The enzyme 6-phospho-β-glucosidase is an important member of the glycoside hydrolase family 1 (GH1). However, its catalytic mechanisms, especially the key residues determining substrate specificity and affinity, are poorly understood. A metagenome-derived gene sequence, encoding a novel 6-phospho-β-glucosidase designated Pbgl25-217, was isolated and characterized. The optimal conditions for enzymatic activity were 37°C and pH 7; Ca(2+), Mg(2+), and Mn(2+) stabilized the activity of Pbgl25-217, whereas Ni(2+), Fe(2+), Zn(2+), Cu(2+), and Fe(3+) inhibited its activity. The Km and Vmax of Pbgl25-217 were 4.8 mM and 1,987.0 U mg(-1), respectively. Seven conserved residues were recognized by multiple alignments and were tested by site-directed mutagenesis for their functions in substrate recognition and catalytic reaction. The results suggest that residues S427, Lys435, and Tyr437 act as "gatekeepers" in a phosphate-binding loop and play important roles in phosphate recognition. This functional identification may provide insights into the specificity of 6-phospho-β-glycosidases in GH1 and be useful for designing further directed evolution.

The thermostable β-glucosidase A (TmBglA) from Thermotoga maritime is a promising biocatalyst for production of isoflavone aglycones. Use of enzymes with high specificity for soy isoflavone conjugates is however essential for efficient hydrolysis. The effect of the amino acids located in the aglycone binding pocket with non-conserved residues between specificity groups in family 1 glycoside hydrolase (GH1) was studied using wild-type TmBglA and 3 exchange mutants (MI-TmBglA, M2-TmBglA, M1M2-TmBglA). Three mutants were expressed in Escherichia coli, purified and characterized. They had shifts in both optimum tem- perature and thermal stability, and their narrowing pH-activity curve caused by removing the ionized side chain in mutation. All mutants demonstrated the decreased catalytic efficiency more effectively revealed with natural glycoside, salicin, than with artificial substrate, p-nitrophenyl-β-D-glucopyranoside, suggesting that' these amino acids are the key residues to determine aglycone specificity. A lower hydrolysis of genistin and daidzin for M2-TmBglA than M1-TmBglA indicated that L400, A407 and E408 being preferable to V170, A171, V173, G 174 and H180 residues of Tm-BglA could be essential for soy isoflavone glycoside binding and catalysis.

Fungal β-glucosidases (BGLs) have unceasingly utilized for industrial applications and recently, they possess a crucial role in bioethanol production. To engineer the BGLs, understanding their structures, intermolecular interactions and molecular docking is requisite, which is carried out in this work based on the glycosyl hydrolase (GH) family. Among 12 BGLs, protein sequence, structure, and conserved sites of GH1 BGLs are evidently diverged to GH3 BGLs. Even biophysical and chemical features of GH1 BGLs are utterly varied from GH3 BGLs, wherein pI, instability index, aliphatic index, surface & buried area, thermostability and thermodynamics are included. On the contrary, aromatic, charged, polar, and hydrophobic residues are significantly higher in GH1 BGLs as compared to that of GH3 BGLs. Moreover, molecular docking of BGLs with 12 substrates and 5 inhibitors revealed that the GH3 BGLs efficiently bound with laminaribose, gentibiose, aryl- and cello-substrates than GH1 BGLs; however, GH3 BGLs are noticeably inhibited by glucose, glucono-δ-lactone, methanetriamine. So, structural insight of BGLs provides an explicit knowledge regarding the catalytic residues, biophysical chemistry and notable binding ligands, which are most important factors for enzyme engineering.

