Optimizing Parameters for Production of Asparaginase Enzyme by Trichoderma Harzianum Under Solid-State Fermentation

Dr. Praveen Kumar Dasari, Kumar Raja Jayavarapu, Esukapalli Krishna Kumari, Dasari Prameela Rani, Pothumarthi Haritha, Pulagam Pavan Kumar Reddy,

doi:10.5281/zenodo.15152233

Research Paper | Open Access
Volume 03 | Issue 04 | Article Id IJPS/250304032

Optimizing Parameters for Production of Asparaginase Enzyme by Trichoderma Harzianum Under Solid-State Fermentation
Dr. Praveen Kumar Dasari* Kumar Raja Jayavarapu Esukapalli Krishna Kumari Dasari Prameela Rani Pothumarthi Haritha Pulagam Pavan Kumar Reddy
Mother Teresa Pharmacy College, Sathupally, Telangana-507303, India.

Abstract

Asparaginase is an enzyme that catalyses the hydrolysis of asparagine, an amino acid vital for protein synthesis and cell growth.Asparaginase is an enzyme, often referred to as L-asparaginase, used in chemotherapy, particularly for acute lymphoblastic leukaemia (ALL), by breaking down asparagine, an amino acid crucial for cell growth, thus inhibiting cancer cell proliferation. The present research work describes the production of L-Asparaginase using Tectona grandis leaves as substrate by Trichoderma harzianum in a solid-state fermentation. In present research, Asparaginase enzyme production was carried out by observing different parameters like time, temperature, moisture content, inoculum level, pH, carbon source and nitrogen source were noted. Different carbon supplements were checked for their influence on enzyme production; they are glucose, sucrose, maltose, lactose and fructose. The incubation time of 72hrs, the temperature of 250C, pH 8.0, inoculum level of 90% v/w and moisture content 70% v/w were observed for asparaginase enzyme production. Among the carbon source, lactose gave better production when compared to other carbon supplements. Nitrogen source like asparaginase with different concentration were checked and 0.3% w/w gave best enzyme production. Final conclusion is that Tectona grandis leaves could be a promising substrate for industrial application since it produces a significant asparaginase (74.32 IU/ml) activity in solid state fermentation.

Keywords

Tectona grandis, Trichoderma harzianum, solid state fermentation.

Introduction

L-asparaginase, an enzyme that hydrolyses asparagine, is produced through biotechnological methods like submerged fermentation (SmF), solid-state fermentation (SSF), and recombinant DNA technology, using various microbial sources. Cancer Therapy: L-asparaginase is used in cancer treatment by depleting asparagine, a crucial amino acid for tumor cell growth. Food Industry: The aspartic acid produced during asparagine hydrolysis can enhance the sour and savory taste of food. L-asparaginase can be used in food processing to reduce acrylamide formation. The mechanism of action of,Asparaginase hydrolyzes serum asparagine to nonfunctional aspartic acid and ammonia, depriving tumor cells of a required amino acid; thus, tumor cell proliferation is blocked by the interruption of asparagine-dependent protein synthesis. Solid state fermentation is a process that takes place on a non-soluble material that acts both as support and a source of nutrients, with a reduced number of water, under the action of fermenting agent. A major amount of researches were conducting research on the asparaginase enzyme production. Asparaginase manufacturewas carried out throughout the world by solid state fermentation and submerged fermentation. In solid state fermentation, the substrate uses solid materials like grains, bran, or other organic residues. Submerged fermentation, while versatile, faces challenges like high equipment costs, a greater risk of contamination, and potentially lower productivity compared to solid-state fermentation. Asparaginase is used as part of a multi-agent chemotherapy regimen for the treatment of acute lymphoblastic leukaemia (ALL). It is also being studied for its potential in treating other types of cancer. Therefore, the aim of the present research work is to discovery of a new asparaginase producer that is serologically different from the previously reported ones, but one that have similar therapeutic effects.

MATERIALS AND METHODS:

Substrate:Tectona grandis leaves were collected from our college garden, Sathupally and dried naturally, powdered, packed and stored until further use.

Microorganism:

Trichoderma harzianum (NCIM 1347) procured from National Collection of Industrial Microorganisms (NCIM), Pune was used for the production of Asparaginase enzyme using Tectona grandis leaves as substrate. Potato dextrose agar medium was used for sub culturing and maintenance of microorganisms.

Preparation of inoculum: streaking was done from the old cultures of Trichoderma harzianum on pure potato dextrose agar medium and incubated them at 36⁰C for 3days.

Development of inoculum: 10ml of sterile distilled water were mixed to 3 days old culture slant, from that 1ml of suspension that contains approximately, 10⁷ cells/ml was used as the inoculums.

Solid state fermentation: solid state fermentation was carried out in 250ml Erlenmeyer flask by taking production medium containing (in g/L): glucose-12.5g, ammonium nitrate-0.26g, ferrous sulphate-0.01g, L-asparagine-0.5g, potassium chloride-0.5g, potassium hydrogen phosphate- 1g. The pH medium was adjusted to 8.0. solid state fermentation was accomplished by taking 10g of substrate in 250ml Erlenmeyer flask, moistening it with 5ml of production medium, mixed thoroughly and placed in an autoclaved at 15lb pressure, 121⁰C for 15min for sterilization. After pasteurize, it was cooled; then the flasks were inoculated with 1ml of inoculum and incubated in an incubator.

