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Chemical Permeabilization of Cells for Intracellular Product Release
Published in Juan A. Asenjo, Separation Processes in Biotechnology, 2020
Thomas J. Naglak, David J. Hettwer, Henry Y. Wang
Triton X-100 has been used to permeabilize yeast cells for enzymatic assays. Lammers and Follmann (1986) employed 0.05% Triton in conjunction with four freeze/thaw cycles to assay deoxyribonucleotide reductase. Although the activity remained entirely with the cell, 12% of overall protein was released. Similarly, Miozzari and coworkers (1977) employed 0.05% Triton and one freeze /thaw cycle to assay tryptophan enzyme activities. Furthermore, Miozzari and coworkers (1978) also found that 0.05% Triton released 20–25% of cellular protein from yeast. Three enzymes smaller than 70,000 MW were released from the cells, but two enzymes larger than 132,000 MW were not, implying the existence of a molecular weight cutoff for protein release by Triton X-100. Detergents have been used in conjunction with other chemicals to treat yeast cells. Serrano and co-workers (1973) used Triton along with toluene and ethanol to render yeast cells permeable for assaying fructose 1,6-biphosphatase. Nucleases have been released from yeast for subsequent purification by following zymolyase digestion with treatment by the nonionic detergent Brij-58 (West et al., 1987; Symington and Kolodner, 1985; West and Korner, 1985).
Cell Disruption
Published in Pau Loke Show, Chien Wei Ooi, Tau Chuan Ling, Bioprocess Engineering, 2019
Different types of enzymes are used to digest cell walls depending on the type of cell wall. Lysozyme is a well-known enzyme used for cell disruption. The enzyme reacts with the peptidoglycan layer directly by hydrolysing the beta 1–4 glycosidic bonds in peptidoglycan. But in the case of gram negative cells, it requires the removal of the outer membrane before treatment with any enzyme. Hence, treatment using enzymes for cell disruption is not very suitable. Zymolyase, chitinase, cellulase, and pectinase are the enzymes used for yeast cell disruption. The addition of enzymes does not cause degradation of proteins or enzymes during extraction. However, enzymes are limited in availability and also very expensive. Hence, their usage is restricted only to labs (Harrison, 1991; Salazar and Asenjo, 2007).
Sustainable Pre-treatment Methods for Downstream Processing of Harvested Microalgae
Published in Kalyan Gayen, Tridib Kumar Bhowmick, Sunil K. Maity, Sustainable Downstream Processing of Microalgae for Industrial Application, 2019
Hrishikesh A. Tavanandi, A. Chandralekha Devi, K. S. M. S. Raghavarao
As discussed, the cell disruption efficiency is affected by the cell wall/membrane composition and location of target biomolecules in the cell. Many times, individual methods fail to offer the desired degree of cell disruption. Under such conditions, the synergistic combination of different methods are preferred (Anand et al. 2007). Most of the literature reported is a combination of mechanical and non-mechanical methods, for example, a combination of chemical and mechanical methods (Anand et al. 2007) or enzymatic hydrolysis with mechanical methods (Baldwin and Robinson 1990; Vogels and Kula 1992) and other relatively newer mechanical methods such as electropermeabilization (Ganeva et al. 2015) and US (Priego-Capote and de Castro 2007; Tavanandi et al. 2019). Baldwin et al. in their study on the disruption of commercially available pressed baker’s yeast observed that mechanical disruption using a high-pressure homogenizer resulted in only 32% disruption at 95 MPa pressure after four passes, whereas combinations of enzymatic hydrolysis by Zymolyase and high-pressure homogenization resulted in 100% disruption after four passes at 95 MPa (Baldwin and Robinson 1990). Similarly, an improvement in extraction efficiency was observed when the combination of heat and/or enzymatic treatment with mechanical methods such as high-pressure homogenization and wet milling was employed for cell disruption of Bacillus cereus (Vogels and Kula 1992). Enzymatic hydrolysis was applied to microwave pre-treated samples. This resulted in lipid recovery of ~97% wherein a five-fold increase in yield was observed compared to enzymatic hydrolysis alone (Jin et al. 2012). The improved yield was attributed to the permeabilization of the cell wall after microwave pre-treatment. The damaged cell wall is expected to be more susceptible to enzyme hydrolysis, and the same observation has been made in other reports also (Okuda et al. 2008; Tan and Lee 2014). Ganeva et al. applied a combination of electropermeabilization and subsequent treatment with a lytic enzyme to achieve the efficient and selective recovery of proteins from yeast (Ganeva et al. 2015). The electropermeabilization facilitated the extraction of a portion of the proteins, and the subsequent addition of lyticase enzyme led to a protein yield of 70%.
In-situ transesterification of single-cell oil for biodiesel production: a review
Published in Preparative Biochemistry & Biotechnology, 2023
Tasneem Gufrana, Hasibul Islam, Shivani Khare, Ankita Pandey, Radha P.
Enzymes such as lysozyme are used in cell wall disruption in microorganisms, especially in gram-positive bacteria. The lysosomes break the cell by degrading the 1,4-glucosidic linkage of peptidoglycan present in the bacterial cell wall. Nevertheless, these enzymes are ineffective against gram-negative bacteria as they differ in structural composition. In the case of yeast and fungal cell walls, zymolyase is an effective enzyme with 1,3-glucanase activity. Other enzymes include cellulases, pectinases, xylanases, and chitinases, which are widely employed to degrade cellular integrity. One of the gentlest approaches is to use enzymes in the cell lysis process. However, the enzyme’s high cost and restricted availability limit its use in large-scale operations.[147]