Using the information available in the sequences of well-characterized transglycosidases found in plants, mutations were introduced in the glycoside hydrolase of the bacterium Thermus thermophilus, with the aim of turning it into an efficient transglycosidase. All mutants happen to have fair catalytic efficiencies, being at worst 25 times less efficient than the wild type. Noteworthy, W120F, one of our high transglycosylation yield (≈ 50%) mutants, is only two times less efficient than the wild type. Interestingly, while in the wild type the sidechain of the acid-base is only found able to sample a pair of equivalent conformations during 0.5-μs-long molecular dynamics simulations, its flexibility is much higher in the case of the high transglycosylation yield mutants. Our results thus suggest that engineering the flexibility of the acid-base of a retaining glycoside hydrolase could be a general way to turn it into an efficient transglycosidase.

The oomycetous phytopathogen Pythium myriotylum secretes cellulases for growth/nutrition of the necrotroph. Cellulases are multi-enzyme system classified into different glycoside hydrolase (GH) families. The present study deals with identification and characterization of GH gene sequence from P. myriotylum by a PCR strategy using consensus primers. Cloning of the full-length gene sequence using genome walker strategy resulted in identification of 1230-bp P. myriotylum GH gene sequence, designated as PmGH1. Analysis revealed that PmGH1 encodes a predicted cytoplasmic 421 amino acid protein with an apparent molecular weight of 46.77 kDa and a theoretical pI of 8.11. Tertiary structure of the deduced amino acid sequence showed typical (α/β)8 barrel folding of family 1 GHs. Sequence characterization of PmGH1 identified the conserved active site residues, viz., Glu 181 and Glu 399, that function as acid-base catalyst and catalytically active nucleophile, respectively. Binding sites for N-acetyl-D-glucosamine (NAG) were revealed in the PmGH1 3D structure with Glu181 and Glu399 positioned on either side to form a catalytic pair. Phylogenetic analysis indicated a closer affiliation of PmGH1 with sequences of GH1 family. Results presented are first attempts providing novel insights into the evolutionary and functional perspectives of the identified P. myriotylum GH.

In this work, we investigated how activity and oligomeric state are related in a purified GH1 β-glucosidase from Spodoptera frugiperda (Sfβgly). Gel filtration chromatography coupled to a multiple angle light scattering detector allowed separation of the homodimer and monomer states and determination of the dimer dissociation constant (K_D ), which was in the micromolar range. Enzyme kinetic parameters showed that the dimer is on average 2.5-fold more active. Later, we evaluated the kinetics of homodimerization, scanning the changes in the Sfβgly intrinsic fluorescence over time when the dimer dissociates into the monomer after a large dilution. We described how the rate constant of monomerization (k_off ) is affected by temperature, revealing the enthalpic and entropic contributions to the process. We also evaluated how the rate constant (k_obs ) by which equilibrium is reached after dimer dilution behaves when varying the initial Sfβgly concentration. These data indicated that Sfβgly dimerizes through the conformational selection mechanism, in which the monomer undergoes a conformational exchange and then binds to a similar monomer, forming a more active homodimer. Finally, we noted that conformational selection reports and experiments usually rely on a ligand whose concentration is in excess, but for homodimerization, this approach does not hold. Hence, since our approach overcomes this limitation, this study not only is a new contribution to the comprehension of GH1 β-glucosidases, but it can also help to elucidate protein interaction pathways.

Keywords: Spodoptera frugiperda; conformational selection; enzyme activity; glycoside hydrolase; homodimer; oligomerization; β-glucosidase.

Familial isolated growth hormone deficiency (GHD) type 1 is characterized by an autosomal recessive pattern of inheritance with varying degrees of phenotypic severity. We report a proband, with isolated GHD (IGHD) with very early growth arrest and undetectable levels of GH. Homozygous complete deletion of the GH1 gene was identified by real-time/quantitative polymerase chain reaction (RT/q-PCR) and confirmed by an independent molecular genetic method; the multiplex ligation-dependent probe amplification (MLPA) technique. Prenatal diagnosis was offered for the subsequent pregnancy in the mother of our proband. Identical heterozygous deletion of the GH1 gene was detected in both parents. The fetus had a similar homozygous deletion of the GH1 gene. We thus report a unique case with a confirmed mutation in GH1 gene in the proband followed by prenatal detection of the same mutation in the amniotic fluid which to our knowledge hitherto has not been documented from India.

