Recombinant production and characterization of chitinase enzyme from Pseudomonas mandelii KGI_MA19

Abstract

Biological catalysts, generally found in protein structure, that catalyze the biochemical reactions necessary for life are called enzymes. They increase the reaction rate by lowering the activation energy of the reaction catalysed. In recent years, enzymes have gained a large share in different industrial areas, like food, agriculture, pharmacy, cosmetics, waste removal, leather processing, detergent, and medical applications. Enzymes can minimize the formation of unwanted by-products, they are environmentally friendly and cheap, and have biodegradable properties. They are also considered safe for the cleaning, health, and food industries. In addition to their preferred properties, enzymes that remain active at low/high temperatures, in the presence of organic solvents, at different salt concentrations and pH values, and with an affinity for different substrates are attracting the attention of the industry. Meeting the increasing demand for the use of biomolecules with different properties in the industry is possible with the development of the physical and biochemical properties of the biocatalysts defined today with the help of protein engineering, metagenomics, advanced DNA technologies, nanotechnology, and finally, the discovery of new biocatalysts. Scientific research has been increasing in this direction, especially because enzymes from living things that live in extreme conditions can be used in a wide range of industries. Chitin (C8H13O5N)n is an inelastic, hard, white, non-elastic biopolymer formed by the bonding of N-Acetyl-D-glucosamine monomers (Glc-NAc) with β-1,4 glycosidic bonds, which ranks second after cellulose among the most abundant biopolymers in nature. It is a nitrogen-containing polysaccharide. Chitin is found in the cell wall of fungi, the outer shells of insects, the shells of sea creatures such as lobster, crab, shrimp, and the mouth areas of cephalopods such as cuttlefish and octopus which have a high annual production in the world. According to the Food and Agriculture Organization (FAO, 2019) data, 10.5 million chitin-rich shellfish (12.3%) are grown in aquaculture. The need for converting chitin wastes, which are rising in quantity by the day, into biologically useful products develops. In recent years, the employment of biocatalysts in the removal of chitin wastes instead of chemical methods, which are poisonous to the environment and expensive, has allowed for a safe conversion for the environment. Chitinase enzymes (EC 3.2.1.14), in the hydrolase class, catalyse the destruction of β-1-4 glycosidic bonds in chitin (C8H13O5N)n and separate N-acetyl-D-glucosamine molecules from the polysaccharide chain. The chitinase enzyme, which is one of the most common hydrolase enzymes in nature, is found in insects, plants, fungi and viruses. It is also commonly found in different bacterial genera such as Aeromonas, Arthrobacter, Bacillus, Chromobacterium, Flavobacterium, Pseudomonas, Sanguibacter, Serratia, and Streptomyces. With the increase in green and environmentally-friendly technology in recent years, the interest in chitinases is increasing day by day. Chitinase enzymes, which have agricultural importance, replace chemicals and pesticides used in agriculture with their ability to be used as biocontrol agents. It creates a positive effect on the marine ecosystem by recycling marine waste. Due to their pharmaceutically antifungal and antibacterial properties, they are used in the production of anti-inflammatory and anti-fungal drugs, anti-cancer and immune-enhancing agents, wound dressings, contact lenses, and surgical sutures. It is a bioprotective additive used to increase shelf life in the food industry. Antarctica with the highest elevation and lowest temperature has recently become a popular research area. 70% of the freshwater resources on the Antarctic continent, which has not been touched by human hands until lately, are in the form of ice. It also features powerful winds, extremely cold temperatures, and is exposed to low temperatures in the winter and high UV rays in the summer. It has a natural and distinct habitat as a result of these factors. Because of the harsh circumstances, it is unavoidable to uncover new species and enzymes and genes. Extremophiles are living systems that can thrive in harsh environments. Biocatalysts derived from extremophilic organisms and demonstrating catalytic activity even under adverse circumstances have been dubbed extremozymes. Psychrophiles and psychrotolerants are extremophilic microorganisms that can live at 0-20 °C and 0-30 °C, respectively. Because of their low energy needs, flexibility, and high catalytic activity, cold-compatible enzymes derived from psychrophilic and psychrotolerant organisms are crucial for commercial applications. Biotechnological approaches help the identification of novel and diverse extremophiles and extremozymes from Antarctica that has not been described in the literature yet. In the study, which was carried out using Antarctic sediment samples collected within the scope of the 2nd National Antarctic Science Expedition (TAE-2), 12 sediment samples taken from 8 different regions were cultured and enriched. Subsequently, freeze-thaw stress was applied to each cultured sample. After the applied freeze-thaw stress, it was observed that five cultures were resistant to stress. Two morphologically different colonies were selected from each stress-resistant sample, DNA isolations were made and 16S rRNA analyzes were carried out. The sample with the lowest similarity rate according to 16S rRNA analysis was sent for whole-genome analysis and according to the results of the analysis, a new strain of Pseudomonas mandelii, Pseudomonas mandelii KGI_MA19, was identified. Phenotypic and biochemical characterizations of the identified KGI_MA19 strain were performed. The next step was the recombinant production and the characterization of the chitinase enzyme of the psychrotolerant Pseudomonas mandelii KGI_MA19. The gene region of the chitinase enzyme of Pseudomonas mandelii KGI_MA19 was amplified by polymerase chain reaction (PCR) by designing gene-specific primers containing SacI and NotI restriction sites. The amplified gene region of interest and the pET-28a (+) vector were cut with SacI and NotI restriction enzymes. T4 DNA ligase enzyme was used for ligation of the cut PCR product and expression vector. Plasmid pET-28a (+) containing the PCR product was transformed into Escherichia coli BL21 (DE3) competent cells. The colony PCR method was applied to determine the colonies containing the relevant gene as a result of the transformation, and plasmid isolation of two colonies selected from among the colonies thought to be positive was performed. Then, to understand whether the colonies contain the gene product or not, the plasmid was cut with the EcoRV endonuclease enzyme, which has a fast-cutting feature. The DNA sequencing results showed that the chitinase gene-specific PCR product was correctly inserted into the pET-28a (+) plasmid. The Magic MediaTM (Invitrogen), which is commercially available and used in the expression of E.coli cells, was used to monitor the expression level of the chitinase gene in E.coli BL21 cells. At the end of Magic Media incubation, cells were treated with lysis (0.1M Tris-HCl, pH 8.0, 0.3M NaCl) buffer, and homogenization was performed by ultrasonication using the M73 probe. A high amount of produced chitinase enzyme was purified by the His-Tag affinity chromatography method using the His-Trap column. The activity of the chitinase enzyme obtained in high purity was determined by the 3,5-Dinitrosalicylic acid (DNS) method which allows one to determine the amount of reducing sugars expected to be released as a result of the reaction performed by the usage of colloidal chitin as a substrate. Biochemical characterization including optimum pH, pH stability, and optimum temperature, thermal stability has been completed. A new, psychrotolerant strain, Pseudomonas mandelii KGI_MA19, was identified from sediment samples from the Antarctic King George Island, and its molecular, phenotypic, morphological, and biochemical characterization was completed. In addition, the chitinase enzyme which has an increasing impact in the industrial field has been successfully produced and biochemically characterized by using recombinant DNA methods. The cold-adaptive chitinase enzyme of the Pseudomonas mandelii KGI_MA19 strain, which was obtained for the first time within the scope of this thesis, is thought to be a promising potential biocatalyst for industrial applications.