Studies on Production and Optimization of Glutaminase and Urease free L-Asparaginase using newly isolated Aspergillus tubingensis IBBL1

Doriya, Kruthi and Devarai, Santhosh Kumar (2019) Studies on Production and Optimization of Glutaminase and Urease free L-Asparaginase using newly isolated Aspergillus tubingensis IBBL1. PhD thesis, Indian institute of technology Hyderabad.

Full text not available from this repository. (Request a copy)

Abstract

L-asparaginase is an amido-hydrolytic enzyme that degrades asparagine, an attribute that makes it as an anti-neoplastic agent and anti-acrylamide agent in therapeutic and food industry, respectively. Clinically available L-asparaginase has glutaminase and urease that may lead to side effects during treatment of Acute Lymphoblastic Leukemia (ALL). L- asparaginase from bacteria causes anaphylaxis and other abnormal sensitive reactions due to low specificity to asparagine. To overcome this, eukaryotic organisms such as fungi has been used for the production of L-asparaginase which is free of glutaminase and urease. For this study, 45 fungal isolates were subjected to screening, with a view to assess the isolates for their ability to utilize different substrates as a nitrogen source. Four isolates have shown the presence of L-asparaginase free of glutaminase and urease using semi-quntitative assay method. Among the four isolates strains C7 (Aspergillus sp.) has shown the highest zone index of 1.57. This strain was further identified as Aspergillus tubingensis IBBL1 strain at molecular level by ITS rRNA gene sequencing (GenBank: MH185806). These results suggest that this Aspergillus strain can be used successfully in developing a biological process for the degradation of L-asparagine. L-asparaginase can be produced either by solid state fermentation (SSF) or submerged fermentation (SmF). Fungal strain, C7, characterised as Aspergillus tubingensis IBBL1 which is an L-asparaginase (free of L-glutaminase and urease)- producing strain has shown highest enzyme activity of 36.1 U/mL with carbon source as glucose; asparagine as nitrogen source at inoculum volume of 5 x 107 cells/mL. Once parameters were optimized at SmF, subsequent studies were performed with SSF. SSF is preferred over submerged fermentation as it is cost effective, eco-friendly and it delivers high yield. In this work, sequential optimization of agricultural solid substrate mixture and process parameters for the production of L-asparaginase were studied using Aspergillus tubingensis IBBL1. In the first stage, through simplex centroid design maximum L-asparaginase activity was observed using a ternary mixture of cotton seed cake (2/3), wheat bran (1/6), and red gram husk (1/6). In the next step, cultivation parameters such as pH, temperature and moisture content were optimized using Box-Behnken design. After 6 days of fermentation using optimized ternary mixture, maximum activity of 34.9 U/gds was obtained at temperature of 35 °C, pH-8 and moisture content 70 %(v/w). The limited use of SSF is due to its disadvantage when considering the large scale extraction and purification of the enzyme. Scaling up of SSF bioreactor is difficult due to heat and mass transfer problem in heterogeneous system. Continuous efforts towards improving design and modeling characteristics led to the development of various bioreactors such as tray, packed bed, rotating drum and fluidized bed. By considering all the limitations of the existing bioreactors used for the SSF process, rotary bioreactor was designed as it can overcome these limitations. Additionally, L-asparaginase production was carried out using modified tray bioreactor and in-house designed and fabricated rotary bioreactor using previously optimized conditions by Aspergillus tubingensis IBBL1. Maximum L-asparaginase activity of 20.58 U/gds was observed using modified tray bioreactor with bed height of 1 cm and by applying intermittent mixing strategy at 120 h of fermentation. In case of in-house designed rotary bioreactor, L-asparaginase activity of 19.96 U/gds was obtained with 1.5 kg substrate loading, 3 min delay time of the drum and on-rotation for 30 sec. These findings demonstrate that in-house designed rotary bioreactor was easy to handle, and has shown comparable results with tray bioreactor. Correlation between growth dynamics and L-asparaginase production was explained by means of Leudeking-Piret equation. Kinetic profile of L-asparaginase production indicated that enzyme production is completely growth associated.

[error in script]

IITH Creators: