Enzymatic Digestion Of Cell Wall Research Articles

An enzyme-based system for extraction of small extracellular vesicles from plants

Plant-derived nanovesicles (NVs) and extracellular vesicles (EVs) are the next generation of nanocarrier platforms for biotherapeutics and drug delivery. EVs exist not only in the extracellular space, but also within the cell wall. Due to the limitations of existing isolation methods, the EVs extraction efficiency is low, and a large amount of plant material is wasted, which is of concern for rare and expensive medicinal plants. We proposed and validated a novel method for isolation of plant EVs by enzyme degradation of the plant cell wall to release the EVs. The released EVs can easily be collected. The new method was used for extraction of EVs from the roots of Morinda officinalis (MOEVs). For comparison, nanoparticles from the roots (MONVs) were extracted using the grinding method. The new method yielded a greater amount of MOEVs, and the vesicles had a smaller diameter compared to MONVs. Both MOEVs and MONVs were readily absorbed by endothelial cells without cytotoxic effect and promoted the expression of miR-155. The promotion of miR-155 by MOEVs was dose-dependent. More importantly, we found that MOEVs and MONVs were enriched toward bone tissue. These results support our hypothesis that EVs in plants could be efficiently extracted by enzymatic cell wall digestion and confirm the potential of MOEVs as therapeutic agents and drug carriers.

Partial enzymatic cell wall disruption of Oocystis sp. for simultaneous cultivation and extraction

A green extraction process is proposed to reduce energy and petroleum solvent’s consumption while maintaining environmental safety and quality that allows multiple product recovery. The goal of the study was to generate a highly permeable cell, through the use of crude fungal enzymes, that could permit simultaneous cultivation and extraction. A novel pretreatment method is developed which combines partial enzymatic digestion of cell walls of microalgae, while maintaining their viability, thus enabling further product recovery from the same biomass and has not been reported in literature yet. Partial digestion of the cell wall was accomplished through regulation of the contact time of crude enzymes with the microalgal cells leading to a highly permeable cell that is viable and actively growing. Experimentally this was achieved through controlling the time of incubation of microalgal cells with the crude enzymes. The effects of enzymatic treatment using crude fungal enzymes from Trichoderma reesei (cellulase 10.98 ± 0.17 U/ml and amylase 20.28 ± 0.13 U/ml) were investigated on the structural properties of marine microalga Oocystis sp. Results showed over 2-fold increase in protein efflux; 41% increase in released sugars and 24% decrease in cell count after 1 h of enzyme treatment at 33% (v/v) enzyme concentration. Controlled hydrolysis of algal cells could be achieved through optimizing the contact time (1 h) of enzymes with microalgal cells which retained their viability. This was validated through FTIR, SEM, XRD and Flow Cytometric analysis. The carotenoid content increased by approximately 9% and lipid content rose by nearly 6.2%.

An Automated Protoplast Transformation System.

Efficient plant protoplast production from cell suspension cultures, leaf, and stem tissue allows for single-cell plant biology. Since protoplasts do not have cell walls, they can be readily transformed to enable rapid assessment of regulatory elements, synthetic constructs, gene expression, and more recently genome-editing tools and approaches. Historically, enzymatic cell wall digestion has been both expensive and laborious. Protoplast production, transformation, and analysis of fluorescence have recently been automated using an integrated robotic system. Here we describe its use for bulk protoplast isolation, counting, transformation, and analysis at very low cost for high-throughput experiments.

The Isolation of Plant Organelles and Structures in the Post-genomic Era.

In this chapter, we provide an overview of the techniques and approaches used in the isolation of plant organelles and structures. This overview shows there is a great diversity of methods currently used for the initial physical disruption of plant tissue before the downstream isolation of a target cellular component. These include hand grinding, high-speed mechanical disruption, and enzymatic digestion of cell walls by a variety of methods. Coupled to these disruption techniques is a wide array of additives included as ingredients in extraction solutions to minimize chemical or physical damage that may occur to target components. These additives are collated into a table outlining their function. We also provide an introduction to some of the history of common approaches used for the isolation plant organelles and structures and a synopsis of the methods used by researchers for assessment of the purity of their isolated structures. This chapter therefore provides an introduction to the following chapters that document the methodology for the isolation of individual plant organelles or structures.

