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An Improved Method for Protein Extraction from Minuscule Quantities of Fungal Biomass.

ABSTRACT: Filamentous fungi are ubiquitous eukaryotes having chitin as a major constituent of the cell wall. Chitin is tough to lyse due to which the intracellular fungal proteins are not readily accessible. The problem is further enhanced when the biomass to be analyzed for protein studies is too little due to the extreme experimental parameters under consideration such as increased or lowered pH, temperature, hydrostatic pressure, nutrients, etc. The method described here is capable of obtaining proteins from minuscule quantities of biomass (~5 mg lyophilized biomass). In this study, different lysing conditions and varied composition of extraction buffers were tried to obtain maximum protein of high quality. Lysis with zirconium beads in a combination buffer system (Tris-MgCl2 buffer, urea buffer I and urea buffer II) was best for extracting proteins from the fungal isolates used. The protocol described here provides for a simple and quick method for extraction of high-quality proteins from very less biomass that could be extended to other tough to lyse biological material also.

SUBMITTER: Krishnaswamy A

PROVIDER: S-EPMC6328415 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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An Improved Method for Protein Extraction from Minuscule Quantities of Fungal Biomass.

Krishnaswamy Akhila A Barnes Natasha N Lotlikar Nikita P NP Damare Samir R SR

Indian journal of microbiology 20180626 1

Filamentous fungi are ubiquitous eukaryotes having chitin as a major constituent of the cell wall. Chitin is tough to lyse due to which the intracellular fungal proteins are not readily accessible. The problem is further enhanced when the biomass to be analyzed for protein studies is too little due to the extreme experimental parameters under consideration such as increased or lowered pH, temperature, hydrostatic pressure, nutrients, etc. The method described here is capable of obtaining protein ...[more]

PMID: 30728637

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Project description:BACKGROUND:Phenolic acids are a major group of secondary metabolites widely distributed in plants. In the case of maize, the major proportion of these metabolites occurs in the edible grain and their antioxidant activities are associated with improvements in human health. However, conventional extraction of secondary metabolites is very time consuming and generates a substantial amount of solvent waste. One approach to resolve these limitations is the use of microscale approaches, which minimize the quantity of solvents required, as well as the sample amounts and processing times. The objective of this work was to develop an improved microscale method for extraction of phenolic acids from maize and to compare it with a conventional extraction method. RESULTS:The improved microscale extraction method, coupled with an HPLC-DAD detection method, allowed identification of ferulic acid, p-coumaric acid in its free and bound form, and some diferulic acids. In its free form, p-coumaric acid ranged in content from 2.4 to 6.5 µg/g dry weight (dw) using the conventional method and 7.7 to 54.8 µg/g dw using the improved microscale method. Free ferulic acid content ranged from 2.6 to 12.9 µg/g dw for the conventional method and 16.8 to 181.7 µg/g dw for the improved microscale method. In its bound form, p-coumaric acid ranged in content from 6.0 to 30.6 µg/g dw for the traditional method and 34.4 to 138.6 µg/g dw for the improved microscale method. Bound ferulic acid ranged from 131.8 to 427.5 µg/g dw for the conventional method and 673.8 to 1702.7 µg/g dw for the improved microscale method. The coefficient of variation associated was lower for the improved microscale method than for the conventional method, thereby assuring the replicability of the process. CONCLUSIONS:The improved microscale method proposed here increases the extraction power and batch capacity, while reducing the sample quantity, solvent amounts and extraction time. It also achieves a better replicability with a lower coefficient of variation than is possible with conventional extraction.

Project description:A key requirement for 13C Metabolic flux analysis (13C-MFA), a widely used technique to estimate intracellular metabolic fluxes, is an efficient method for the extraction of intermediate metabolites for analysis via liquid chromatography mass spectrometry (LC/MS). The 13C isotopic labeling results in further distribution of an already sparse pool of intermediate metabolites into isotopologues, each appearing as a separate chromatographic feature. We examined some of the reported solvent systems for the extraction of polar intracellular metabolites from three strains of cyanobacteria of the genus Synechococcus, viz., Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 7942, and a newly isolated Synechococcus elongatus PCC 11801 (manuscript under review). High resolution-LC/MS was used to assess the relative abundance of the extracted metabolites. The different solvent systems used for extraction led to statistically significant changes in the extraction efficiency for a large number of metabolites. While a few hundred m/z features or potential metabolites were detected with different solvent systems, the abundance of over a quarter of all metabolites varied significantly from one solvent system to another. Further, the extraction methods were evaluated for a targeted set of metabolites that are important in 13C-MFA studies of photosynthetic organisms. While for the strain PCC 7002, the reported method using methanol-chloroform-water system gave satisfactory results, a mild base in the form of NH4OH had to be used in place of water to achieve adequate levels of extraction for PCC 7942 and PCC 11801. While minor changes in extraction solvent resulted in dramatic changes in the extraction efficiency of a number of compounds, certain metabolites such as amino acids and organic acids were adequately extracted in all the solvent systems tested. Overall, we present a new improved method for extraction using a methanol-chloroform-NH4OH system. Our method improves the extraction of polar compounds such as sugar phosphates, bisphosphates, that are central to 13C-MFA studies.

Dataset Information

An Improved Method for Protein Extraction from Minuscule Quantities of Fungal Biomass.

Publications

An Improved Method for Protein Extraction from Minuscule Quantities of Fungal Biomass.

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