An environment‐friendly approach to isolate and purify glucan from spent cells of recombinant <i>Pichia pastoris</i> and the bioactivity characterization of the purified glucan

Xing, Yan; Chen, Chaonan; Sun, Wenlong; Zhang, Bowei; Sang, Yuanbin; Jiang, Lili; Dong, Yuesheng

doi:10.1002/elsc.201700125

Cited by 9 publications

(13 citation statements)

References 25 publications

(54 reference statements)

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“…β-glucan mass and yeast cell mass weight can be used to determine the percentage of β-glucan produced based on the obtained cell mass [35]. β-glucan from Pichia pastoris produced a yield of 11.7% with a purity of 85.3% [71]. The percentage of β-glucan to cell mass obtained from Saccharomyces cerevisiae was 6-12% [72].…”

Section: Production Of β-Glucan From Tofu Wastewater By Native Yeast Isolatementioning

confidence: 99%

“…One of the factors that affects the amount of yield produced is the treatment at the autolysis stage and the β-glucan extraction method. Cell autolysis is a key step for isolating polysaccharides and inactivating yeast cells [71]. At this stage, yeast cells are under stress conditions that are influenced by their environment so that endogenous enzymes will be activated to degrade proteins and other macromolecules in the cell.…”

Section: Production Of β-Glucan From Tofu Wastewater By Native Yeast Isolatementioning

confidence: 99%

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Antifungal and Aflatoxin-Reducing Activity of β-Glucan Isolated from Pichia norvegensis Grown on Tofu Wastewater

Utama

Suraloka

Rialita

et al. 2021

Foods

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Yeast can be isolated from tofu wastewater and the cell wall in the form of β-glucan can act as a natural decontaminant agent. This study aimed to isolate and characterize native yeast from tofu wastewater, which can be extracted to obtain β-glucan and then identify the yeast and its β-glucan activity regarding antifungal ability against Aspergillus flavus and aflatoxin-reducing activity towards aflatoxin B1 (AFB1) and B2 (AFB2). Tofu wastewater native yeast was molecularly identified, and the growth observed based on optical density for 96 h and the pH also measured. β-glucan was extracted from native yeast cell walls with the acid-base method and then the inhibition activity towards A. flavus was tested using the well diffusion method and microscopic observation. AFB1 and AFB2 reduction were identified using HPLC LC-MS/MS. The results showed that the native yeast isolated was Pichia norvegensis with a β-glucan yield of 6.59%. Pichia norvegensis and its β-glucan showed an inhibition zone against Aspergillus flavus of 11.33 ± 4.93 and 7.33 ± 3.51 mm, respectively. Total aflatoxin-reducing activity was also shown by Pichia norvegensis of 26.85 ± 2.87%, and β-glucan of 27.30 ± 1.49%, while AFB1- and AFB2-reducing activity by Pichia norvegensis was 36.97 ± 3.07% and 27.13 ± 1.69%, and β-glucan was 27.13 ± 1.69% and 32.59 ± 4.20%, respectively.

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Section: Production Of β-Glucan From Tofu Wastewater By Native Yeast Isolatementioning

confidence: 99%

Section: Production Of β-Glucan From Tofu Wastewater By Native Yeast Isolatementioning

confidence: 99%

Antifungal and Aflatoxin-Reducing Activity of β-Glucan Isolated from Pichia norvegensis Grown on Tofu Wastewater

Utama

Suraloka

Rialita

et al. 2021

Foods

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“…Other constituents of the cell wall are mannans accounting for up to 30% of its mass, chitin 1–2%, proteins up to 30% and lipids around 9%. Mannans, chitin and proteins attach to glucans through branched β-1,6-glucan molecules [ 14 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. Due to the function of cell walls, molecules within are highly organized and linked together, which makes the isolation of β-glucan difficult, time and energy consuming multistep process [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

“…Cell lysis and cell wall breaking is an essential step in glucan isolation. Different isolation methods affect the mass and branching of the β-glucans, which can lead to a wide range of molecular mass distribution, and level of branching, yield and purity of the final product can lead to structural differences of extracted β-glucans and, as a result, differences in their biological activity [ 1 , 16 , 23 , 25 , 26 , 27 , 28 ].…”

