Background and objective Invertase, one of the most important enzymes used in industrial and pharmaceutical applications, has been produced from different Aspergillus spp. Statistical and mathematical tools [response surface methodology (RSM)] have been carried out to optimize culture conditions to facilitate maximum enzyme production. This investigation aims to screen the ability of some Aspergillus spp. to produce invertase enzyme, to optimize the culture parameters, and to detect the genetic diversity between them. Materials and methods A total of five fungal strains were tested for the production of invertase. The most promising strain was selected for optimization. Optimization experiments were carried out to obtain maximum enzyme production, and a molecular marker (RAPD-PCR) was used to differentiate between these strains. Results and conclusion Some Aspergillus spp. were examined to produced invertase enzyme after 2 days of cultivation. Aspergillus niger recorded the highest enzyme production (6.8 U/ml). Ten RAPD-PCR primers were used to differentiate between Aspergillus spp. A total of 131 bands were amplified in the five strains of Aspergillus spp. Based on RAPD-PCR analysis, the highest similarities obtained between A. parasiticus and A. niger showed 75%. Moreover, the RAPD-PCR technique is a good tool for differentiation between the Aspergillus spp. To optimize culture condition, RSM was used to increase invertase production by A. niger. Temperature, pH, and incubation time dramatically affect invertase yield. A combination of these factors (pH 5, 35°C, and incubation for 72 h) was optimum for maximum production of invertase (8.2 U/ml). Our results employed RSM to elucidate the combination of effective factors such as temperature, pH, and incubation time to obtain a high yield of an important enzyme, invertase, from A. niger, used as a promising candidate for industrial applications.

Background and objective: In this work, isolation, identification (morphological and chemical), and molecular characterization were done of local isolates of some pectinase-producing microorganisms such as bacteria, actinomycetes, fungi, and yeast. Materials and methods: A total of 22 local bacterial isolates were obtained from various sources and were assayed for pectinolytic activity after optimization of conditions for pectinase production. Isolate no. 19 showed the highest pectinase-specific activity (6.73 U/ml) on glucose-supplemented medium, whereas isolate no. 5 gave the lowest pectinase productivity (3.21 U/ml). The identification of isolate no. 19 revealed that it belonged to the genus Bacillus based on morphological and biochemical characteristics. Based on molecular identification (16 S rRNA technique), isolate no. 19 was named Bacillus sp. strain NRBANKI-4 (with 99% similarity), with Gene Bank accession number OM540351. Results and conclusion: A total of 14 local actinomycete isolates were obtained from soil samples. Isolate no. 13 showed the highest pectinase-specific activity (6.48 U/ml), whereas sample no. 10 gave the lowest pectinase-specific activity (3.07 U/ml). Based on molecular identification (16 S rRNA technique), isolate no. 13 was named Streptomyces sp. KP 12 (90.63% similarity), with Gene Bank accession number OM403596. A total of 10 fungal isolates were obtained from crop waste soil. Isolate no. 2 gave the highest pectinase productivity (21.20 U/ml). Based on molecular identification (internal transcribed spacer-PCR technique), isolate no. 2 was named Aspergillus niger F8121 (99.47% similarity), with Gene Bank accession number OM392061. Following the same trend, 10 yeast isolates were isolated from crop waste soil. The isolate that gave the highest pectinase productivity was no. 7, which gave 22.03 U/ml. The isolate that gave the lowest was no. 9 (20.74 U/ml). Isolate no. 7 was named Pichia barkeri Y1 (90.91% similarity), with Gene Bank accession number OM392066.

Background and objective: Keratinases are gaining considerable momentum in green technology because of their endowed robustness and multifaceted application potentials, such as valorization of keratinous agro-waste. Therefore, the production of novel keratinases from relative yeasts grown in agro-waste formulated medium is cost-effective and imperative for the sustainability of thriving bioeconomy. Materials and methods: A total of 51 yeast isolates were isolated from 10 different poultry farms and assayed for keratinase-specific activity. Molecular identification of the high-efficiency keratinase-producing yeast isolate was done by PCR amplification, employing sequencing of internal transcribed spacer regions of yeast. Mutagenesis induction with ethidium bromide, ultraviolet, and ethyl methane sulfonate (EMS) was done in a multistep mutation-induction process for creating super keratinase-productive mutants. Response surface methodology optimization of culture conditions for high-productive mutant was carried out using different parameters such as incubation time, pH, carbon sources, and nitrogen sources to test keratinase activity. Inter-simple sequence repeat (ISSR-PCR) was applied to study the genetic diversity of isolated Pichia kudriavzevii YK46 compared with their five mutants. Results and conclusion: The results indicated that the isolate with the highest keratinase activity was isolate no. 46, which recorded 164.04 U/ml. It was identified as P. kudriavzevii and was submitted to NCBI under accession number ‘OK092586’. It was named as P. kudriavzevii YK46. Results of mutagenesis showed that the best keratinolytic efficiency mutant was designated as EMS-37, which showed an activity of 211.90 U/ml. After response surface methodology optimization of culture conditions for mutant EMS-37, the maximum keratinase activity was noted after an optimized condition at pH 5, 72 h of incubation time, 2.5% glucose, and 2.5% beef extract (as carbon and nitrogen sources), with an activity of 240.172 U/ml (Run3). Inter-simple sequence repeat showed that the highest total and polymorphic with unique bands were revealed in the mutant EMS-37, with 82 and 54 bands, respectively, whereas the mutant EMS-56 showed 72 and 44 bands, respectively, compared with the wild-type strain P. kudriavzevii YK46, with 86 and 58 bands, respectively. The data obtained showed that mutant EMS-37 was the highest producer of keratinase enzyme. It had seven unique bands. These bands might be related to the increase in the productivity of keratinase enzyme.