Darshan H. Patel

A thermostable l-asparaginase was produced from Bacillus licheniformis UDS-5 (GenBank accession number, OP117154). The production conditions were optimized by the Plackett Burman method, followed by the Box Behnken method, where the enzyme production was enhanced up to fourfold. It secreted l-asparaginase optimally in the medium, pH 7, containing 0.5% (w/v) peptone, 1% (w/v) sodium chloride, 0.15% (w/v) beef extract, 0.15% (w/v) yeast extract, 3% (w/v) l-asparagine at 50 °C for 96 h. The enzyme, with a molecular weight of 85 kDa, was purified by ion exchange chromatography and size exclusion chromatography with better purification fold and percent yield. It displayed optimal catalysis at 70 °C in 20 mM Tris–Cl buffer, pH 8. The purified enzyme also exhibited significant salt tolerance too, making it a suitable candidate for the food application. The l-asparaginase was employed at different doses to evaluate its ability to mitigate acrylamide, while preparing French fries without any prior treatment. The salient attributes of B. licheniformis UDS-5 l-asparaginase, such as greater thermal stability, salt stability and acrylamide reduction in starchy foods, highlights its possible application in the food industry.

Background: Autism spectrum disorder (ASD) affects 1 in 100 children globally with a rapidly increasing prevalence. To the best of our knowledge, no data exists on the genetic architecture of ASD in India. This study aimed to identify the genetic architecture of ASD in India and to assess the use of whole exome sequencing (WES) as a first-tier test instead of chromosomal microarray (CMA) for genetic diagnosis. Methods: Between 2020 and 2022, 101 patient-parent trios of Indian origin diagnosed with ASD according to the Diagnostic and Statistical Manual, 5th edition, were recruited. All probands underwent a sequential genetic testing pathway consisting of karyotyping, Fragile-X testing (in male probands only), CMA and WES. Candidate variant validation and parental segregation analysis was performed using orthogonal methods. Results: Of 101 trios, no probands were identified with a gross chromosomal anomaly or Fragile-X. Three (2.9%) and 30 (29.7%) trios received a confirmed genetic diagnosis from CMA and WES, respectively. Amongst diagnosis from WES, SNVs were detected in 27 cases (90%) and CNVs in 3 cases (10%), including the 3 CNVs detected from CMA. Segregation analysis showed 66.6% (n = 3 for CNVs and n = 17 for SNVs) and 16.6% (n = 5) of the cases had de novo and recessive variants respectively, which is in concordance with the distribution of variant types and mode of inheritance observed in ASD patients of non-Hispanic white/ European ethnicity. MECP2 gene was the most recurrently mutated gene (n = 6; 20%) in the present cohort. Majority of the affected genes identified in the study cohort are involved in synaptic formation, transcription and its regulation, ubiquitination and chromatin remodeling. Conclusions: Our study suggests de novo variants as a major cause of ASD in the Indian population, with Rett syndrome as the most commonly detected disorder. Furthermore, we provide evidence of a significant difference in the diagnostic yield between CMA (3%) and WES (30%) which supports the implementation of WES as a first-tier test for genetic diagnosis of ASD in India.

The strengthening and retrofitting of concrete structures are becoming an essential part of the building and its structures. When the building comes of age, there is a need to increase the structural members' strength, life, and resistivity against unfavorable environmental conditions. In the current scenario, ferrocement composites are used for structural strengthening and rehabilitation. Therefore, an experimental investigation is needed to evaluate the effectiveness of the strengthening on the square and circular reinforced concrete columns by ferrocement and micro concrete materials. The experimental results showed that the ferrocement and micro concrete techniques enhanced the stress-strain behavior and displacement limit of the square and circular RC column. The strengthening circular RC column by micro concrete gives a better performance in stress-strain behavior and displacement limit. In addition, the ferrocement and micro concrete strengthening technique is a promising and economical alternative compared to the other strengthening techniques. Also, test results were compared with ACI and CNR–DT representative load-carrying models to verify each model's reliability and accuracy. Furthermore, the TOPSIS method was adopted to find out the best solution for square and circular columns, and cost analysis was carried out. HIGHLIGHTS RC columns were strengthened using ferro cement and micro concrete RC Columns under compressive concentric load were tested Micro concrete strengthening controls deflection and enhances the load capacity of columns The analytical approach of TOPSIS was used to find the best solution GRAPHICAL ABSTRACT

Industrial processing of enzymes requires higher heating that affects the thermal stability of the enzyme and increases the production cost. In this study, xylanase–phytase (XP) fusion protein was generated via co-expression in a single vector with a cold-shock promoter, leading to improved activity at optimal pH, temperature and the thermal behaviour of the protein. Xylanase–phytase (XP) fusion and phytase proteins were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The XP fusion was thermally stable up to 124 °C, higher than phytase which was steady up to 113.5 °C. XP fusion exhibits higher stability at its thermal transition midpoint (T_m) 108 °C, higher than the T_m value of phytase which is 90 °C. Industrially efficient and environment-friendly proteins with low production cost and higher stability can be generated by ‘fusion protein’ technology.

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. 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 (K_m 19.34 ± 0.72 mg/ml; k_cat 6449.12 ± 155.37 min^− 1 and k_cat/K_m 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. 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.

A thermostable keratinase designated as KBALT was purified from Bacillus altitudinis RBDV1 from a poultry farm in Gujarat, India. The molecular weight of the native KBALT (nKBALT) purified using ammonium sulfate and ion exchange and gel permeation chromatography with a 40% yield and 80-fold purification was estimated to be ~ 43 kDa. The gene for KBALT was successfully cloned, sequenced and expressed in Escherichia coli. Recombinant KBALT (rKBALT) when purified using a single step Ni–NTA His affinity chromatography achieved a yield of 38.20% and a 76.4-fold purification. Comparison of the deduced amino acid sequence of rKBALT with known proteases of Bacillus species and inhibitory effect of PMSF suggest that rKBALT was a subtilisin-like serine protease. Both native and rKBALT exhibited higher activity at 85 °C and pH 8.0 in the presence of Mg²⁺, Mn²⁺, Zn²⁺, Ba²⁺ and Fe³⁺ metal ions. Interestingly, 70% of their activity was retained at temperatures ranging from 35 to > 95 °C. The keratinolytic activity of both nKBALT and rKBALT was enhanced in the presence of reducing agents. They exhibited broad substrate specificity towards various protein substrates. KBALT was determined for its kinetic properties by calculating its K_m (0.61 mg/ml) and V_max (1673 U/mg/min) values. These results suggest KBALT as a robust and promising contender for enzymatic processing of keratinous wastes in waste processing plants.

Energy security, climate change, and economic development are the driving forces behind the increasing focus on biofuel production worldwide. Lignocellulose can provide ample raw materials for biofuel production, via its hydrolysis and the fermentation of the released sugars. Currently, lignocellulases are often used to catalyze this step, which hydrolyze the β-1, 4 glycosidic linkages in lignocellulose. These enzymes offer a good starting point for enhancing the overall economics of biofuel production. To reduce cost in biofuel production, there is a need for enzymes with improved activity and broader substrate range. The rapid development of a variety of protein engineering techniques has facilitated the development of novel biocatalysts with improved properties. A range of specific properties can be enhanced in enzymes, including the catalytic activity, thermostability, and specificity, under industrial conditions.