The somatotropic axis consists of genes that are involved in muscular development. These genes are potential regions of study to identify possible QTL for economically important traits in beef cattle. The aim of this study was to verify the existence of GH1, POU1F1, and GHR polymorphisms in Nellore cattle to verify the influence of selection in these mutations and to analyse the association between molecular markers and body weight at different ages, yearling hip height, carcass fat thickness and loin eye area. Six hundred forty-five animals from the Centro APTA Bovinos de Corte, were genotyped by PCR-RFLP techniques. The association analyses were performed with general mixed models taking into consideration the effect of one marker, and other model taking into consideration interactions between two molecular markers. Only the molecular markers rs81109601 on GH1 and rs109136815 on GHR were polymorphic; however, they were not found to be under selection. The association of the GHR rs109136815 marker and loin eye area was observed (p<0.05), as well as the effect of interaction between the markers and the female body weight at 550 days of age (p<0.04). The interaction effect should be considered in situations where the interactivity between two genes is known.

Incubation of pituitary GH1 cells with N'-methylnicotinamide, nicotinamide and 3-acetylpyridine which inhibit nuclear ADP-ribosylation and/or the cellular concentration of its substrate NAD+ reduced the amount of nuclear thyroid hormone receptors in a time- and dose-dependent manner without altering the affinity of the receptors for the hormone. A transient activation of poly(ADP-ribose)polymerase by methyl methanesulfonate, ultraviolet irradiation or spermine caused a rapid depletion of cellular NAD+ content and was followed by a strong inhibition of ADP-ribosylation. These agents also produced a very rapid and marked reduction of receptor numbers. The decrease of receptors caused by the different compounds is not secondary to a generalized inhibition of protein synthesis or to an alteration in hormone availability. The abundance of c-erbA alpha and beta mRNAs, which encode thyroid hormone receptors, was reduced in cells treated with the compounds that decrease receptor number, thus suggesting that this effect is caused by a decrease in the expression of c-erbA genes.

Background: Cellulose degradation by cellulase is brought about by complex communities of interacting microorganisms, which significantly contribute to the cycling of carbon on a global scale. β-Glucosidase (BGL) is the rate-limiting enzyme in the cellulose degradation process. Thus, analyzing the expression of genes involved in cellulose degradation and regulation of BGL gene expression during composting will improve the understanding of the cellulose degradation mechanism. Based on our previous research, we hypothesized that BGL-producing microbial communities differentially regulate the expression of glucose-tolerant BGL and non-glucose-tolerant BGL to adapt to the changes in cellulose degradation conditions.

Results: To confirm this hypothesis, the structure and function of functional microbial communities involved in cellulose degradation were investigated by metatranscriptomics and a DNA library search of the GH1 family of BGLs involved in natural and inoculated composting. Under normal conditions, the group of non-glucose-tolerant BGL genes exhibited higher sensitivity to regulation than the glucose-tolerant BGL genes, which was suppressed during the composting process. Compared with the expression of endoglucanase and exoglucanase, the functional microbial communities exhibited a different transcriptional regulation of BGL genes during the cooling phase of natural composting. BGL-producing microbial communities upregulated the expression of glucose-tolerant BGL under carbon catabolite repression due to the increased glucose concentration, whereas the expression of non-glucose-tolerant BGL was suppressed.

Conclusion: Our results support the hypothesis that the functional microbial communities use multiple strategies of varying effectiveness to regulate the expression of BGL genes to facilitate adaptation to environmental changes.

Keywords: Carbon catabolite repression; Compost; Differential expression; Functional microbial community; β-Glucosidase.

Here the statistics concerning X-ray data processing and structure refinement are given, together with the substrate preference analysis for ThBgl1 and ThBgl2. Finally, the analysis of the influence of temperature and pH on the activities of both enzymes are shown.

