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Selection and Improvement of Industrial Organisms for Biotechnological Applications
Published in Nduka Okafor, Benedict C. Okeke, Modern Industrial Microbiology and Biotechnology, 2017
Nduka Okafor, Benedict C. Okeke
Some chemical mutagens, such as nitrous acid and nitrosoguanidine, work by causing chemical modifications of purine and pyrimidine bases that alter their hydrogen-bonding properties. For example, nitrous acid converts cytosine to uracil which then forms hydrogen bonds with adenine rather than guanine. These chemicals act on the non-dividing cell and include nitrous acid, alkylating agents, and nitrosoguanidine (NTG, also known as MNNG).Nitrous acid: This acid is rather harmless and the mutation can be easily performed by adding 0.1 to 0.2 M of sodium nitrate to a suspension of the cells in an acid medium several times. The acid is neutralized after suitable intervals by the addition of appropriate amounts of sodium hydroxide. The cells are plated out subsequently.Alkylating agents: These are compounds with one or more alkyl groups which can be transferred to DNA or other molecules. Many of them are known but the following have been routinely used as mutagens: EMS (ethyl methane sulfonate), EES (ethyl ethane sulfonate), and DES (Diethyl sulfonate). They are liquids and easy to handle. Cells are treated in solutions of about 1% concentration and allowed to react from ¼ hour to ½ hour, and thereafter are plated out. Experimentation has to be done to decide the amount of kill that will provide a suitable amount of mutation. While some are carcinostatic (i.e. stop cancers), some are carcinogenic and must be handled carefully.NTG—nitrosoguanidine: also known as 1-methyl-3-nitro-1-nitrosoguanidine— MNNG: It is one of the most potent mutagens known and should be handled with care. Amounts ranging from 0.1 to 3.0 mg/ml have been used, but for most mutations, the lower quantity is used. It is reported to induce mutation in closely linked genes. It is widely used in industrial microbiology.Nitrogen mustards: The most commonly used of this group of compounds is methyl- bis (Beta-chlorethyl) amine also referred to as ‘HN2’. Nitrogen mustards were used for chemical warfare in World War I. Other members of the group are ‘HN’, ‘HN1’, or ‘HN3’ from the wartime code name for mustard gas, H. The number after the H denotes the number of 2-chloroethyl groups which have replaced the methyl groups in tri-methylamine. A spore or cell suspension is made in HN2 (methyl-bis [Beta-chlorethyl amine]), and after exposure to various concentrations for about 30 minutes each, the reaction is ended by a decontaminating solution containing 0.7% NaHCO3 and 0.6% glycine. The solution is then plated out to identify survivors. Between 0.05 and 0.1% HN2 solutions in 2% sodium bicarbonate solutions have been found satisfactory for Streptomyces.
Isolation and identification of high-yielding alkaline phosphatase strain: a novel mutagenesis technique and optimization of fermentation conditions
Published in Preparative Biochemistry & Biotechnology, 2023
Le Bo, Xin Kang, Zuohui Chen, Yue Zhao, Si Wu, Jie Li, Shuang Bao
Microbial breeding by genetic mutation is of great importance for the commercialization of enzyme formulations. Traditional microbial mutagenesis breeding methods include physical mutagenesis methods (e.g., γ-rays, x-rays, ultraviolet light, particle radiation) and chemical mutagenesis methods (such as alkylating agents, azides, diethyl sulfate, ethyl methanesulfonate),[12] however, these traditional methods lack diversity in mutagenesis. A novel cold atmospheric plasma (CAP)-based mutagenesis breeding method was developed, but requires ultra-low temperatures and a vacuum environment. Atmospheric dielectric barrier discharge (DBD) plasma breeding methods improve extreme experimental conditions, however, due to the dielectric barrier layer between the DBD generator electrodes, DBD breakdown and discharge voltages are always high and large amounts of ozone are generated.[13] (ARTP) is a novel microbial mutation breeding method that has been recently developed in recent years based on this. This technique can effectively mutate microalgae, bacteria, fungi, yeasts and actinomycetes.[14] Uniformly distributed high concentrations of neutral active particles can alter microbial genetic characteristics.[15] ARTP can also cause direct mutations of molecular structures at the nucleotide level, such as DNA damage, reversals, conversions, transfers, insertions and deletions or openings.[16] Breaking or fragmenting circular plasmids[17] or causing DNA double- or single-stranded breaks are inevitable outcomes of ARTP.[18] Furthermore, ARTP can act on cells to indirectly affect intracellular genetic materials. Its use also damages whole cells, thereby initiating multiple DNA repair mechanisms, such as SOS repair mechanisms. These repair mechanisms can then trigger complex regulatory networks in biologically responsive cells, resulting in changes of genetic materials and metabolic pathways.[19] The mutant strains obtained from ARTP-mediated mutagenesis using ARTP technology have good genetic stability.[20,21] ARTP has been applied to mutate common strains, ARTP can screen and mutagenize Bacillus amylolyticus and can produce vitamin K2 from maize flour hydrolysate, reducing substrate costs.[22] ARTP can significantly increase the yield of recombinant proteins in Bacillus subtilis. At the same time, the fermentation optimization strategy effectively promoted the expression of recombinant alkaline amylase in B. subtilis 168 mut16 #.[23] ARTP mutagenesis could also be combined with high throughput screening methods to improve uridine production, showing good uridine production and genetic stability.[24,25] ARTP has been successfully applied to the mutation breeding of more than 40 microorganisms including bacteria, fungi and microalgae.[13] However, only few reports exist on the role of ARTP in improving the activity of ALPs.