Determination of enzyme activity:

Enzyme extraction: The enzyme extraction was carried out at a temperature of 36⁰C for 24hrs interval. The solid-state fermentation material corresponding to one Erlenmeyer flask was mixed with 10ml of sodium phosphate buffer and rotated for 45mins with the help of Rotator shaker. After 45min the extraction was filtered in Whatman filter paper, from that 2ml of the extract was placed in centrifugal tube and centrifuged at 10,000rpm for 10mins.

Enzyme assay: Asparaginase enzyme activity was detected by measuring the amount of ammonia formed by nesslerization. The free suspension in centrifugal tube of 0.5ml was taken into centrifugal tube and add with 0.5ml of L-asparagine and followed by the addition of 1ml sodium borate. It was incubated for 10mins to liberate the ammonia, 0.5ml of 15% trichloroacetic acid was added to the centrifugal tube and centrifuged for 10mins at 10,000rpm. From the supernatant liquid 1.0ml was taken and mixed with 1.0ml of Nessler’s reagent to detect liberated ammonia at 480nm in UV equipment. One unit (U) of asparaginase was the amount of enzyme which liberates the 1 micromole of ammonia in 1min at 37⁰C.

RESULTS AND DISCUSSION

To determine the effect of fermentation time on enzyme production, the medium incubate at different time intervals, after completion of every 24hrs, enzyme extraction process was done and the maximum asparaginase activity was noted at 72hrs. after 72hrs, it was decline due to depletion of nutrients in the medium. Asparaginase enzyme at different time intervals was shown in fig. 1.

Fig.1.Effect of time on enzyme production

The temperature was crucial in solid state fermentation because it ultimately affects the growth of the microorganism. To determine the effect of fermentation temperature on enzyme production, the medium was incubated at different temperatures, after completion of 72hrs, enzyme extraction process was done. The maximum production of asparaginase enzyme was noted at 25⁰C temperature fig.2.

Fig. 2. Effect of temperature on enzyme production

Every enzyme has an optimum pH when it was more effective. An increasing or decreasing pHreduces enzyme activity by changing the ionization. To determine the effect of pH, the nutrientmedium was adjusted to different pH ranges 6, 7, 8, 9 and 10. The maximum enzyme productionof L-asparaginase was noted at pH 8 fig.3

Fig. 3. Effect of pH on enzyme production

When inoculum size was increased from 5 to 10% there was increase in enzyme production but thereafter the enzyme activity was decreased, because depletion of nutrients by the enhanced biomass, which resulted diminishing in metabolic activity. To determine inoculum size, different inoculum levels were prepared for the production of enzyme 60%, 70%, 80%, 90% and 100%, v/w. The maximum enzyme production was noted at 90% v/w of inoculum fig.4.

Fig. 4. Effect of inoculum level on enzyme production

Moisture content in solid state fermentation is plays crucial role in the production of enzymes. High moisture content results in decreasing the substrate porosity, which may turn reduction in penetration, it may cause contamination. To determine the moisture content on the enzyme production, various moisture content was prepared like 60%, 70%, 80%, 90% and 100% v/w were taken in different conical flask. The maximum activity was noted at70% v/w of the moisture content fig.5.

Fig.5. Effect of moisture content on enzyme production

To determine the effect of carbon source on enzyme production, five different carbon supplements were screened for the production of L-asparaginase enzyme which is sucrose, maltose, glucose, fructose, and lactose. The nutrient medium was enriched with different carbon concentrations % w/w. The result noted that lactose supplementation gave better improved enzyme production than other supplementations fig.6.

Fig.6. Effect of carbon source on enzyme production

To determine the effect of nitrogen source on the production of enzyme, the production medium was made with different concentrations of potassium nitrate like 0.2%, 0.3%, 0.4%, 0.5%, 0.6% and 0.7% w/w were dispersed in 250ml conical flasks. The results indicate that maximum enzyme production was noted at 0.3% w/w of potassium nitrate concentration fig.7.

Fig.7. Effect of nitrogen source on enzyme production

CONCLUSION:

Finally, we conclude that Tectona grandis leaves were a promising agent for the production of asparaginase enzyme. Asparaginase production research highlights its potential as a therapeutic agent, particularly in leukaemia treatment, while also exploring its applications in food processing to reduce acrylamide formation, with ongoing efforts focused on optimizing production and identifying novel sources. It gave a significant asparaginase enzyme production (74.32 IU/ml) in solid state fermentation using Trichoderma harzianum. Tectona grandis leaves is low-cost substrate and easily available in our local areas and showing suitability for the solid-state cultivation of microbes, it was suggested as a potential substrate for optimizing the parameters of asparaginase production under solid state fermentation.

ACKNOWLEDGEMENT

The authors express their sincere thanks to the management, Mother Teresa Pharmacy College, Sathupally, Khammam, Telangana for providing the necessary facilities to carry out the research work. The authors express sincere thanks to National Collection of Industrial Microorganisms (NCIM), Pune for providing microorganism to carry out the research work.

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