The in-silico study of the structural changes in the Arthrobacter globiformis choline oxidase induced by high temperature.

BACKGROUND: Choline oxidase, a flavoprotein, is an enzyme that catalyzes the reaction which converts choline into glycine betaine. Choline oxidase started its journey way back in 1933. However, the impact of the high temperature on its structure has not been explored despite the long history and availability of its crystal structure. Both choline oxidase and its product, glycine betaine, have enormous applications spanning across multiple industries. Understanding how the 3D structure of the enzyme will change with the temperature change can open new ways to make it more stable and useful for industry. PROCESS: This research paper presents the in-silico study and analysis of the structural changes of A. globiformis choline oxidase at temperatures from 25 °C to 60 °C. A step-wise process is depicted in Fig. 1. RESULTS: Multiple sequence alignment (MSA) of 11 choline oxidase sequences from different bacteria vs Arthrobacter globiformis choline oxidase showed that active site residues are highly conserved. The available crystal structure of A. globiformis choline oxidase with cofactor Flavin Adenine Dinucleotide (FAD) in the dimeric state (PDB ID: 4MJW)1 was considered for molecular dynamics simulations. A simulated annealing option was used to gradually increase the temperature of the system from 25 °C to 60 °C. Analysis of the conserved residues, as well as residues involved in Flavin Adenine Dinucleotide (FAD) binding, substrate binding, substate gating, and dimer formationwas done. At high temperatures, the formation of the inter-chain salt bridge between Arg50 and Glu63 was a significant observation near the active site of choline oxidase. CONCLUSION: Molecular dynamics studies suggest that an increase in temperature has a significant impact on the extended Flavin Adenine Dinucleotide (FAD) binding region. These changes interfere with the entry of substrate to the active site of the enzyme and make the enzyme inactive.

Enzyme engineering of choline oxidase for improving stability.

Functioning as a flavoprotein, choline oxidase facilitates the transformation of choline into glycine betaine. Notably, choline oxidase and its resultant product, glycine betaine, find extensive applications across various industries and fields of study. However, its high sensitivity and tendency to lose functional activity at high temperatures reduces its industrial usage. MD simulation and mutation studies have revealed the role of certain residues responsible for the enzyme's thermal instability. This study focuses on inducing thermal stability to choline oxidase of A. globiformis through computational approaches at a maximum temperature of 60 °C. MD simulation analysis showed that Trp 331, Val 464 and Ser 101 contribute to structural instability, leading to the instability at 60 °C. Mutation of these residues with phenylalanine residues and simulation of the mutated enzyme at 60 °C exhibited thermostability and insignificant residual fluctuation. The re-docking and MM/GBSA analyses further validated the mutated enzyme's binding affinity and catalytic activity.Communicated by Ramaswamy H. Sarma.

Thermo-alkali stable bacterial xylanase for deinking of copier paper.

BACKGROUND: The bleach-boosting capability of xylanases is well-known. The use of xylanase pre-treatment before the application of chemical bleach has multiple advantages including (i) lesser use of polluting chemicals of the traditional bleaching process; (ii) less damage to the cellulosic fibers, therefore better recyclability; and (iii) better brightness of chemical bleach. The major impediment in the application is the availability of commercial enzymes that are active at the elevated temperature and pH that exist during the industrial pulping process. In the present paper, xylanase having suitability for application in deinking is reported. RESULTS: The xylanase used showed high deinking potential. Optimal deinking was obtained at the xylanase dosing of 20U/g of the dried pulp at 60℃ for a treatment time of 1h. It could bring about a 50% reduction in the usage of chemical bleach that was applied after xylanase pre-treatment. The comparison of FTIR spectra showed changes in intensity without significant changes in the functional group signatures implying that there is negligible damage to the fiber strength in the xylanase pre-treatment process as compared to the chemical bleach process. CONCLUSION: The xylanase used in this study was effective in deinking paper pulp and can be used for bio-bleaching of recycled paper.

Phyto-microbiome to mitigate abiotic stress in crop plants.

Plant-associated microbes include taxonomically diverse communities of bacteria, archaebacteria, fungi, and viruses, which establish integral ecological relationships with the host plant and constitute the phyto-microbiome. The phyto-microbiome not only contributes in normal growth and development of plants but also plays a vital role in the maintenance of plant homeostasis during abiotic stress conditions. Owing to its immense metabolic potential, the phyto-microbiome provides the host plant with the capability to mitigate the abiotic stress through various mechanisms like production of antioxidants, plant growth hormones, bioactive compounds, detoxification of harmful chemicals and toxins, sequestration of reactive oxygen species and other free radicals. A deeper understanding of the structure and functions of the phyto-microbiome and the complex mechanisms of phyto-microbiome mediated abiotic stress mitigation would enable its utilization for abiotic stress alleviation of crop plants and development of stress-resistant crops. This review aims at exploring the potential of phyto-microbiome to alleviate drought, heat, salinity and heavy metal stress in crop plants and finding sustainable solutions to enhance the agricultural productivity. The mechanistic insights into the role of phytomicrobiome in imparting abiotic stress tolerance to plants have been summarized, that would be helpful in the development of novel bioinoculants. The high-throughput modern approaches involving candidate gene identification and target gene modification such as genomics, metagenomics, transcriptomics, metabolomics, and phyto-microbiome based genetic engineering have been discussed in wake of the ever-increasing demand of climate resilient crop plants.

Gut microbiome influences incidence and outcomes of breast cancer by regulating levels and activity of steroid hormones in women.

