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Comprehensive Analysis of L-Asparaginase: Structure, Purification, and Functional Insights

Introduction: Comprehensive Analysis of L-Asparaginase: Structure, Purification, and Functional Insights

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L-Asparaginase is a hydrolyzed product from the conversion reaction of L-asparagine to its respective acid. It was named the first-ever enzyme which was used as a cancer-preventing agent in early clinical practices. Due to its high chemical value, the structural and functional properties of this enzyme have been studied and researched over the years since last thirty to forty years. This helps to provide the perfect structural and biochemical description for the enzyme. There have at least 100 functional models of L-Asparaginase which have been considered in the study and research purpose through the years stored in protein data banks. The principal objective of this report is to critically analyze the role and structure of the enzyme and the importance of purification of the enzyme.

L-asparaginase helps to catalyze the degradation of asparagine, which is an important amino acid source for leukemic cells. It breaks down into aspartate and ammonia. It has also a role in the inhibition of the biosynthesis of the protein complex in lymphoblast. L-asparaginase is applied for the treatment of lymphoblastic leukaemia. Various types of structural isomers of this enzyme have been isolated from different varieties of organisms which include many marines as well as terrestrial organisms. For some organisms like Escherichia coli, it has been identified some residues which are important for the catalytic activities of the organisms. In the human body, the lack of l-asparaginase enzyme can be demonstrated as the production from the aspartic acid pathway and from glutamine by the synthesis of asparagine. The reduction of l-asparagine from body plasma results in the inhibitory activities of RNA and DNA synthesis. The various other economic roles of this enzyme can be said such as it is used as a food processing agent. It helps to deplete the level of acrylamide in carbohydrates and starchy food. It doesn't change the taste and flavour of the food; moreover, it provides the fried brown appearance of food. It has a wide application as a drug to treat diseases like lymphoblastic leukaemia and in the treatment of some tumour cells.

The asparaginase enzymes have tertiary structures and also have active site pockets in their structural configuration. In the below demonstration, the structural breakdown of the enzyme has been shown.

Structure of L-Asparaginase

Figure 1: Structure of L-Asparaginase

A protomer of the enzyme consists of two domains, a larger and a smaller. The larger domain is connected with a covalent bond with the smaller one. The larger domain is an N-terminal and the smaller domain is the C-terminal domain, which has an amino acid linkage of 20 to 25 amino acids. The N terminal domain contains 8 stranded beta-sheets, which have a switch point between the first and third strand with alpha-helices. Two alpha helices, the first and the fourth of this N-terminal domains, are seen to have been placed in the solvent exposure side of the beta strands. The other two strands, the second and the third, are complimentary in their position. While the seventh and eighth strands are antiparallel to each other. A beta hairpin is formed between the strands which are placed towards the inside location of the tetramer. A bridge is seen to have composed of two sulphide bonds at the parallel segments of the N-terminal strands. The C-terminal domain is constructed of four beta sheets which are parallel to each other. The first and the fourth strand are placed on the outer side while the rest of the two lies on the inner side.

The main importance of the purification of this enzyme is to evaluate its anti-carcinogenic activity of it. It helps in the treatment related to lymphoblastic leukaemia and lymphoma. So the purification of this enzyme has deep importance in the clinical aspect.

Materials and methods

The steps which are being followed here for the purification and then isolation of the above-mentioned enzyme is the Polymerase Chain Reaction and then Western Blotting Technique. After that, the results have been observed and interpreted to get the outcome from a Km, Vmax graph to know the kinetics and velocity of the whole reaction (Lubkowski et al. 2020). To perform the PCR technique isolated samples of genetic material have been collected which contain the target sequence, the DNA primer or base sequence which are specific to the targeted sequence, the Primer of DNA, nucleotides and DNA polymerase enzyme with the buffer solution of PCR. The PCR steps have been performed by following four primary steps first the heat denaturation of the double-stranded DNA from the collected sample. It helps to break the sample into two individual strands which are performed at a temperature of more than 90-degree centigrade (Brumano et al. 2019). The hydrogen bonding between the two strands hereby breaks. Then, the targeted sequence is needed to be added with the primer before performing replication. This is called annealing of targeted sequence with the DNA sequence which is complimentary to the DNA strand, such as adenine to thymine and guanine to cytosine. Annealing takes place at a lower temperature than denaturation. Here the primers which have been borrowed are –

Forward :( 5’-GCCATATGTTTAACGCATTATTCGTTGTTG-3) (NdeI)

Reverse :( 5’-GCGGATCCCAAGCGATTAATAAGCGTGGAAG-3) (BamHI)

After following the annealing process the temperature is thus increased and the DNA polymerase enzyme is applied to make polymers of the DNA strands, which means the replication process. This replication procedure is repeated multiple times until the targeted sequences are not generated (Castro et al. 2021). After the results of the PCR technique, the protein sequence is incorporated in a different source and then the western blotting technique is performed to identify whether the demanded protein is the one which is received or not. To perform WBT, sample buffer and western blotting paper are required. A buffer used to block is used for this process which can be made from the BSA or skimmed milk. The blotting paper is incubated with this blocking buffer at a temperature of 4'c overnight (Qin et al. 2022). Also, a secondary antibody is taken to apply with it. After performing WBT the Km Vmax is derivates for the reaction which helps to distinguish the kinetic behaviours of the targeted enzyme.

