Screening of a Novel Polysaccharide Lyase Family 10 Pectate Lyase from Paenibacillus polymyxa KF-1: Cloning, Expression and Characterization

Pectate lyase (EC 4.2.2.2) catalyzes the cleavage of α-1,4-glycosidic bonds of pectin polymers, and it has potential uses in the textile industry. In this study, a novel pectate lyase belonging to polysaccharide lyase family 10 was screened from the secreted enzyme extract of Paenibacillus polymyxa KF-1 and identified by liquid chromatography-MS/MS. The gene was cloned from P. polymyxa KF-1 genomic DNA and expressed in Escherichia coli. The recombinant enzyme PpPel10a had a predicted Mr of 45.2 kDa and pI of 9.41. Using polygalacturonic acid (PGA) as substrate, the optimal conditions for PpPel10a reaction were determined to be 50 °C and pH 9.0, respectively. The Km, vmax and kcat values of PpPel10a with PGA as substrate were 0.12 g/L, 289 μmol/min/mg, and 202.3 s−1, respectively. Recombinant PpPel10a degraded citrus pectin, producing unsaturated mono- and oligogalacturonic acids. PpPel10a reduced the viscosity of PGA, and weight loss of ramie (Boehmeria nivea) fibers was observed after treatment with the enzyme alone (22.5%) or the enzyme in combination with alkali (26.3%). This enzyme has potential for use in plant fiber processing.

Download Full-text

A Novel PL9 Pectate Lyase from Paenibacillus polymyxa KF-1: Cloning, Expression, and Its Application in Pectin Degradation

International Journal of Molecular Sciences ◽

10.3390/ijms20123060 ◽

2019 ◽

Vol 20 (12) ◽

pp. 3060 ◽

Cited By ~ 3

Author(s):

Ye Yuan ◽

Xin-Yu Zhang ◽

Yan Zhao ◽

Han Zhang ◽

Yi-Fa Zhou ◽

...

Keyword(s):

High Performance ◽

Pectate Lyase ◽

Apparent Molecular Weight ◽

Paenibacillus Polymyxa ◽

Single Step ◽

Water Soluble ◽

Citrus Pectin ◽

Pectin Degradation ◽

Pharmacologically Active

Pectate lyases play an important role in pectin degradation, and therefore are highly useful in the food and textile industries. Here, we report on the cloning of an alkaline pectate lyase gene (pppel9a) from Paenibacillus polymyxa KF-1. The full-length gene (1350 bp) encodes for a 449-residue protein that belongs to the polysaccharide lyase family 9 (PL9). Recombinant PpPel9a produced in Escherichia coli was purified to electrophoretic homogeneity in a single step using Ni2+-NTA affinity chromatography. The enzyme activity of PpPel9a (apparent molecular weight of 45.3 kDa) was found to be optimal at pH 10.0 and 40 °C, with substrate preference for homogalacturonan type (HG) pectins vis-à-vis rhamnogalacturonan-I (RG-I) type pectins. Using HG-type pectins as substrate, PpPel9a showed greater activity with de-esterified HGs. In addition, PpPel9a was active against water-soluble pectins isolated from different plants. Using this lyase, we degraded citrus pectin, purified fractions using Diethylaminoethyl (DEAE)-sepharose column chromatography, and characterized the main fraction MCP-0.3. High-performance gel permeation chromatography (HPGPC) analysis showed that the molecular mass of citrus pectin (~230.2 kDa) was reduced to ~24 kDa upon degradation. Ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS) and monosaccharide composition analyses demonstrated that PpPel9a worked as an endo-pectate lyase, which acted primarily on the HG domain of citrus pectin. In vitro testing showed that the degradation product MCP-0.3 significantly promotes the growth of Lactobacillus plantarum and L. rhamnosus. In this regard, the enzyme has potential in the preparation of pharmacologically active pectin products.

Download Full-text

Preparation of low-molecular-weight citrus pectin by recombinant Bacillus subtilis pectate lyase and promotion of growth of Bifidobacterium longum

Catalysis Communications ◽

10.1016/j.catcom.2018.01.017 ◽

2018 ◽

Vol 107 ◽

pp. 39-42 ◽

Cited By ~ 5

Author(s):

Ming-qi Liu ◽

Wen-kang Huo ◽

XianJun Dai ◽

Ya-hui Dang

Keyword(s):

Molecular Weight ◽

Bacillus Subtilis ◽

Bifidobacterium Longum ◽

Pectate Lyase ◽

Low Molecular Weight ◽

Citrus Pectin ◽

Recombinant Bacillus Subtilis

Download Full-text

The first structure of pectate lyase belonging to polysaccharide lyase family 3

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444901014482 ◽

2001 ◽

Vol 57 (12) ◽

pp. 1786-1792 ◽

Cited By ~ 31

Author(s):

Masatake Akita ◽

Atsuo Suzuki ◽

Tohru Kobayashi ◽

Susumu Ito ◽

Takashi Yamane

Keyword(s):

