Background The techniques of amplifying genetic materials have enabled the extensive study of several biological activities outside the biological milieu of living systems. More recently, this approach has been extended to amplify population of genes, from evolutionarily related gene family for detection and evaluation of microbial consortial with several unique potentialities (e.g., enzymatic degradability). Conceivably, primer mixtures containing substitutions of different bases at specific sites (degenerate primers) have enabled the amplification of these genes in PCR reaction. However, the degenerate primer design problem (DPD) is a constraint to designing this kind of primer. To date, different algorithms now exist to solve various versions of DPD problem, many of which, only few addresses and satisfy the criteria to design primers that can extensively cover high through-put sequences while striking the balance between specificity and efficiency. The highly degenerate primer (HYDEN) design software program primarily addresses this variant of DPD problem termed “maximum coverage-degenerate primer design (MC-DPD)” and its heuristics have been substantiated for optimal efficiency from significant successes in PCR. In spite of the premium presented for designing degenerate primers, literature search has indicated relatively little use of its heuristics. This has been thought to result from the complexity of the program since it is run only by command-line, hence limiting its accessibility. To solve this problem, researchers have optionally considered the manual design of degenerate primers or design through software programs that provides accessibility through a graphical user interface (GUI). Realizing this, we have attempted in this study to provide a user-friendly approach for researchers with little or no background in bioinformatics to design degenerate primers using HYDEN Results Virtual Tests of our designed degenerate primer pair through in silico PCR substantiated the correspondence between efficiency and coverage with the target sequences as pre-defined by the initial HYDEN output, thereby validating the potentials of HYDEN to effectively solve the MC-DPD problem. Additionally, the designed primer-pair mechanistically amplified all sequences used as a positive control with no amplification observed in the negative controls. Conclusion In this study, we provided a turnkey protocol to simplify the design of degenerate primers using the heuristics of the HYDEN software program.

The ceaseless quest for economical cellulase, an enzyme that hydrolyzes cellulose, has led to exploring diverse environments, such as insect guts. In this study, we report the optimization of cellulase production and isolation, purification, and characterization of cellulose-degrading enzymes from Aspergillus awamori AFE1. Aspergillus awamori AFE1 was screened for its cellulase-degrading ability, and molecular and phylogenetic analyses of the isolate were performed. Two activity peaks were observed during ion exchange chromatography. A final purification fold of 0.86 and 1.86 with a recovery of 0.18% and 0.44% were achieved for cellulase A and B, respectively; molecular weight of 48.5 KDa and 36.5 KDa for A and B, respectively. The optimum pH of 5.0 was observed for both purified cellulases, and both were stable at an acidic pH of 4.0. An optimum temperature of 60 oC for CA and dual optimum temperatures of 60 and 70 oC were obtained for CB, while both were stable at 30 oC with 63 and 61% residual activity after 2 h, respectively. Fe2+ stimulated both cellulase activity, whereas Zn2+, Cu2+, Mn2+, K+, and Na+ inhibited cellulase activity. Similarly, urea, ascorbic acid, and EDTA inhibited the enzyme. The enzymes were stable in the presence of some organic solvents. The Km and Vmax values were found to be 3.86 mM and 0.3159 mg/ml/min, 4.12 mM, and 0.223 mg/ml/min for the enzyme. The remarkable and unique physicochemical properties of cellulases from Aspergillus awamori AFE1 could be exploited for industrial and biotechnological applications.