Results

Forward “5’-GCCATATGTTTAACGCATTATTCGTTGTTG-3” NdeI
Reverse “5’-GCGGATCCCAAGCGATTAATAAGCGTGGAAG-3” BamHI

Table 1: Primer design for PCR of L-asparaginase

The table above shows the primers that had been designed to amplify the l-asparaginase gene in the PCR reaction. NdeI restriction enzyme has been used to bind to the antisense strand and BamH1 has been used to bind to the sense strand.

PCR result of L-asparaginase

Figure 2: PCR result of L-asparaginase

The gene has been successfully amplified in PCR with designed primers and the size of a PCR product has been measured with a DNA ladder. The size of each fragment has been found to be almost 1000bp, as shown in the figure above.

Western blotting result of L-asparaginase

Figure 3: Western blotting result of L-asparaginase

The obtained PCR product has been introduced into a host, followed by which proteins have been isolated from the host to test the translational activity of the gene. The protein has been detected by western blotting. The image above displays the results obtained after performing western blotting on the protein and the purified product has been used for functional studies.

Kinetic activity of L-asparaginase

Figure 4: Kinetic activity of L-asparaginase

Gel electrophoresis has been conducted on the purified product and its results have not been displayed. The kinetic activity of L-asparaginase has been tested on the purified product. The activity of the enzyme against each substrate concentration has been pointed out in the graph above.

Discussion

PCR is conducted to produce multiple copies of a gene specifically when it is available in limited amounts. The PCR reaction involves three stages, namely denaturation, annealing and extension (Duryodhan et al. 2020). Denaturation involves breaking the two DNA stands to make them two single strands such that two primers can bind to each one of them individually. Each primer binds to each strand, from, which one strand is synthesised, making it a double strand again, and the process continues. As mentioned by Rahbari et al. (2021), the main criterion for designing primers for PCR reactions is that the size does not exceed 18-30 bases. The length of the primer taken in this experiment is 30 to 35 bases and it has been aimed that the ends have GC. Each restriction enzyme recognises a specific site based on which they are selected to bind to the primer strands. NdeI recognises TATG sites and BamH1 recognises GATCC sites. Based on the sequence of the L-asparaginase gene, the primer has been designed accordingly.

These enzymes have been selected because obtained 5’ and 3’ sites in the sense and antisense strands are recognised by them. As opined by Bernardo et al. (2020), the main reason behind adding restriction enzymes to the primer is to preserve reading frames present at both ends of the strands. Both NdeI and BamH1 are among the most commonly used restriction enzymes in scientific studies because their recognition sites are commonly present in most genomes. Apart from that, their commercial availability and wide range of uses make them a preferable choice and that is why these have been chosen in this study. A DNA ladder is a biological tool that is used to measure or confirm the size of an obtained DNA product (Li et al. 2019). In this case, it has been used to detect the size of obtained PCR products, which has been found to be 1000 bp, a standard PCR product size.

One of the objectives on which this experiment has been based is to detect the functional activity of the enzyme L-asparaginase. As said by Sharma et al. (2022), L-asparaginase catalyses the activity of L-asparagine in the human body, which triggers neoplastic cells to function and prevent the occurrence of leukaemia. Hence, the obtained PCR product had been introduced into a host to detect its activity. Followed by insertion into the host, it has been re-collected and subjected to western blotting. As mentioned by Butler et al. (2019), western blotting is used for the detection of a specific protein in the sample. The enzyme would translate in the host body and produce a protein having specific activity and identifying this protein has been the main purpose of this part of the experiment. Western blotting has helped in identifying the protein based on the numbers marked on top. The enzyme has been made to interact with the substrate in the blotting plate and the chemical change has been observed.

The western blotting result shows that chemiluminescence has been observed in columns 2, 3, 4 and 5 which have led to the confirmation of L-asparaginase being the enzyme. Followed by that, the purified product has been taken to test the kinetic activity of the enzyme, the reason why purified products are preferred in this process is that the presence of impurities might alter the results (Costa-Silva et al. 2020). The graph obtained reveals that the kinetic activity of L-asparaginase has increased gradually with an increase in substrate concentration. The optimum substrate concentration required to help the enzyme demonstrate its maximum activity has also been displayed in the graph. It has been found that the kinetic activity of the enzyme has increased consistently up until 125 micro-M, which makes 125 micro-M the optimum substrate concentration where enzyme activity is maximum. A further decline would indicate that increasing the concentration of the substrate beyond this limit restricts L-asparaginase activity.