The active site residues in GH1 β-glycosidases are compartmentalized into 3 functional regions, involved in catalysis or binding of glycone and aglycone motifs from substrate. However, it still remains unclear how residues outside the active site modulate the enzymatic activity. To tackle this question, we solved the crystal structure of the GH1 β-glycosidase from Spodoptera frugiperda (Sfβgly) to systematically map its residue contact network and correlate effects of mutations within and outside the active site. External mutations neighbouring the functional residues involved in catalysis and glycone-binding are deleterious, whereas mutations neighbouring the aglycone-binding site are less detrimental or even beneficial. The large dataset of new and previously characterized Sfβgly mutants supports that external perturbations are coherently transmitted to active site residues possibly through contacts and specifically disturb functional regions they interact to, reproducing the effects observed for direct mutations of functional residues. This allowed us to suggest that positions related to the aglycone-binding site are preferential targets for introduction of mutations aiming to further improve the hydrolytic activity of β-glycosidases.

A novel aerobic bacterial strain, designated ZS60^T, with long, rod-shaped, Gram-staining-negative, aerobic cells was isolated from the soil in the Tianshan Mountains, Xinjiang, China. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain ZS60^T was affiliated with the genus Lysobacter, and was most closely related to Lysobacter daejeonensis GH1-9^T (96.9 %), Lysobacter caeni BUT-8^T (96.8 %) and Lysobacter ruishenii CTN-1^T (96.7 %). The average nucleotide identity values between strain ZS60^T, L. daejeonensis GH1-9^T and L. ruishenii CTN-1^T were 78.14 and 78.39 %, respectively. The DNA-DNA relatedness between strain ZS60^T, L. daejeonensis GH1-9^T and L. caeni BUT-8^T were 44.8 and 39.1 %, respectively. The genomic DNA G+C content of the strain ZS60^T was 67.7 mol% (draft genome sequence), and Q-8 was the predominant ubiquinone. The major cellular fatty acids of strain ZS60^T were iso-C15 : 0 (23.4 %), iso-C17 : 0 (17.2 %) and iso-C17 : 1 ω9c (12.6 %). On the basis of genotypic, phenotypic and biochemical data, strain ZS60^T is considered to represent a novel species of the genus Lysobacter, for which the name Lysobacterpsychrotolerans sp. nov. is proposed. The type strain is ZS60^T (=CGMCC 1.15509^T=NBRC 112614^T).

BACKGROUND

Lactulose has various uses in the food and pharmaceutical fields. Thermostable enzymes have many advantages for industrial exploitation, including high substrate solubilities as well as reduced risk of process contamination.

RESULTS

Enzymatic synthesis of lactulose employing a transgalactosylation reaction by a recombinant thermostable glycoside hydrolase (GH1) from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167 was investigated. The optimal pH for lactulose production was found to be 4.5, while the optimal temperature was 85 °C, before it dropped moderately to 83% at 90 °C. However, the relative activity for lactulose synthesis dropped sharply to 35% at 95 °C. At optimal reaction conditions of 70% (w/w) initial sugar substrates with molar ratio of lactose to fructose of 1:4, 15 U mL-1 enzyme concentration and 85 °C, the time course reaction produced a maximum lactulose concentration of 108 g L-1 at 4 h, corresponding to a lactulose yield of 14% and 27 g L-1 h-1 productivity with 84% lactose conversion. The transgalactosylation reaction for lactulose synthesis was greatly influenced by the ratio of galactose donor to acceptor.

CONCLUSIONS

This novel GH1 may be useful for process applications owing to its high activity in very concentrated substrate reaction media and promising thermostability. © 2017 Society of Chemical Industry.

