Comparative Screening and Integrative Identification of Indigenous, High Yielding, Glutaminase Producing Aspergillus Strains Isolated from Spoilt Rice

Abstract

Fungi, an abundant eukaryote, show diverse characteristics and produce many valuable biomolecules with wide industrial applications, making accurate identification essential in bioprocesses. A combination of morphological, microscopic features and molecular characterization guarantee some confidence in fungal identification compared to the morphological and microscopic features alone. L-glutaminase (L-glutamine amidohydrolase E.C 3.5.1.2) is an endopeptidase that specifically catalyses the breakdown of L-glutamine to L-glutamic acid and ammonia. The enzyme can be obtained from many sources but fungal sources prove to be more sustainable. The current study is aimed at unveiling the glutaminase production potentials of strains of Aspergillus species isolated from spoilt rice and efficiently identify the best producer using a combination of morphological, microscopy features and molecular identification tools. Spoilt rice samples were inoculated onto modified Czapek Dox (MCD) plates for obtaining isolates and characterizing their morphological features. Glutaminase production potential was evaluated by inoculating the isolates into MCD broth, incubating for 5 days, and measuring absorbance at 540nm. Scanning electron microscopy was employed for microscopic characterization. The DNA of the best producer was extracted using Presto™ Mini gDNA Yeast Kit (Geneaid, GBYB100) and amplified using PCR and sequenced employing Sanger’s DNA sequencing. BLAST search was made using the sequences obtained in order to ascertain the identity of the organism at hand, phylogenetic tree was constructed via neighbour joining. A total of seven fungal isolates were obtained from spoilt rice among which isolate SR05 exhibited the highest glutaminase activity (0.544), followed by SR02 (0.503), while SR04 showed the lowest (0.172). The observed differences were not statistically significant (p 0.3220). BLAST analysis identified SR02 and SR05 as A. oryzae, whereas SB04 was identified as A. niger. In conclusion, isolated and fully characterized indigenous strain of A. oryzae isolate possess the highest glutaminase production potential of 0.544 thus, recommended for further study.