Molecular insights into cold active polygalacturonase enzyme for its potential application in food processing. J Food Sci Technol, 2015, 52(9): 5484-5496.

[25] Saharan R, Sharma KP. Production, purification and characterization of pectin lyase from Bacillus subtilis isolated from moong beans leaves (Vigna radiata). Biocatal Agric Biotechnol, 2019, 21: 101306-101306.

[26] Tang YM, Wu P, Jiang SJ, Selvaraj JN, Yang SH, Zhang GM. A new cold-active and alkaline pectate lyase from Antarctic bacterium with high catalytic efficiency. Appl Microbiol Biotechnol, 2019, 103(13): 5231-5241.

[27] Wang H, Li X, Ma Y, Song J. Characterization and high-level expression of a metagenome-derived alkaline pectate lyase in recombinant Escherichia coli. Process Biochem, 2014, 49(1): 69-76.

[28] Wang X, Lu Z, Xu T, Selvaraj JN, Yi L, Zhang G. Improving the specific activity and thermo-stability of alkaline pectate lyase from Bacillus subtilis 168 for bioscouring. Biochem Eng J, 2018, 129: 74-83.

[29] Wang Z, Xu B, Luo H, Meng K, Tu T. Production pectin oligosaccharides using Humicola insolens Y1-derived unusual pectate lyase. J Biosci Bioeng, 2019, 129(1): 16-22.

[30] Weikert T, Niehues A, Cord-Landwehr S, Hellmann MJ, Moerschbacher BM. Reassessment of chitosanase substrate specificities and classification. Nat Commun, 2017, 8(1): 1698.

[31] Wu P, Yang S, Zhan Z, Zhang G. Origins and features of pectate lyases and their applications in industry. Appl Microbiol Biotechnol, 2020, 104(48): 7247-7260.

[32] Yadav S, Yadav PK, YadavD YKDS. Purification and characterization of an alkaline pectin lyase from Aspergillus flavus. Process Biochem, 2008, 43(5): 547-552.

[33] Yang G, Chen W, Tan H, Li K, Li J, Yin H. Biochemical characterization and evolutionary analysis of a novel pectate lyase from Aspergillus parasiticus. Int J Biol Macromol, 2020, 152: 180-188.

[34] Yoder MD, Keen NT, Jurnak F. New domain motif: the structure of pectate lyase C, a secreted plant virulence factor. Science, 1993, 260(5113): 1503.

[35] Yuan P, Meng K, Wang Y, Luo H, Shi P, Huang H, Yao B. A low-temperature-active alkaline pectate lyase from Xanthomonas campestris ACCC 10048 with high activity over a wide pH range. Appl BiochemBiotechnol, 2012, 168(6): 1489-1500.

[36] Yuan Y, Zhang XY, Zhao Y, Zhang H, Zhou YF, Gao J. A novel PL9 pectate lyase from Paenibacillus polymyxa KF-1: cloning, expression, and its application in pectin degradation. Int J Mol Sci, 2019, 20: 3060.

[37] Zhan PW, Ye JN, Lin XP, Zhang F, Lin D, Zhang Y, Tang K. Complete genome sequence of Echinicola rosea JL3085, a xylan and pectin decomposer. Mar Genomics, 2020

[38] Zhang S, Hu H, Wang L, Liu F, Pan S. Preparation and prebiotic potential of pectin oligosaccharides obtained from citrus peel pectin. Food Chem, 2018, 244: 232-237.

[39] Zhao Y, Yuan Y, Zhang XY, Li YM, Li Q, Zhou YF, Gao J. Screening of a novel polysaccharide lyase family 10 pectate lyase from Paenibacillus polymyxa KF-1: cloning, expression and characterization. Molecules, 2018, 23(11): 2774.

[40] Zheng L, Xu Y, Li Q, Zhu B. Pectinolytic lyases: a comprehensive review of sources, category, property, structure, and catalytic mechanism of pectate lyases and pectin lyases. Bioresour Bioprocess, 2021, 8(1): 79.

[41] Zhou C, Xue Y, Ma Y. Characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic Bacillus licheniformis with potential in ramie degumming. Process Biochem, 2017, 54: 49-58.

[42] Zhu B, Ni F, Sun Y, Ning L, Yao Z. Elucidation of degrading pattern and substrate recognition of a novel bifunctional alginate lyase from Flammeovirga sp. NJ-04 and its use for preparation alginate oligosaccharides. Biotechnol Biofuels, 2019

[43] Zhuge B, Du GC, Shen W, Zhuge J, Chen J. Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris. Biochem Eng J, 2008, 40(1): 92-98.

Funding

National Natural Science Foundation of China(31601410)