Improvement of oxidative stability of menhaden fish oil by microencapsulation within biocapsules formed of yeast cells

Fish oil, rich in polyunsaturated fatty acids, is a valuable addition to human diet. Its composition makes it difficult to handle as it is prone to oxidation and its strong smell is repelling to many. Encapsulation in yeast cells is a possible solution to these issues. The aim of this study was to determine the best pretreatment approach, optimize the process of microencapsulation and verify the stability of the oil encapsulated in Saccharomyces cerevisiae. Pretreatment by autolysis, chemically facilitated autolysis and enzymatic cell wall digestion was tested. Autolysis at 55°C supported by addition of 1.5% ethyl acetate was an effective pretreatment method. Process parameter values (temperature, stirring rate and emulsifier addition) were optimized, improving efficiency to ca. 90%. The encapsulated oil was shown to be stable when stored for 30days at relative humidity below 70%. Additional coating with hydroxypropyl methyl cellulose allowed to improve storage stability.

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.

Effects of red yeast inclusions in diets for salmonids and extrusion temperature on pellet tensile strength: Weibull analysis

Enhancement of fish feed extrusion-technology and improvements of technical pellet quality requires knowledge about causal connections amongst variables of processing condition and raw materials, and how these connections can be elucidated by measurements. Weibull analysis of feed pellet tensile strengths may represent one possible measurement tool for this purpose; this analysis is evaluated here and compared with the Holmen durability test. Diets added pre-treated red yeast Xanthophyllomyces dendrorhous (Basidiomycetes) that differ in the degree of enzymatic cell wall digestion (45, 70 and 97% CWD) were extruded at two temperatures, 100 and 140 °C. Pellet expansion ratio (32%) was independent of both temperature and yeast CWD. Bulk density and sinking velocity corroborated accordingly, with only marginal difference in density (640 kg m −3) and no effect of the independent variables on sinking velocity (10.7 ms −1). Average Holmen durability was significant higher (P<0.01) for pellets extruded at 100 °C (97.2%) than at 140 °C (96.4%), and the difference increased disproportional for testing times of 90 and 120 s. Tensile stresses at failure were Weibull distributed with values for the modulus m in the range 4.3–6.6, and the scale parameter σ 0 in the range 360–430 kPa. Inclusions of yeast cells with digested cell walls (97% CWD) improved tensile strengths significantly, and the positive effect was evident at both extrusion temperatures. Pellets extruded at the highest temperature showed significant lower strength. Weibull analysis suggests that the effect of temperature is caused by differences in anisotropy in these feeds.

DNA isolation protocol for seaweeds

We report a DNA isolation protocol for red seaweeds. Recovering DNA of high quality and quantity is a prerequisite for ensuring suitable applications, such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and sequencing. Isolation of DNA from seaweeds has proven difficult because of coprecipitation of polysaccharides. Our process minimizes this contamination, which is mostly due to the highly hydrocolloidal content of algal cell walls. This protocol, using 2 steps, is based on a preliminary enzymatic digestion of cell wall with specific enzymes (Novozymes) followed by centrifugation, allowing isolation of DNA on the pellet. This provides a higher yield of DNA, in the range of 40 μg (Palmaria palmata) and 18 μg (Gracilaria verrucosa) from 50 mg of fresh frozen pellet.

An Improved Method for Examining Meiotic Chromosomes in Musa L.

Meiotic studies in Musa L. have been hampered by: 1) time-consuming efforts required to find the correct stages of cell division; 2) rigidity of the microsporocyte cell wall that makes preparation of smears difficult; and 3) poor staining of prophase chromosomes. This study describes an improved technique to examine meiosis in Musa. The procedure involves dissection of microsporocytes from the anthers, centrifugation to obtain large number of microsporocytes, enzymatic digestion of cell walls and treatment of cells with acetic-alcohol that results in spontaneous bursting of the protoplasts and release of chromosomes. Previous meiotic studies in Musa used acetocarmine that stained only highly condensed metaphase and anaphase chromosomes easily but not the relaxed prophase stages. In this study, we found that silver nitrate, Giemsa and Leishmans' stain were also effective for staining Musa chromosomes. Silver staining was most effective for the less contracted prophase chromosomes. By providing an improved procedure to examine all the meiotic stages in Musa, this technique will be useful to develop pachytene karyotypes, characterize new hybrids and identify nuclear restitution mechanisms that are important in breeding schemes.

