A Simple Method for Removal of the Chlamydomonas reinhardtii Cell Wall Using a Commercially Available Subtilisin (Alcalase)

2018 ◽  
Vol 28 (4) ◽  
pp. 169-178 ◽  
Author(s):  
Hyun-Ju Hwang ◽  
Yong Tae Kim ◽  
Nam Seon Kang ◽  
Jong Won Han
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Algal Cell ◽  
Chemical Properties ◽  
Transformation Method ◽  
Lytic Enzyme ◽  
Higher Plant ◽  
Time Saving ◽  
Simple Method ◽  
Cell Wall Disruption

The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for <i>Chlamydomonas reinhardtii</i> using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of <i>C. reinhardtii</i>. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of <i>C. reinhardtii</i>, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans­formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

scholarly journals A Simple Method for the Determination of Grown Mycelial Content in Ricekoji using Commercial Cell Wall Lytic Enzyme, Yatalase

1992 ◽  
Vol 87 (10) ◽  
pp. 757-759 ◽  
Author(s):  
Fumiko FUJII ◽  
Kenji OZEKI ◽  
Akihiro KANDA ◽  
Masaaki HAMACHI ◽  
Yataro NUNOKAWA
Keyword(s):  
Cell Wall ◽  
Lytic Enzyme ◽  
Simple Method

scholarly journals The Effect of High-Intensity Ultraviolet Light to Elicit Microalgal Cell Lysis and Enhance Lipid Extraction

Metabolites ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 65
Author(s):  
Thomas Sydney ◽  
Jo-Ann Marshall-Thompson ◽  
Rahul Kapoore ◽  
Seetharaman Vaidyanathan ◽  
Jagroop Pandhal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Uv Irradiation ◽  
Algal Cell ◽  
Lipid Extraction ◽  
Downstream Processing ◽  
Ultraviolet B ◽  
Cell Counting ◽  
Small Scale ◽  
Bead Beating ◽  
Cell Wall Disruption

Currently, the energy required to produce biofuel from algae is 1.38 times the energy available from the fuel. Current methods do not deliver scalable, commercially viable cell wall disruption, which creates a bottleneck on downstream processing. This is primarily due to the methods depositing energy within the water as opposed to within the algae. This study investigates ultraviolet B (UVB) as a disruption method for the green algae Chlamydomonas reinhardtii, Dunaliella salina and Micractinium inermum to enhance solvent lipid extraction. After 232 seconds of UVB exposure at 1.5 W/cm2, cultures of C. reinhardtii (culture density 0.7 mg/mL) showed 90% disruption, measured using cell counting, correlating to an energy consumption of 5.6 MJ/L algae. Small-scale laboratory tests on C. reinhardtii showed bead beating achieving 45.3 mg/L fatty acid methyl esters (FAME) and UV irradiation achieving 79.9 mg/L (lipids solvent extracted and converted to FAME for measurement). The alga M. inermum required a larger dosage of UVB due to its thicker cell wall, achieving a FAME yield of 226 mg/L, compared with 208 mg/L for bead beating. This indicates that UV disruption had a higher efficiency when used for solvent lipid extraction. This study serves as a proof of concept for UV irradiation as a method for algal cell disruption.

scholarly journals AN INSIGHT ON ALGAL CELL DISRUPTION FOR BIODIESEL PRODUCTION

2018 ◽  
Vol 11 (2) ◽  
pp. 21 ◽  
Author(s):  
Aarthy A ◽  
Smita Kumari ◽  
Prachi Turkar ◽  
Sangeetha Subramanian
Keyword(s):  
Cell Wall ◽  
Enhanced Recovery ◽  
Algal Cell ◽  
Lipid Extraction ◽  
Algal Species ◽  
Cost Effective ◽  
Downstream Processing ◽  
Cell Disruption ◽  
Biodiesel Production ◽  
Cell Wall Disruption

 Objective: This review article deals with the effect that various cell disruption techniques have on the efficiency of lipid extraction. We have reviewed existing algal cell disruption techniques that aid the biodiesel production process.Methods: Current rise in demand for energy has led the researcher to focus on the production of sustainable fuels, among which biodiesel has received greater attention. This is due to its larger lipid content, higher growth rate, larger biomass production, and lower land use. Extraction of lipid from algae (micro and macro) for the production of biodiesel involves numerous downstream processing steps, of which cell wall disruption is a crucial step. Bead milling, high-pressure homogenization, ultra-sonication, freeze-drying, acid treatment, and enzymatic lysis are some methods of cell disruption. The cell disruption technique needs to be optimized based on the structure and biochemical composition of algae.Result: The lipid extraction efficiency varies depending on the algal species and the cell disruption technique used.Conclusion: In-depth research and development of new techniques are required to further enhance the cell disruption of the algal cell wall for the enhanced recovery of lipids. In addition, the operating costs and energy consumption should also be optimized for the cost-effective recovery.

scholarly journals Development of a pVEC peptide-based ribonucleoprotein (RNP) delivery system for genome editing using CRISPR/Cas9 in Chlamydomonas reinhardtii

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Seongsu Kang ◽  
Seungjib Jeon ◽  
Seungcheol Kim ◽  
Yong Keun Chang ◽  
Yeu-Chun Kim
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Genome Editing ◽  
Delivery System ◽  
Cell Biology ◽  
Algal Cell ◽  
Cell Penetrating Peptide ◽  
Simple Method ◽  
Cell Penetrating ◽  
Technical Advances ◽  
A Cell

AbstractRecent technical advances related to the CRISPR/Cas9-based genome editing system have enabled sophisticated genome editing in microalgae. Although the demand for research on genome editing in microalgae has increased over time, methodological research has not been established to date for the delivery of a ribonucleoprotein (Cas9/sgRNA complex) using a cell penetrating peptide into microalgal cell lines. Here, we present a ribonucleoprotein delivery system for Chlamydomonas reinhardtii mediated by the cell penetrating peptide pVEC (LLIILRRRIRKQAHAHSK) which is in a non-covalent form. Using this technically simple method, the ribonucleoprotein was successfully delivered into C. reinhardtii. Gene Maa7 and FKB12 were disrupted, and their distinguishing patterns of Indel mutations were analyzed with the observation of several insertions of sequences not originating from the genome DNA, such as chloroplast DNA, into the expected loci. In addition, the cytotoxicity of Cas9 and the ribonucleoprotein was investigated according to the concentration and time in the algal cells. It was observed that Cas9 alone without the sgRNA induces a more severe cytotoxicity compared to the ribonucleoprotein. Our study will not only contribute to algal cell biology and its genetic engineering for further applications involving various organisms but will also provide a deeper understating of the basic science of the CRISPR/Cas9 system.

Purification and characterization of cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii

FEBS Letters ◽  
1984 ◽  
Vol 166 (2) ◽  
pp. 293-297 ◽  
Author(s):  
Yoshihiro Matsuda ◽  
Atsuko Yamasaki ◽  
Saito Tatsuaki ◽  
Tetsuya Yamaguchi
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Lytic Enzyme ◽  
Purification And Characterization
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