Pectate Lyase ◽

Polysaccharide Lyase

Download Full-text

Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04

Marine Drugs ◽

10.3390/md17060323 ◽

2019 ◽

Vol 17 (6) ◽

pp. 323 ◽

Cited By ~ 7

Author(s):

Qian Li ◽

Fu Hu ◽

Benwei Zhu ◽

Yun Sun ◽

Zhong Yao

Keyword(s):

Biochemical Characterization ◽

Alginate Lyase ◽

Ionization Mass ◽

Action Pattern ◽

Polysaccharide Lyase ◽

Glycosidic Bonds ◽

Alginate Oligosaccharides ◽

Degradation Pattern ◽

Alginate Lyases ◽

Layer Chromatography

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of −1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.

Download Full-text

Detection of the Optimal Conditions for Pectate lyase Productivity and Activity by Erwiniachrysanthemi

Journal of Medical and Bioengineering ◽

10.12720/jomb.4.3.184-191 ◽

2015 ◽

Vol 4 (3) ◽

pp. 184-191 ◽

Cited By ~ 1

Author(s):

Sahira N. Muslim ◽

Israa M.S. AL_Kadmy ◽

Alaa N. Mahammed ◽

Hadeel K. Musafer ◽

Sraa N. Muslim

Keyword(s):

Pectate Lyase ◽

Optimal Conditions

Download Full-text

A novel low-temperature active alkaline pectate lyase from Klebsiella sp. Y1 with potential in textile industry

Process Biochemistry ◽

10.1016/j.procbio.2011.06.023 ◽

2011 ◽

Vol 46 (10) ◽

pp. 1921-1926 ◽

Cited By ~ 16

Author(s):

Peng Yuan ◽

Kun Meng ◽

Huiying Luo ◽

Pengjun Shi ◽

Huoqing Huang ◽

...

Keyword(s):

Low Temperature ◽

Textile Industry ◽

Pectate Lyase ◽

Alkaline Pectate Lyase

Download Full-text

Characteristics of pectate lyase formation by Hypomyces solani f. sp. cucurbitae

Canadian Journal of Microbiology ◽

10.1139/m70-013 ◽

1970 ◽

Vol 16 (2) ◽

pp. 69-74 ◽

Cited By ~ 9

Author(s):

Joseph G. Hancock ◽

Colleen Eldridge ◽

Michal Alexander

Keyword(s):

Fusarium Solani ◽

Culture Medium ◽

Pectate Lyase ◽

Extracellular Enzyme ◽

Citrus Pectin ◽

Inorganic N ◽

Inoculum Level ◽

Active Growth ◽

Initial Ph ◽

Enzyme Formation

Pectate lyase, produced by Hypomyces (Fusarium) solani f. sp. cucurbitae, is considered a normal extracellular enzyme in that most of the enzyme was found in the culture medium during early stages of enzyme production and before extensive lysis. This enzyme was produced during active growth in the presence of citrus pectate but not citrus pectin. Enzyme yield was highest when the initial pH of the medium was between 5.5 and 7.5 and the temperature was between 25 and 29 C. Production of the enzyme was reduced when individual amino acids or inorganic N-sources were substituted for casein hydrolyzates. Glucose repressed enzyme formation when it exceeded 0.025%, while arabinose and xylose had little effect or stimulated greater production at 0.1%. Pectate lyase yield varied considerably, depending on fungus strain and inoculum level used. A delay in enzyme formation was reduced substantially when small amounts of agar were present in the liquid medium or the medium was solidified with agar.

Download Full-text

Factorial experimental design for the removal of disperse dyes using hydroxyapatite prepared from Moroccan phosphogypsum

Mediterranean Journal of Chemistry ◽

10.13171/mjc811902219mb ◽

2019 ◽

Vol 8 (1) ◽

pp. 1-9

Author(s):

Meryeme Joudi ◽

Jihan Mouldar ◽

Houyem Hafdi ◽

Hamid Nasrellah ◽

Badreddine Hatimi ◽

...

Keyword(s):

Experimental Design ◽

Azo Dyes ◽

Textile Industry ◽

Contact Time ◽

Disperse Dyes ◽

Synthetic Dyes ◽

Optimal Conditions ◽

Factorial Experimental Design ◽

Full Factorial Experimental Design ◽

Full Factorial

Azo dyes are the major group of synthetic dyes known and have given rise to many water and soil environmental problems, the most of this azo dyes were used in textile industry. The aim of this study is the removal of Disperse Blue 79 (DB 79) and Disperse Blue 165 (DB 165) as azo dyes by Hydroxyapatite (HAP). The adsorption experiments were carried out to investigate the factors that influence the dyes uptake by hydroxyapatite, such as the contact time under agitation, adsorbent dosage, initial dye concentration and size of HAP. To reduce the number of experiments, full factorial experimental design at two levels (24) was used to achieve optimal conditions for the removal of DB 79 and DB 165 from aqueous solutions.

Download Full-text