Light and Electron Microscope Immunocytochemistry in Freeze-Substituted Plant and Fungal Cells

In efforts to better understand the cell biology of fungi and their pathogenic interactions with plants, we have pursued the use of cryo-preparative techniques for specimen preservation and immunocytochemistry at the light and electron microscope level. The chemically diverse composition of plant and fungal walls has proven very challenging for immunofluorescence using confocal microscopy. Typically, to allow probe access in these organisms, immunolabeling has required sectioning or enzymatic digestion of cell walls. The ideal method would preserve the spatial fidelity and antigenicity of the target molecule as well as maintain the integrity of the surrounding tissue. Our early work using methacrylate de-embedment, as described for sectioned plant material, was very effective for freeze-substituted fungi. Further improvements, using a combination of slow freezing and freeze-substitution followed by methacrylate de-embedment, permitted whole mount fluorescence antibody labeling of a variety of phylogenetically diverse fungi without enzymatic digestion or sectioning (Fig.l).

Laser Microsurgery Permits Fungal Plasma Membrane Single-Ion-Channel Resolution at the Hyphal Tip

A method for formation of high-electrical-resistance seals on the Neurospora crassa plasma membrane, allowing resolution of single-ion-channel activity by patch clamp electrophysiology, is reported. Laser microsurgery permits access to the hyphal apex without enzymatic cell wall digestion and loss of morphological polarity. Cell wall reformation is delayed by brefeldin. This method can allow full characterization of apical plasma membrane channels, which are implicated in tip growth.

Protoplast Isolation from Shoots of Asparagus Cultures

Experiments to maximize the isolation and purification of viable protoplasts from shoot cultures of asparagus (Asparagus oficitzalis L.) were conducted. Important factors for high yield of viable protoplasts included: the use of in vitro etiolated shoots as source material; 0.6 M glucose as an osmoticum in a modified KM medium; a combination of pectinase, cellulase, and hemicellulase, each at 1% (wlv) for enzymatic digestion of cell walls; and physical factors such as the volume of enzyme solution and speed of gyratory shaking. Protoplasts were purified by suspending digested etiolated shoot tissue in 0.6 M sucrose, overlaid with KMG medium and centrifugation at 650 g. The asparagus genotype had a marked influence on protoplast yield, with some genotypes yielding up to 18.4 X 10h protoplastslg fresh etiolated shoot tissue with 90% viability.

A method for direct soil extraction and PCR amplification of endomycorrhizal fungal DNA

DNA from endomycorrhizal fungi was extracted directly from a weathered soil (alfisol) mixed with sand. Mycorrhizae were established in a greenhouse culture of Glomus clarum with Sudan grass (Sorghum vulgare var. sudanense) host plants. The extraction procedure included enzymatic digestion of cell walls, sodium dodecyl sulfate lysis of cells, polyvinylpolypyrrolidone absorption of organic compounds, and ethanol precipitation of the DNA. DNA in the extracts was amplified by the polymerase chain reaction using primers from the nuclear 17S rRNA sequence that were general to fungi or were specific to endomycorrhizae.

RAPD and other PCR-based analyses of plant genomes using DNA extracted from small leaf disks.

A nondestructive, early DNA diagnostic system to implement marker-assisted selection in plant breeding programs has been developed. The main components of the system are a rapid and simple DNA microextraction method and fast DNA polymorphism analyses based on site-specific or arbitrary DNA amplification. A small disk (5 mm diameter) is collected from one cotyledon or the first leaf of a young seedling using a common paper punch. Disruption of plant tissues is done by enzymatic digestion of cell walls. This ensures protection from sample-to-sample contamination and uniform DNA yield. DNA isolated from the resulting protoplasts is sufficient to perform a minimum of five and a maximum of 20 PCR reactions/sample. Total DNA, nuclear DNA, and RNA can be analyzed selectively. The system has been tested successfully with eight major crops. Amplification products generated with DNA prepared with this quick procedure are equivalent to those obtained from CsCl-purified DNA. Up to 120 plants can be treated in 2 days and the procedure lends itself to automation. Potential applications in plant breeding will be discussed.

