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Improvement of Useful Enzymes by Protein Engineering
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Since the introduction of recombinant DNA methodology, genes can be removed from their normal environment in an intact genome and isolated as DNA fragments on cloning vectors. The availability of purified genes in vitro in microgram amounts has dramatically expanded the potential for inducing mutations. In the controlled environment of the test tube, it is now possible to alter, efficiently and systematically, the sequence of nucleotides in a segment of DNA. The new methods of in vitro mutagenesis are divided into three broad categories: (1) methods that restructure segments of DNA, (2) localized random mutagenesis, and (3) site-directed (oligonucleotide-directed) mutagenesis. This classification emphasizes the practical aspects of each method’s application. The outlines of the second and third strategies are summarized in Figures 5 and 6, respectively.
Recombinant DNA Technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Depending on the viral vector, the typical maximum length of an allowable DNA insert in a replication-defective viral vector is usually about 8–10 kb. Although this limits the introduction of many genomic sequences, most cDNA sequences can still be accommodated. The primary drawback to the use of retroviruses such as the Moloney retrovirus involves the requirement for cells to be actively dividing for transduction. As a result, cells such as neurons are very resistant to infection and transduction by retroviruses. There is a concern for insertional mutagenesis because of the integration into the host genome, which can lead to cancer or leukemia.
Next-Generation Immunoassays
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Valerie Fitzgerald, Paul Leonard
An excellent example of this is provided by Korpimäki and colleagues, at the University of Turku, where they converted an antisulphonamide mAb obtained from a hybridoma and modified its specificity to bind up to 15 sulphonamide derivatives, significantly improving the capabilities of the immunoassay [26–29]. Altering the amino acid sequence of antibodies using gene engineering allows for an improvement in characteristics such as affinity, specificity, stability and immunogenicity [30]. Mutagenesis strategies can be divided into the following categories, random mutagenesis, site-directed mutagenesis and DNA recombination.
Enhanced biodiesel properties of Auxenochlorella sp. using chemical mutagenesis and Tralkoxydim
Published in International Journal of Green Energy, 2023
Random cell mutagenesis and adaptation techniques have been proposed by researchers to improve the microalgal strain with improved properties for the aim of biodiesel (Gnouma et al. 2018; Trovão et al. 2022; Wass et al. 2021). Random mutagenesis can be applied using chemical mutagens or physical mutagens. While de Jaeger et al. (2014) investigated the starch biosynthesis pathways of Scenedesmus obliquus using UV mutation at 254 nm and nitrogen depletion conditions; Choi et al. (2014) used radiation breeding for a mutant generation. Besides, chemical mutagens can be used to produce mutant microalgae such as Kawaroe et al. (2015) study. In that study, they used ethyl methane sulfonate (EMS), which attacks the guanine base of DNA molecules (Arora et al. 2022). The reverse mutation can stimulate the high lipid accumulation for random mutants. In another study, for Nannochloropsis sp., 60 mins of exposure to 0.5 M EMS had the highest lipid content, about 18% (control one is 5.53%). Hu et al. (2013) selected a Desmodesmus sp. mutant with higher photosynthetic efficiency and lipid content by using a gamma irradiation technique. Liu et al. (2015) also used gamma irradiation to create Scenedesmus sp. mutant with high lipid capacity. Sun et al. (2020) demonstrated that ARTP (Atmospheric and Room Temperature Plasma) exposure resulted in Desmodesmus sp. mutant with a 2-fold higher lipid productivity compared to the wild-type strain.
Improved bioethanol production using genome-shuffled Clostridium ragsdalei (DSM 15248) strains through syngas fermentation
Published in Biofuels, 2021
Siddhi Patankar, Amol Dudhane, A. D. Paradh, Sanjay Patil
Genome shuffling, in general, involves six steps: (i) development of promising mutants, (2) protoplast preparation, (iii) protoplast fusion, (iv) protoplast inactivation, (v) protoplast regeneration, and (vi) screening of desired fusants [17]. Promising mutants are generally derived from random mutagenesis using suitable physical or chemical mutagenic agents. Random mutagenesis is desired for the alteration of metabolic or phenotypical characteristics. Two or more promising strains can be further subjected to protoplast fusion. For protoplast formation, lytic enzyme and antibiotic treatment have been reported [25]. To enhance re-generation efficiency, the protoplast formed can be further subjected to inactivation using heat or UV treatment [21]. In later stages, fusion can be carried out using fusogenic agents such as glycol or using the electro-fusion technique. After protoplast fusion, regeneration of protoplasts can be further performed using suitable media. Screening of mutants with desired characteristics is an important step in GS, which can be carried out using fermentation studies and monitoring enhancement in metabolite production, metabolite tolerance and increase in enzyme activity or improved phenotypic characteristics [20].