6-phospho-β-glucosidases and 6-phospho-β-galactosidases are enzymes that hydrolyze the β-glycosidic bond between a terminal non-reducing glucose-6-phosphate (Glc6P) or galactose-6-phosphate (Gal6P), respectively, and other organic molecules. Gan1D, a glycoside hydrolase (GH) belonging to the GH1 family, has recently been identified in a newly characterized galactan-utilization gene cluster in the bacterium Geobacillus stearothermophilus T-1. Gan1D has been shown to exhibit bifunctional activity, possessing both 6-phospho-β-galactosidase and 6-phospho-β-glucosidase activities. We report herein the complete 3D crystal structure of Gan1D, together with its acid/base catalytic mutant Gan1D-E170Q. The tertiary structure of Gan1D conforms well to the (β/α)8 TIM-barrel fold commonly observed in GH enzymes, and its quaternary structure adopts a dimeric assembly, confirmed by gel-filtration and small-angle X-ray scattering results. We present also the structures of Gan1D in complex with the putative substrate cellobiose-6-phosphate (Cell6P) and the degradation products Glc6P and Gal6P. These complexes reveal the specific enzyme-substrate and enzyme-product binding interactions of Gan1D, and the residues involved in its glycone, aglycone, and phosphate binding sites. We show that the different ligands trapped in the active sites adopt different binding modes to the protein, providing a structural basis for the dual galactosidase/glucosidase activity observed for this enzyme. Based on this information, specific mutations were performed on one of the active site residues (W433), shifting the enzyme specificity from dual activity to a significant preference toward 6-phospho-β-glucosidase activity. These data and their comparison with structural data of related glucosidases and galactosidases are used for a more general discussion on the structure-function relationships in this sub-group of GH1 enzymes.

Atomic coordinates of Gan1D-wild-type (WT)-P1, Gan1D-WT-C2, Gan1D-E170Q, Gan1D-WT-Gal6P, Gan1D-WT-Glc6P, and Gan1D-E170Q-Cell6P have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, under accession codes 5OKB, 5OKJ/5OKH, 5OKA/5OK7, 5OKQ/5OKK, 5OKS/5OKR, and 5OKG/5OKE, respectively.

Sperm cryopreservation could entail DNA damage, promoting base oxidization and strand breaks. In a previous work we showed that trout DNA damaged sperm is able to fertilize leading to embryo loss when the repair system of the oocyte is inhibited. Here we have analysed the later effects on embryo and larvae of fertilizing trout oocytes with cryopreserved DNA-damaged spermatozoa. Fish have weak sperm selection mechanisms, are very prolific and have external embryo development, being convenient models for this type of study. We cryopreserved rainbow trout semen using extenders containing egg yolk or their low density lipoprotein fraction to obtain samples with different degrees of DNA damage. DNA fragmentation was evaluated using the Comet assay and telomere length using quantitative-PCR. Fertilization trials were performed and the transcription at different developmental stages of telomerase reverse transcriptase (Tert) and eight genes related with embryo growth and development (Igf1, Igf2, Igfr1a, Igfr1b, Gh1, Gh2, Ins1 and Ins2) were analyzed using quantitative-PCR in surviving embryos and larvae. Results showed an increase in sperm DNA fragmentation after both cryopreservation procedures as well as a decrease in sperm telomere length. Larvae obtained with damaged sperm showed longer telomeres and Tert overexpression. The transcription of the analyzed genes in these embryos and larvae was also modified with respect to the control, most of them as an increase at hatch. We conclude that fertilization with cryopreserved DNA-damaged spermatozoa significantly affects offspring performance, detectable as an increase in telomere length as well as some alterations in gene expression in surviving embryo and larvae.