The Effect of Bovine Serum Albumin and Cytohelicase on Surface-Spread Synaptonemal Complexes of Rye (Secale Cereale)

Synaptonemal complexes of rye meiocytes were spread on plastic coated slides for electron microscopic observation. Two proteins generally used in synaptonemal complex spreading techniques, bovine serum albumin and cytohelicase, were applied separately or in combination in an isotonic protoplast medium at concentrations of 0.1-5%. At high concentrations these proteins proved to enhance notably the ultimate number of cells with synaptonemal complexes in the preparations. Also under this condition, centromere structures became stainable with silver nitrate in both the synaptonemal complexes of pollen mother cells and in interphase nuclei of other cell types. Since the true action of cytohelicase under appropriate spreading conditions was uncertain, the putative enzymatic digestion of cell walls was determined in a series of experiments using the fluorochrome calcofluor white as a stain of callose walls. Obvious breakdown of the cell walls was not observed before 8 min of treatment under standard conditions. This made it plausible that the prime effect of cytohelicase is that of a nonspecific protein interacting with the chromatin and improving the adhesion of synaptonemal complexes to the hydrophobic plastic film. The differential staining of the centromere structures in the presence of bovine serum albumin and cytohelicase probably reflects a reduced spreading of these structures due to preferential binding between these proteins and centromeric proteins.

A method for genetic transformation of nonprotoplasted Streptococcus lactis

Plasmid transformation of whole cells of Streptococcus lactis LM0230 was demonstrated. The procedure required polyethylene glycol and incubation in hypertonic media, but did not require enzymatic cell wall digestion. Conditions were optimized, yielding 5 X 10(5) transformants per micrograms of pSA3 DNA. Variables tested for effect on transformation efficiency included molecular weight, concentration, and pH of polyethylene glycol; cell density; plating media; DNA concentration; heat shock; and incubation of cells in hypertonic buffer. DNAs transformed included pSA3, pVA856, pTV1, and c2 phi. Transformation from DNA-DNA ligation mixes, with DNA not purified through density gradients, and with previously frozen cells was also achieved. The method described here for transformation of nonprotoplasted cells of LM0230 is unique, and to date has not been applied successfully to other lactic acid bacteria.

Inhibition of melanin synthesis in the black yeast Phaeococcomyces sp. by growth on low pH ascorbate medium: production of spheroplasts from "albinized" cells

The black yeast Phaeococcomyces sp. produces pentaketide-based melanin constitutively, resulting in a green–black colony colour on commonly used isolation and culture maintenance agar media. Pentaketide melanins are produced by a multi-enzyme pathway, beginning with the cyclization of conjoined acetate molecules and ending with the polymerization of the final phenolic precursor, dihydroxynapthalene, into melanin. A low pH medium, which contains 0.1% ascorbic acid, causes complete inhibition of melanin formation, resulting in a white colony apearance similar to that of albino mutants of Phaeococcomyces sp. Melanized Phaeococcomyces sp. cells resisted enzymatic cell wall digestion, and spheroplasts could not be formed. In contrast, spheroplasts were produced from cells "albinized" by growth on the low pH medium. Inhibition of melanogenesis in other fungi, by growth on low pH ascorbate medium, was found to be specific to pentaketide melanin producers.

Investigation of the enzymatic digestion of plant cell walls using reflectance Fourier Transform Infrared spectroscopy

A method has been developed to determine the reflectance Fourier Transform Infrared spectra of plant cells grown in vitro and of the protoplasts released from such cells by enzymatic digestion. It is demonstrated that there is a smooth and reproducible transition in spectral detail as enzymatic digestion procedes. Reflectance Fourier Transform Infrared spectroscopy has been used to monitor the progress of protoplast release during enzymatic digestion of cell wall material.

Ornithine- and Putrescine-Supported Divisions and Cell Colony Formation in Leaf Protoplasts of Alders (Alnus Glutinosa and A. Incana)

Summary Mesophyll protoplasts were isolated from leaves of Alnus glutinosa (L.) Gaertn. and A. incana (L.) Moensch using enzymatic digestion of cell walls. The protoplasts slowly synthesized new cell walls and 5–10% of the cells divided after 3 weeks. Ornithine (0.025 mM) and putrescine (0.05 and 0.1 mM) were found to stimulate cell division and colony formation in both Alnus species tested. A low concentration of spermidine (0.025 mM) induced slight cell division in A. glutinosa , but spermine and higher concentrations of spermidine were inhibitory and no cell colony formation was observed. The induction of sustained cell divisions and of cell colony formation also seemed to depend on the physiological condition of the seedlings and on the progenies used as starting material for the isolation of protoPlasts. After the cell colonies had been transferred to agar media with the same composition as the media inducing cell divisions, but lacking mannitol, the protoplasts of two A. glutinosa progenies formed actively growing calluses, which turned green in light.