BACKGROUND: Breast cancer, the leading cancer type in women worldwide, is affected by reproductive and nonreproductive factors. Estrogen and progesterone influence the incidence and progression of breast cancer. The microbiome of the gut, a complex organ that plays a vital role in digestion and homeostasis, enhances availability of estrogen and progesterone in the host. Thus, an altered gut microbiome may influence the hormone-induced breast cancer incidence. This review describes the current understanding of the roles of gut microbiome in influencing the incidence and progression of breast cancer, with an emphasis on the microbiome-induced metabolism of estrogen and progesterone. RECENT FINDINGS: Microbiome has been recognized as a promising hallmark of cancer. Next-generation sequencing technologies have aided in rapid identification of components of the gut microbiome that are capable of metabolizing estrogen and progesterone. Moreover, studies have indicated a wider role of the gut microbiome in metabolizing chemotherapeutic and hormonal therapy agents and reducing their efficacy in patients with breast cancer, with a predominant effect in postmenopausal women. CONCLUSION: The gut microbiome and variations in its composition significantly alter the incidence and therapy outcomes of patients with breast cancer. Thus, a healthy and diverse microbiome is required for better response to anticancer therapies. Finally, the review emphasizes the requirement of studies to elucidate mechanisms that may aid in improving the gut microbiome composition, and hence, survival outcomes of patients with breast cancer.

Partial purification and characterization of a thermostable xylanase from Bacillus licheniformis isolated from hot water geyser.

BACKGROUND: Thermo-alkali stable xylanases were purified from the extracellular broth of newly isolated Bacillus licheniformis strain produced in a 5-L stirred-tank bioreactor with wheat bran as a carbon source. RESULTS: A high degree of purity was achieved using size exclusion chromatography resulting in 16-fold purification and 69% recovery for fraction 5 which had the highest activity. The recovery obtained after pooling fractions 5 and 6 was 99%. The Km value of xylanase was calculated as 0.05 mM, and Vmax was 125 µmol/min/mg protein. CONCLUSION: Purified xylanase had a high thermal and pH stability. Xylanase was found to be suitable for application in the de-inking of paper and for saccharification of lignocellulosic waste biomass.

Production of Thermo-Alkali-Stable Xylanase from Bacillus licheniformis Isolated from Natural Hot Water Geyser.

CONTEXT: Xylanase constitutes 20% of world enzyme market. Significantly, they are used in poultry feed, paper pulp, bakery, and textile industries. In view of the increasing demand of the enzyme, it is vital to develop indigenous strains and scalable technologies for the production of industrial enzymes. AIMS: The objective of the present paper was to isolate a high-yielding strain of thermo-alkali-stable xylanase-producing bacteria for potential application in paper and pulp and biofuel industry. METHODS: Sampling for prospecting of suitable organism was carried out from the places with dead and decaying lignocellulosic waste, and then Congo red screening was employed for the primary isolation of xylanase producers. RESULTS: We report the isolation of 18 different strains of xylanase producer bacteria from natural hot water geyser of Sohna, Haryana, India. Subsequently, two of these isolates were chosen for further studies based on xylanase yield and desirable properties such as thermostability and alkali stability of xylanase produced. CONCLUSION: Isolate B2 was later identified as Bacillus licheniformis, whereas isolate Y3 was identified as Brevibacillus borstelensis. This strain when cultured at 35°C for 72 h showed xylanase production at 128 U/ml. The molecular weight of xylanase was determined to be 25 kDa. The production was scaled up in a 5-L stirred-tank bioreactor which led to high xylanase concentration of 380 U/ml in the first 48 h of culture.

Taguchi optimization and scale up of xylanase from Bacillus licheniformis isolated from hot water geyser.

BACKGROUND: Xylanase is one of the widely applied industrial enzymes with diverse applications. Thermostability and alkali tolerance are the two most desirable qualities for industrial applications of xylanase. In this paper, we reveal the statistical Taguchi optimization strategy for maximization of xylanase production. The important process parameters pH, temperature, concentration of wheat bran, and concentration of yeast extract were optimized using the Taguchi L8 orthogonal array where the 4 factors were considered at 2 levels (high and low). RESULTS: The optimized conditions given by model were obtained as follows: (i) pH 6, (ii) culture temperature 35 °C, (iii) concentration of xylan 2% w/v, (iv) concentration of wheat bran 2.5% w/v. The production was scaled upto 2.5 L bioreactor using optimized process parameters. A high xylanase titer of 400 U/ml could be achieved in less than 60 h of culture in the reactor. CONCLUSION: Optimization was successful in achieving about threefold increase in the yield of xylanase. The optimized conditions resulted in a successful scale up and enhancement of xylanase production.

A rare case of mosaic 45,X/47,XX,+13 in 28-year-old women with secondary amenorrhoea: A case report and literature review.

In the present paper we report an extremely rare case of mosaicism of 45,X/47,XX,+13 in a 28-year-old women. The patient was referred for cytogenetic evaluation for secondary amenorrhoea. The patient was found to have some mild characteristic features of Turner syndrome such as wide carrying angle and short stature. Ultrasound examination revealed the presence of a small sized uterus and bilateral streak ovaries. G-banded chromosome analysis revealed a mosaic female karyotype involving two different cell lines. One cell line (72% of analysed metaphases) presented monosomy of X while the remaining 28% of cells showed trisomy of chromosome 13. Fluorescence in situ hybridization (FISH) with locus specific probe for trisomy 13 and CEP X for monosomy X substantiated the results obtained from karyotyping.