Sequential pretreatments for sugarcane bagasse (scb) by NaOH followed by organosolv under mild conditions were evaluated for cellulose recovery and dilignification. The best-optimized sequential pretreatment of scb was obtained at 10% (w/v) of raw scb loading at 1% (w/v) NaOH (50 °C, 2 h) followed by treatment with organosolv (85%, v/v phosphoric acid, 50 °C, 1 h) with chilled acetone. This sequentially pretreated scb showed cellulose recovery, 66.1% (w/w) and delignification, 83.2% (w/w). NaOH or organosolv pretreated scb showed lower cellulose recovery 47.4% (w/w) or 54.5% (w/w) with lower delignification, 61% (w/w) or 56% (w/w), respectively. Pretreated solid residue of sequentially pretreated scb was enzymatically saccharified by chimera (β-glucosidase and endoglucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) cloned from Clostridium thermocellum. Enzymatic hydrolysate of best sequentially pretreated scb gave total reducing sugar (TRS) yield, 230 mg/g and glucose yield, 137 mg/g pretreated scb. Only organosolv pretreated scb gave TRS yield, 112.5 mg/g and glucose yield, 72 mg/g of pretreated scb. Thus, sequentially pretreated scb resulted in 37% higher enzymatic digestibility than only orgnaosolv pretreated scb. Higher enzymatic digestibility was supported by higher crystallinity index CrI (45%) than those obtained with only organosolv pretreated (38%) or raw scb (25%). Field Emission Scanning Electron Microscope (FESEM) and Fourier-transform infrared (FT-IR) analyses showed enhanced cellulose exposure in sequentially pretreated scb. Preliminary investigation of bioethanol production at small scale by separate hydrolysis and fermentation (SHF) of enzymatic hydrolysate from best sequentially pretreated scb by Saccharomyces cerevisiae gave maximum ethanol yield of 0.42 g/g of glucose.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-020-02600-y.

Keywords: Bioethanol; Enzymatic hydrolysis; Enzyme cocktail; Lignocellulosic biomass.

Gene duplication is one of the main mechanisms of formation of new genetic material in evolution. The occurrence of a gene duplication is believed to relax selection pressure on one of the copies. Consequently, this gene accumulates mutations at a higher rate, and over time it acquires a new function. As a result of several rounds of polyploidization, many genes in salmon are duplicated, including the growth hormone gene. The analysis of nucleotide diversity in the paralogous genes of growth hormone, gh1 and gh2, demonstrated that the level of variability in their introns was higher than in the exons. In addition, the variability of each exon weakly correlated with its length, and seems to be determined by the functional significance of the protein region encoded. The level of variability in the exons of the gh2 gene was higher than that in the gh1 one, which was probably due to the current process of gene subfunctionalization.

Keywords: Salmonidae; Salvelinus; gene duplication; negative selection; nucleotide polymorphism; rowth hormone genes.

Background: Estrogen controls the pubertal growth spurt, growth plate closure, and accretion of bone mineral density (BMD) of long bones after biding estrogen receptor (ER). There are two subtypes of ER, ERα and ERβ. If each ER subtype has different effects, we may control those actions by manipulating the estrogen binding intensity to each ER subtype and increase the final adult height without markedly reducing BMD or impairing reproductive functions. The purpose of our study was to compare these effects of ERα and ERβ on long bones in ovariectomized rats.

Methods: Thirty female rats were ovariectomized and randomly divided into 3 groups. The control, propylpyrazole triol (PPT), and 2,3-bis (4-hydroxyphenyl) propionitrile (DPN) groups were subcutaneously injected for 5 weeks with sesame oil, PPT as an ERα agonist, and DPN as an ERβ agonist, respectively. The crown-lump length and body weight were measured weekly. BMD, serum levels of growth hormone (GH) and estradiol were checked before and after 5 weeks of injections. Pituitary GH1 expression levels were determined with quantitative real-time polymerase chain reaction, the proximal tibias were dissected, decalcified and stained with hematoxylin-eosin, and the thicknesses of epiphyseal plates including proliferative and hypertrophic zones were measured in 20-evenly divided sites after 5 weeks of injections. Comparisons for auxological data, serum hormone and pituitary GH1 expression levels, BMD, and epiphyseal plate thicknesses among 3 groups before and after injections were conducted.

Results: There was no significant difference in body lengths among 3 groups. The body weights were significantly lower, but, serum GH, pituitary GH1 expression levels, and BMDs were higher in PPT group than the other 2 groups after 5 weeks of injections. There was no significant difference in the thicknesses of the total epiphyseal plate, proliferative, and hypertrophic zone among 3 groups.