Kinetic activity calculations help reveal the activation of the component that is catalysed by that enzyme. In this case, these results might help in predicting the hydrolysis activity of asparagine because asparagine production is catalysed by this enzyme. A change in the value of the kinetic constant reveals the activation of asparagine (Noma et al. 2021). The Km and Vmax values in this experiment have been calculated by Michaelis-Menten plot where the substrate concentration has been plotted on X-axis and enzyme activity on the Y-axis. The formula that is used to calculate the Vmax and Km values is “v = Vmax / (1+ (km/[S])))”. Using the formula, the value of Vmax is 52.21 and Km is 22.27 as per the results. As mentioned by Contreras-Baeza et al. (2019), a low Km value indicates high affinity. However, the Km value obtained in this experiment is significantly high, implying low affinity.

References

Bernardo, A., St. Amand, P., Le, H.Q., Su, Z. and Bai, G., 2020. Multiplex restriction amplicon sequencing: a novel next?generation sequencing?based marker platform for high?throughput genotyping. Plant biotechnology journal18(1), pp.254-265.

Brumano, L.P., da Silva, F.V.S., Costa-Silva, T.A., Apolinário, A.C., Santos, J.H.P.M., Kleingesinds, E.K., Monteiro, G., Rangel-Yagui, C.D.O., Benyahia, B. and Junior, A.P., 2019. Development of L-asparaginase biobetters: current research status and review of the desirable quality profiles. Frontiers in bioengineering and biotechnology, 6, p.212.

Butler, T.A., Paul, J.W., Chan, E.C., Smith, R. and Tolosa, J.M., 2019. Misleading westerns: common quantification mistakes in western blot densitometry and proposed corrective measures. BioMed research international2019.

Castro, D., Marques, A.S.C., Almeida, M.R., de Paiva, G.B., Bento, H.B., Pedrolli, D.B., Freire, M.G., Tavares, A.P. and Santos-Ebinuma, V.C., 2021. L-asparaginase production review: bioprocess design and biochemical characteristics. Applied microbiology and biotechnology, 105, pp.4515-4534.

Contreras-Baeza, Y., Sandoval, P.Y., Alarcón, R., Galaz, A., Cortés-Molina, F., Alegría, K., Baeza-Lehnert, F., Arce-Molina, R., Guequén, A., Flores, C.A. and San Martín, A., 2019. Monocarboxylate transporter 4 (MCT4) is a high affinity transporter capable of exporting lactate in high-lactate microenvironments. Journal of Biological Chemistry294(52), pp.20135-20147.

Costa-Silva, T.A., Costa, I.M., Biasoto, H.P., Lima, G.M., Silva, C., Pessoa, A. and Monteiro, G., 2020. Critical overview of the main features and techniques used for the evaluation of the clinical applicability of L-asparaginase as a biopharmaceutical to treat blood cancer. Blood Reviews43, p.100651.

Duryodhan, V.S., Singh, S.G. and Agrawal, A., 2020. The concept of making on-chip thermal cycler for RT-PCR using conjugate heat transfer in diverging microchannel. Transactions of the Indian National Academy of Engineering5, pp.221-223.

Li, Q., Liu, L., Mao, D., Yu, Y., Li, W., Zhao, X. and Mao, C., 2019. ATP-triggered, allosteric self-assembly of DNA nanostructures. Journal of the American Chemical Society142(2), pp.665-668.

Lubkowski, J. and Wlodawer, A., 2021. Structural and biochemical properties of L?asparaginase. The FEBS Journal, 288(14), pp.4183-4209.

Lubkowski, J., Vanegas, J., Chan, W.K., Lorenzi, P.L., Weinstein, J.N., Sukharev, S., Fushman, D., Rempe, S., Anishkin, A. and Wlodawer, A., 2020. Mechanism of Catalysis by l-Asparaginase. Biochemistry, 59(20), pp.1927-1945.

Noma, S.A.A., Acet, Ö., Ulu, A., Önal, B., Odaba??, M. and Ate?, B., 2021. l-asparaginase immobilized p (HEMA-GMA) cryogels: A recent study for biochemical, thermodynamic and kinetic parameters. Polymer Testing93, p.106980.

Qin, X., Costa-Silva, T.A., Pessoa, A. and Long, P.F., 2022. A scoping review to compare and contrast quality assurance aspects of L-asparaginase biosimilars. International Journal of Pharmaceutics, p.122523.

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Sharma, A., Kaushik, V. and Goel, M., 2022. Insights into the Distribution and Functional Properties of l-Asparaginase in the Archaeal Domain and Characterization of Picrophilus torridus Asparaginase Belonging to the Novel Family Asp2like1. ACS omega7(45), pp.40750-40765.

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