Cloning, Secretory Expression and Characterization of a Unique pH-Stable and Cold-Adapted Alginate Lyase

Wang, Zhipeng; Cao, Min; Li, Bing; Ji, Xiaofeng; Zhang, Xinyue; Zhang, Yueqi; Wang, Haiying

doi:10.3390/md18040189

Cited by 35 publications

(52 citation statements)

References 33 publications

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“…The specific activity of purified Alyw202 toward sodium alginate was 1926.4 U/mg, while those toward polyG blocks and polyM blocks were 2132.2 and 1074.6 U/mg, respectively. These specific activities were much higher than those of Alyw201 [ 13 ]. The results indicated that Alyw202 may be a more efficient tool for alginate degradation.…”

Section: Resultsmentioning

confidence: 97%

“…As mentioned in our previous study, Vibrio sp. W2 was screened from the viscera of abalone using alginate as the sole carbon source at 25 °C [ 13 ]. During the 48 h fermentation in ASC liquid medium at 25 °C, alginate lyase activity was observed in culture supernatants, while no alginate-degrading activity was detcted in the collected intact cells.…”

Section: Resultsmentioning

confidence: 99%

“…To investigate further, it is necessary to clone the gene of Alyw202. The genome DNA of this strain was sequenced as mentioned earlier [ 13 ]. Sequence analysis revealed the presence of a single putative gene ALYW202 (MT424751) encoding an alginate lyase without the signal peptide, consistent with the intracellular-enzyme characteristic.…”

Section: Resultsmentioning

confidence: 99%

“…So far, many extracellular alginate lyases secreting from bacteria have been well characterized for potential industrial applications [ 13 , 14 , 15 , 16 , 17 , 18 ]. However, few studies of intracellular alginate lyases have been conducted, which are also potential candidates for commercial use of alginate lyases as important as the extracellular ones.…”

Section: Introductionmentioning

confidence: 99%

“…As reported previously, we have isolated a cold-adapted alginate lyase-producing strain Vibrio sp. W2, and the recombinant extracellular alginate lyase was also well characterized [ 13 ]. In this work, a new intracellular alginate lyase with pH-stable and high salt-tolerant properties has been identified and characterized from Vibrio sp.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of a New Intracellular Alginate Lyase with Metal Ions-Tolerant and pH-Stable Properties

Zhang

et al. 2020

Marine Drugs

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Alginate lyases play an important role in alginate oligosaccharides (AOS) preparation and brown seaweed processing. Many extracellular alginate lyases have been characterized to develop efficient degradation tools needed for industrial applications. However, few studies focusing on intracellular alginate lyases have been conducted. In this work, a novel intracellular alkaline alginate lyase Alyw202 from Vibrio sp. W2 was cloned, expressed and characterized. Secretory expression was performed in a food-grade host, Yarrowia lipolytica. Recombinant Alyw202 with a molecular weight of approximately 38.3 kDa exhibited the highest activity at 45 °C and more than 60% of the activity in a broad pH range of 3.0 to 10.0. Furthermore, Alyw202 showed remarkable metal ion-tolerance, NaCl independence and the capacity of degrading alginate into oligosaccharides of DP2-DP4. Due to the unique pH-stable and high salt-tolerant properties, Alyw202 has potential applications in the food and pharmaceutical industries.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Characterization of a New Intracellular Alginate Lyase with Metal Ions-Tolerant and pH-Stable Properties

Zhang

et al. 2020

Marine Drugs

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Characterization and enhanced extracellular overexpression of a new salt‐activated alginate lyase

et al. 2021

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BACKGROUND Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance. RESULTS A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L–1 glycine and 10 mmol L–1 calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine‐related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL−1. CONCLUSION A new salt‐activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. © 2021 Society of Chemical Industry

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Cloning, expression, and characterization of a novel endo‐type alginate lyase from Microbulbifer sp. BY17

et al. 2022

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BACKGROUND: Alginate oligosaccharides (AOS), with various physiological effects, have been widely used in the food, agricultural, and pharmaceutical industries. The biological enzymatic method of preparing AOS, using alginate lyase, has more advantages compared with physical and chemical methods. Cloning and heterologously expressing alginate lyase are therefore very important.RESULTS: A novel alginate lyase, BY17PV7, from Microbulbifer sp. BY17, isolated from Gracilaria, was cloned and expressed in Escherichia coli BL21(DE3). BY17PV7 was about 27 KDa. BY17PV7 showed the greatest activity (150.42 ± 3.32 U/mg) at 43 °C and pH 8.9. It could be activated by Ca 2+ , Mn 2+ , Co 2+ , Fe 3+ , Na + , and inhibited by Mg 2+ , Zn 2+ , Ba 2+ , Cu 2+ , sodium dodecyl sulfate (SDS), ethylene diamine tetraacetic acid (EDTA). BY17PV7 had a wide range of substrate specificity and good degradation effects for poly ⊎-D-mannuronate (polyM) and poly ⊍-L-guluronate (polyG), demonstrating that it is a bifunctional alginate lyase. The kinetic parameters showed that BY17PV7 had a greater affinity for polyG. BY17PV7 released AOS with a degree of polymerization (DP) of 3-4 in an endolytic manner from sodium alginate. Alginate oligosaccharides showed strong antioxidant ability of reducing Fe 3+ and scavenging radicals such as hydroxyl, 2,2-azion-bia (3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH).CONCLUSION: A novel bifunctional alginate lyase, BY17PV7, was expressed and characterized in Escherichia coli BL21(DE3). The results were helpful for the analysis of the molecular mechanisms of degrading patterns in the polysaccharide lyase (PL) family.

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Cloning, Secretory Expression and Characterization of a Unique pH-Stable and Cold-Adapted Alginate Lyase

Cited by 35 publications

References 33 publications

Characterization of a New Intracellular Alginate Lyase with Metal Ions-Tolerant and pH-Stable Properties

Characterization of a New Intracellular Alginate Lyase with Metal Ions-Tolerant and pH-Stable Properties

Characterization and enhanced extracellular overexpression of a new salt‐activated alginate lyase

Cloning, expression, and characterization of a novel endo‐type alginate lyase from Microbulbifer sp. BY17

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