Section: Introductionmentioning

confidence: 99%

FT-IR Method Limitations for β-Glucan Analysis

et al. 2022

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β-glucans are known as biological response modifiers. However, different sources can result in structural differences and as a result differences in their biological activity. The hot water extraction method allows to obtain, high molecular weight β-glucans without altering their structure by using strong chemicals, such as alkalis or acids. Analysis of β-glucans by FT-IR and NMR spectroscopy in solid state is superior to analysis in solution as it allows researchers to study the preserved structure of the extracted polysaccharides. FT-IR spectroscopy was used in this study to make side-by-side comparison analysis of hot water extracted β-glucans from different yeast sources. NMR spectroscopy was used to confirm findings made by FT-IR spectroscopy. Extracted β-glucans exhibit characteristic structure of β-1,3/1,6-linked glucans with noticeable levels of proteins, possibly in a form of oligopeptides, chitin and other impurities. β-glucans obtained from C. guilliermondii, P. pastoris and S. pastorianus exhibited higher protein content. Differences in mannan, chitin and α-glucan content were also observed; however, the species-specific structure of obtained β-glucans could not be confirmed without additional studies. Structural analysis of high molecular weight β-glucans in solid state by FT-IR spectroscopy is difficult or limited due to band intensity changes and overlapping originating from different molecules.

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“…A cold-induced promoter driving the endo-beta-1,3-glucanase gene ( eng ) was constructed and transformed into P. pastoris , and the transformant underwent autolysis after a cold-shock, which could reduce the protein recovery cost [ 6 ]. A NaCl-induced autolysis was performed to isolate and purify glucan from the cell wall of the P. pastoris [ 7 ]. Although autolysis is an important cellular process which can be prevented or applied in the protein expression system to improve efficiency and reduce cost, the mechanism of this process in P. pastoris has not been totally illuminated.…”

Section: Introductionmentioning

confidence: 99%

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris

Wang

Sheng

et al. 2022

Molecules

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Autolysis is a common physiological process in eukaryotic cells that is often prevented or applied, especially in yeast expression systems. In this study, an antimicrobial peptide from chicken (AMP) was recombinantly expressed in the Pichia pastoris expression system, which induced a series of cellular autolysis phenotypes after methanol treatment, such as the aggregated, lysed, irregular, and enlarged cell morphology, while the cells expressing a recombinant aflatoxin-detoxifizyme (ADTZ) were not autolyzed. A comparative transcriptomic analysis showed that the transcriptomic profiles of cells derived from the autolysis and non-autolysis groups were well discriminated, suggesting that the mechanisms of autolysis were at the transcriptional level. A further differential expression gene (DEG) analysis showed that the DEGs from the two groups were involved mainly in autophagy, the MAPK signaling pathway, transcriptional factors, the central carbon metabolism, anti-stress functions, and so on. In the autolysis group, the cell activity was significantly reduced with the MAPK signaling pathway, the central carbon metabolism was down-regulated, and components of the cytoplasm-to-vacuole targeting (CVT) and mitophagy pathways were up-regulated, suggesting that the autophagy involved in the trafficking of intracellular molecules in the vacuole and mitochondrion contributed to autolysis, which was regulated by transcriptional factors and signal pathways at the transcriptional level. This study provides a theoretical basis for genetic modifications to prevent or utilize cell autolysis in the recombinant protein expression system.

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An environment‐friendly approach to isolate and purify glucan from spent cells of recombinant Pichia pastoris and the bioactivity characterization of the purified glucan

Cited by 9 publications

References 25 publications

Antifungal and Aflatoxin-Reducing Activity of β-Glucan Isolated from Pichia norvegensis Grown on Tofu Wastewater

Antifungal and Aflatoxin-Reducing Activity of β-Glucan Isolated from Pichia norvegensis Grown on Tofu Wastewater

FT-IR Method Limitations for β-Glucan Analysis

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris

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