Conclusion: ERα is more involved in pituitary GH secretion and bone mineral deposition than ERβ. Weight gain might be prevented with the ERα agonist.

Keywords: Bone Density; Estrogen Receptor Alpha; Estrogen Receptor Beta; Growth.

Glycoside hydrolase family 1 (GH1) β-glucosidases (BGLUs) are encoded by a large number of genes, and are involved in many developmental processes and stress responses in plants. Due to their importance in plant growth and development, genome-wide analyses have been conducted in model plants (Arabidopsis and rice) and maize, but not in Brassica species, which are important vegetable crops. In this study, we systematically analyzed B. rapaBGLUs (BrBGLUs), and demonstrated the involvement of several genes in pollen development. Sixty-four BrBGLUs were identified in Brassica databases, which were anchored onto 10 chromosomes, with 10 tandem duplications. Phylogenetic analysis revealed that 64 genes were classified into 10 subgroups, and each subgroup had relatively conserved intron/exon structures. Clustering with Arabidopsis BGLUs (AtBGLUs) facilitated the identification of several important subgroups for flavonoid metabolism, the production of glucosinolates, the regulation of abscisic acid (ABA) levels, and other defense-related compounds. At least six BrBGLUs might be involved in pollen development. The expression of BrBGLU10/AtBGLU20, the analysis of co-expressed genes, and the examination of knocked down Arabidopsis plants strongly suggests that BrBGLU10/AtBGLU20 has an indispensable function in pollen development. The results that are obtained from this study may provide valuable information for the further understanding of β-glucosidase function and Brassica breeding, for nutraceuticals-rich Brassica crops.

BACKGROUND

In recent times, focus on plant research has improved all over the world and essential parts of plants provide bioactive compounds in human diet. The bael (Aegle marmelos) has enormous traditional uses in the treatment of chronic diarrhea, dysentery, peptic ulcers and as a laxative. The main focus of this study was characterization of bael leaf extract for its bioactive constituents, antihypercholestrolemic and antilipidemic perspectives.

METHODS

After proximate composition of bael powder, the aqueous extract of bael leaf was used for phytochemical profiling (alkaloids, total phenolic content and total flavonoid content). Afterwards, normal rats group G0 was administrated basal diet while G1 and G2 normal rat groups were fed diets containing bael leaf extract 125 mg and 250 mg, respectively for consecutive 60 days. In a similar way, hyperlipidemic rats group Gh0 was administrated basal diet while Gh1 and Gh2 hyperlipidemic rat groups were fed diets containing bael leaf extract 125 mg and 250 mg, respectively for consecutive 60 days. The blood drawn on day 0, day 30 and day 60 was analyzed for serum parameters, such as total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides concentration and free and ester cholesterol.

RESULTS

Bael leaf powder is a rich source of crude fiber (14.50 ± 0.10 g/100 g). Aqueous extract of bael leaf contains alkaloids (15.58 ± 0.05 mg/g), flavonoids (64.00 ± 0.05 mg/g), phenolics (30.34 ± 0.01 GAEmg/g). From the In vivo studies, the lowest weight gain was observed in group G2 and in Gh2 as compared to control of both groups. The decrease in serum TC for G1-15.06%, G2-17.27% while in Gh1-22.46% and Gh2-34.82% after day 60, respectively. The maximum decrease was observed in group G2 (- 14.33%) and in Gh2 (- 24.79%) for triglycerides after 60 days. For HDL-cholesterol, significant increase (11.20%) in G2 and (49.83%) in Gh2 was observed of after 60 days. A trend in decrease of serum LDL-cholesterol in G2 (- 9.63%) and in Gh2 (- 44.65%) was also observed at day 60, and - 19.05% and - 30.06% decrease was noted in G2 and Gh2, respectively and decreasing trend was observed in free and total cholesterol - 22.30% and - 81.49% for groups G2 and Gh2 after day 60.

