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Background: The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis. These residues are highly conserved and play an important role in substrate binding through steric hindrance. The substitution of these residues with alanine allows the enzyme to accommodate nonspecific substrates. Results: Wild type and mutated genes were cloned and overexpressed in BL21. Optimum pH and temperature of rBAxn exhibited pH 9.0 and 50 °C respectively and it was stable up to 215 h. Along with the physical properties of rBAxn, kinetic parameters (Km 19.34 ± 0.72 mg/ml; kcat 6449.12 ± 155.37 min− 1 and kcat/Km 333.83 ± 6.78 ml min− 1 mg− 1) were also compared with engineered enzymes. Out of five mutations, W63A, Y128A and W144A lost almost 90% activity and Y124A and W187A retained almost 40–45% xylanase activity. Conclusions: The site-specific single mutation, led to alteration in substrate specificity from xylan to CMC while in case of double mutant the substrate specificity was altered from xylan to CMC, FP and avicel, indicating the role of aromatic residues on substrate binding, catalytic process and overall catalytic efficiency. [ABSTRACT FROM AUTHOR]

Aim/objectives: This study examines the in vitro impact of an ethanolic extract derived from Bryonia laciniosa seeds on the Gir bull ( Bos indicus ) spermatozoa. The objective is to thoroughly assess the effects of the seed extract on the physiological parameters of bull spermatozoa, followed by evaluating its effects on X and Y-bearing spermatozoa and its impact on gene expression through transcriptome profiling., Material Method: For this study, one Gir bull was selected, and 12 ejaculates were collected at one-week time intervals. Sperm cells were isolated from each ejaculate and incubated with varying concentrations of the ethanolic extract. The physiological parameters of the spermatozoa were assessed using Computer Assisted Semen Analysis (CASA) and compared with control groups to evaluate the extract's effects on sperm quality and motility., Results and Discussion: At a concentration of 18 mg/mL B. laciniosa extract, we noticed a statistically significant 16.4% increase in sperm motility ( p  = 0.0065). In order to understand the specific effect on X and Y-bearing spermatozoa, motile and non-motile sperm separated by glass wool column method and further evaluated for quantification of X and Y-bearing sperm in all samples by ddPCR. To understand the effect of B. laciniosa extract on spermatozoa at the molecular level, whole transcriptome profiling was carried out using Illumina MiSeq. Transcriptome profiling revealed 81 genes that were expressed differently between the group treated with the extract and the control group. The current investigation revealed an increase in the expression of TLX1, CRYGB, KLF13, and ZAR1 transcripts, which play a role in embryonic development. In addition, several genes have been identified that are involved in sperm motility, such GSK3B, LAPRS, MAPK1, CAMK2B, and AQP7. The findings exhibited the therapeutic effectiveness of B. laciniosa seeds in augmenting fertility through a synergistic blend of activities, including enhanced sperm motility and positive influence on embryogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Italiya, Panchal, Jakhesara, Joshi and Koringa.)