High-Level Expression and Biochemical Properties of A Thermo-Alkaline Pectate Lyase From Bacillus sp. RN1 in Pichia pastoris With Potential in Ramie Degumming

Zheng, Xueyun; Zhang, Yimin; Liu, Xiaoxiao; Cheng, Li; Lin, Ying; Liang, Shuli

doi:10.3389/fbioe.2020.00850

Cited by 20 publications

(13 citation statements)

References 40 publications

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“…However, it was observed that Cu 2+ ions could significantly enhance the mannanase activity of AfMan5A. A similar phenomenon was also observed in other studies regarding enzymes, such as amylase [28], pectate lyase [29] and laccase [30]. We speculate that this is likely caused by the structure changes of AfMan5A in the presence of Cu 2+ ions.…”

Section: Discussionsupporting

confidence: 88%

Characterization of a Novel Thermophilic Mannanase and Synergistic Hydrolysis of Galactomannan Combined with Swollenin

Chen

et al. 2021

Catalysts

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Aspergillus fumigatus HBFH5 is a thermophilic fungus which can efficiently degrade lignocellulose and which produces a variety of glycoside hydrolase. In the present study, a novel β-mannanase gene (AfMan5A) was expressed in Pichia pastoris and characterized. AfMan5A is composed of 373 amino acids residues, and has a calculated molecular weight of 40 kDa. It has been observed that the amino acid sequence of AfMan5A showed 74.4% homology with the ManBK from Aspergillus niger. In addition, the recombined AfMan5A exhibited optimal hydrolytic activity at 60 °C and pH 6.0. It had no activity loss after incubation for 1h at 60 °C, while 65% of the initial activity was observed after 1 h at 70 °C. Additionally, it maintained about 80% of its activity in the pH range from 3.0 to 9.0. When carob bean gum was used as the substrate, the Km and Vmax values of AfMan5A were 0.21 ± 0.05 mg·mL−1 and 15.22 ± 0.33 U mg−1·min−1, respectively. AfMan5A and AfSwol showed a strong synergistic interaction on galactomannan degradation, increasing the reduction of the sugars by up to 31%. Therefore, these findings contribute to new strategies for improving the hydrolysis of galactomannan using the enzyme cocktail.

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Section: Discussionsupporting

confidence: 88%

Characterization of a Novel Thermophilic Mannanase and Synergistic Hydrolysis of Galactomannan Combined with Swollenin

Chen

et al. 2021

Catalysts

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“…pastoris GS115 strain containing the pPICHKAPel vector was used for the heterologous expression of pectate lyase (Pel from Bacillus sp. RN1) under control of the AOX1 promoter G/Pel ( Zheng et al, 2020 ). The P .…”

Section: Methodsmentioning

confidence: 99%

“…Pectate lyase activity was determined according to the method described by Zheng et al (2020) , with one modification: measuring the absorbance change at 235 nm with 2 mg PGA ml-1 as the substrate in 50 mM glycine–NaOH (pH 10.0) buffer containing 1 mM CaCl 2 for 40°C, 30 min. One unit (U) of pectin lyase activity was defined as the amount of enzyme that is required to produce unsaturated oligogalacturonide equivalent to 1 μmol of unsaturated diglucuronide min–1 using a molecular extinction coefficient of 4,600 M −1 cm −1 at 235 nm ( Zheng et al, 2020 ).…”

Section: Methodsmentioning

confidence: 99%

“…The P. pastoris GS115 strain containing the pPICHKAPel vector was used for the heterologous expression of pectate lyase (Pel from Bacillus sp. RN1) under control of the AOX1 promoter G/Pel (Zheng et al, 2020). The P. pastoris GS115 strain containing the pPICZαAphy vector was used for heterologous expression of phytase (Phy from C. amalonaticus CGMCC 1696) under the control of the AOX1 promoter G/Phy (Weidner et al, 2010).…”

Section: Experimental Strains and Mediamentioning

confidence: 99%

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Green Process: Improved Semi-Continuous Fermentation of Pichia pastoris Based on the Principle of Vitality Cell Separation

Wang

Zhang

et al. 2021

Front. Bioeng. Biotechnol.

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The large-scale fermentation of Pichia pastoris for recombinant protein production would be time consuming and produce a large amount of waste yeast. Here we introduce a novel semi-continuous fermentation process for P. pastoris GS115 that can separate vitality cells from broth and recycle the cells to produce high-secretory recombinant pectate lyase. It is based on differences in cell sedimentation coefficients with the formation of salt bridges between metal ions and various cell states. Compared to batch-fed cultivation and general semi-continuous culture, the novel process has significant advantages, such as consuming fewer resources, taking less time, and producing less waste yeast. Sedimentation with the addition of Fe3+ metal ions consumed 14.8 ± 0.0% glycerol, 97.8 ± 1.3% methanol, 55.0 ± 0.9 inorganic salts, 81.5 ± 0.0% time cost, and 77.0 ± 0.1% waste yeast versus batch-fed cultivation to produce an equal amount of protein; in addition, the cost of solid–liquid separation was lower for cells in the collected fermentation broth. The process is economically and environmentally efficient for producing recombinant proteins.

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“…Nevertheless, its industrial applications are limited. Under some of the required processing conditions, its stability and enzyme activity is decreased, and its efficiency in degumming ramie, cotton textiles, and pulp processing is not ideal (Xu et al 2021 ; Zheng et al 2020 ; Cheng et al 2019 ; Chiliveri and Linga 2014 ). Therefore, an alkaline pectin lyase with increased stability, heat resistance, and enzymatic activity is imperative to industrial processing.…”

Section: Introductionmentioning

confidence: 99%

Modification and application of highly active alkaline pectin lyase

Wei

et al. 2022

AMB Expr

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Alkaline pectate lyase has developmental prospects in the textile, pulp, paper, and food industries. In this study, we selected BacPelA, the pectin lyase with the highest expression activity from Bacillus clausii, modified and expressed in Escherichia coli BL21(DE3). Through fragment replacement, the catalytic activity of the enzyme was significantly improved. The optimum pH and temperature of the modified pectin lyase (PGLA-rep4) were 11.0 and 70 °C, respectively. It also exhibited a superior ability to cleave methylated pectin. The enzyme activity of PGLA-rep4, measured at 235 nm with 0.2% apple pectin as the substrate, was 554.0 U/mL, and the specific enzyme activity after purification using a nickel column was 822.9 U/mg. After approximately 20 ns of molecular dynamics simulation, the structure of the pectin lyase PGLA-rep4 tended to be stable. The root mean square fluctuation (RMSF) values at the key catalytically active site, LYS168, were higher than those of the wildtype PGLA. In addition, PGLA-rep4 was relatively stable in the presence of metal ions. PGLA-rep4 has good enzymatic properties and activities and maintains a high pH and temperature. This study provides a successful strategy for enhancing the catalytic activity of PGLA-rep4, making it the ultimate candidate for degumming and various uses in the pulp, paper, and textile industries.

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High-Level Expression and Biochemical Properties of A Thermo-Alkaline Pectate Lyase From Bacillus sp. RN1 in Pichia pastoris With Potential in Ramie Degumming

Cited by 20 publications

References 40 publications

Characterization of a Novel Thermophilic Mannanase and Synergistic Hydrolysis of Galactomannan Combined with Swollenin

Characterization of a Novel Thermophilic Mannanase and Synergistic Hydrolysis of Galactomannan Combined with Swollenin

Green Process: Improved Semi-Continuous Fermentation of Pichia pastoris Based on the Principle of Vitality Cell Separation

Modification and application of highly active alkaline pectin lyase

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