CONCLUSIONS

The results of the present study demonstrated that the extract contents of bael leaf provide protective role against hypercholesterolemic and hyperlipidemic conditions.

Cellulose, the most abundant polysaccharide in nature, is a rich source of renewable energy and sustains soil nutrients. Among the microorganisms known to degrade cellulose, bacteria are less studied compared to fungi. In the present work, we have investigated the culturable bacteria actively involved in cellulose degradation in forest and crop field soils. Based on clear zone formation and enzyme activity assay, we identified 7 bacterial strains positive for cellulose degradation. Of these, two most efficient strains (Bacillus cereus strains BHU1 and BHU2) were selected for whole genome sequencing, annotation, and information regarding GC content, number of genes, total subsystems, starch, and cellulose degradation pathways. Average nucleotide identity (ANI) showed more than 90% similarity between both the strains (BHU1 and BHU2) and with B. cereus ATCC 14579. Both the strains have genes and enzyme families like endoglucanase and β-glucosidase as evident from whole genome sequence. Cellulase containing gene families (GH5, GH8, GH1), and many other carbohydrate-degrading enzymes, were present in both the bacterial strains. Taken together, the results suggest that the strains were efficient in cellulose degradation, and can be used for energy generation and production of value-added product.

This study aimed to recover metagenome-assembled genomes (MAGs) from human fecal samples to characterize the glycosidase profiles of Bifidobacterium species exposed to different prebiotic oligosaccharides (galacto-oligosaccharides, fructo-oligosaccharides and human milk oligosaccharides, HMOs) as well as high-fiber diets. A total of 1806 MAGs were recovered from 487 infant and adult metagenomes. Unsupervised and supervised classification of glycosidases codified in MAGs using machine-learning algorithms allowed establishing characteristic hydrolytic profiles for B. adolescentis, B. bifidum, B. breve, B. longum and B. pseudocatenulatum, yielding classification rates above 90%. Glycosidase families GH5 44, GH32, and GH1B. bifidum. The presence or absence of GH1, GH2, GH5 and GH20 was characteristic of B. adolescentis, B. breve and B. pseudocatenulatum, while families GH1 and GH30 were relevant in MAGs from B. longum. These characteristic profiles allowed discriminating bifidobacteria regardless of prebiotic exposure. Correlation analysis of glycosidase activities suggests strong associations between glycosidase families comprising HMOs-degrading enzymes, which are often found in MAGs from the same species. Mathematical models here proposed may contribute to a better understanding of the carbohydrate metabolism of some common bifidobacteria species and could be extrapolated to other microorganisms of interest in future studies.

Keywords: bifidobacteria; glycosidase; machine learning; metagenome-assembled genomes; metagenomics; prebiotics.

Genetic disorders have been shown to co-occur in individual patient. A Thai boy with features of osteogenesis imperfecta (OI) and combined pituitary hormone deficiency (CPHD) was identified. The causative mutations were investigated by whole exome and Sanger sequencing. Pathogenicity and pathomechanism of the variants were studied by luciferase assay. The proband was found to harbor a novel de novo heterozygous missense mutation, c.1531G > T (p.G511C), in COL1A2 leading to OI and a heterozygous missense variant, c.364C > T (p.R122W), in LHX4. The LHX4 p.R122W has never been reported to cause CPHD. The variant was predicted to be deleterious and found in the highly conserved LIM2 domain of LHX4. The luciferase assays revealed that the p.R122W was unable to activate POU1F1, GH1, and TSHB promoters, validating its pathogenic effect in CPHD. Moreover, the variant did not alter the function of wild-type LHX4, indicating its hypomorphic pathomechanism. In conclusion, the novel de novo heterozygous p.G511C mutation in COL1A2 and the heterozygous pathogenic p.R122W mutation in LHX4 were demonstrated in a patient with OI and CPHD. This study proposes that the mutations in two different genes should be sought in the patients with clinical features unable to be explained by a mutation in one gene.