The conformational itineraries taken by carbohydrate residues in the catalytic subsite of retaining glycoside hydrolases (GHs), harness the link between substrate conformation and reactivity. GHs' active sites may be described as a combination of subsites dedicated to the binding of individual sugar residues and to catalysis. The three-dimensional structure of GH:carbohydrate complexes has demonstrated that carbohydrate ring conformation changes in an ordered manner during catalysis. Here we demonstrate in silico that a link exists between subsite binding dynamics and substrate specificity for β-galactosidases from clan GH-A families GH1, GH2, GH35, GH42 and GH59. Different oligosaccharides were docked in the active site of reference β-galactosidase structures using Vina-Carb. Subsequent molecular dynamics (MD) simulations revealed that these enzymes favor a high degree of flexibility and ring distortion of the substrate the lytic subsite -1. Although the β-galactosidase families examined are structurally and mechanistically related, distinct patterns of ring distortion were unveiled for the different families. For β-galactosidases, three different family-dependent reaction itineraries (¹S₃ → ⁴H₃^‡ → ⁴C₁, ^1,4B → ⁴H₃/ ⁴E^‡ → ⁴C₁, and ¹S₅ → ⁴E/ ⁴H₅^‡ → ⁴C₁) were identified, all compatible with the antiperiplanar lone pair hypothesis (ALPH) for the hydrolysis of β-glycosides. This comparative study reveals the fuzzy character of the changes in carbohydrate ring geometry prior to carbohydrate hydrolysis.

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus: a GH1 β-gluco-/β-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of β-gluco-/β-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan.

Keywords: Saccharolobus solfataricus; archaea; glycoside hydrolases; polysaccharide degradation; xyloglucan.

Antibacterial peptides or bacteriocins produced by many strains of lactic acid bacteria have been used as food preservatives for many years without any known adverse effects. Bacteriocin titres can be modified by altering the physiological and nutritional factors of the producing bacterium to improve the production in terms of yield and productivity. The effects of culture conditions (initial pH, inoculum age and inoculum size) and medium compositions (organic and inorganic nitrogen sources; carbon sources) were assessed for the production of bacteriocin-like inhibitory substances (BLIS) by Lactococcus lactis Gh1 in shake flask cultures. An inoculum of the mid-exponential phase culture at 1% (v/v) was the optimal age and size, while initial pH of culture media at alkaline and acidic state did not show a significant impact on BLIS secretion. Organic nitrogen sources were more favourable for BLIS production compared to inorganic sources. Production of BLIS by L. lactis Gh1 in soytone was 1.28-times higher as compared to that of organic nitrogen sources ((NH₄)2SO₄). The highest cell concentration (X_mX = 0.69 ± 0.026 g·L^-1) and specific growth rate (μ_max = 0.14 h^-1) were also observed in cultivation using soytone. By replacing carbon sources with fructose, BLIS production was increased up to 34.94% compared to BHI medium, which gave the biomass cell concentration and specific growth rate of 0.66 ± 0.002 g·L^-1 and 0.11 h^-1, respectively. It can be concluded that the fermentation factors have pronounced influences on the growth of L. lactis Gh1 and BLIS production. Results from this study could be used for subsequent application in process design and optimisation for improving BLIS production by L. lactis Gh1 at larger scale.

Keywords: Lactococcus lactis; bacteriocin; culture conditions; fermentation; medium compositions.

Determination of a microbial strain for the joining into sustenance items requires both in vitro and in vivo assessment. A newly isolated bacteriocin-like inhibitory substance (BLIS) producing lactic acid bacterium, Lactococcus lactis Gh1, was isolated from a traditional flavour enhancer and evaluated in vitro for its potential applications in the food industry. Results from this study showed that L. lactis was tolerant to NaCl (≤ 4.0%, w/v), phenol (≤ 0.4%, w/v), 0.3% (w/v) bile salt, and pH 3. BLIS from L. lactis showed antimicrobial activity against Listeria monocytogenes ATCC 15313 and was susceptible to 10 types of antibiotics. The absence of haemolytic activity and the presence of acid phosphatase and naphthol-AS-BI-phosphohydrolase were observed in L. lactis. L. lactis could coagulate milk and showed a negative response to amylolytic and proteolytic activities and did not secrete β-galactosidase. The antimicrobial activity of BLIS was completely abolished at 121 °C. The BLIS was conserved at 4 °C in BHI and MRS medium up to 6-4 months, respectively. BLIS activity was more stable in BHI as compared to MRS after four freeze-thaw cycles and was not affected by a wide range of pH (pH 4-8). BLIS was sensitive to proteinase k and resistant to catalase and trypsin. The antimicrobial activity was slightly reduced by acetone, ethanol, methanol, and acetonitrile at 10% (v/v) and also towards Tween-80, urea, and NaCl 1% (v/v). Results from this study have demonstrated that L. lactis has a vast potential to be applied in the food industry, such as for the preparation of starter culture, functional foods, and probiotic products.

Keywords: Bacteriocins; Cell-free supernatant; Characterization; Food industry; Lactic acid bacteria; Lactococcus lactis.

Woody biomass represents an important source of carbon on earth, and its global recycling is highly dependent on Agaricomycetes fungi. White-rot Basidiomycetes are a very important group in this regard, as they possess a large and diverse enzymatic repertoire for biomass decomposition. Among these enzymes, the recently discovered lytic polysaccharide monooxygenases (LPMOs) have revolutionized biomass processing with their novel oxidative mechanism of action. The strikingly high representation of LPMOs in fungal genomes raises the question of their functional versatility. In this work, we studied an AA9 LPMO from the white-rot basidiomycete Pycnoporus sanguineus, PsAA9A. Successfully produced as a recombinant secreted protein in Pichia pastoris, PsAA9A was found to be a C1-specific LPMO active on cellulosic substrates, generating native and oxidized cello-oligosaccharides in the presence of an external electron donor. PsAA9A boosted cellulolytic activity of glysoside hydrolases from families GH1, GH5, and GH6.This study serves as a starting point towards understanding the functional versatility and biotechnological potential of this enzymatic family, highly represented in wood decay fungi, in Pycnoporus genus. KEY POINTS: • PsAA9A is the first AA9 from P. sanguineus to be characterized. • PsAA9A has activity on cellulose, producing C1-oxidized cello-oligosaccharides. • Boosting activity with GH1, GH5, and GH6 was proven.

Keywords: AA9; CELLULOSE; LPMO; Pycnoporus.

Despite intensive searches, few proteins involved in telomere homeostasis have been identified in plants. Here, we used pull-down assays to identify potential telomeric interactors in the model plant species Arabidopsis (Arabidopsis thaliana). We identified the candidate protein GH1-HMGA1 (also known as HON4), an uncharacterized linker histone protein of the High Mobility Group Protein A (HMGA) family in plants. HMGAs are architectural transcription factors and have been suggested to function in DNA damage repair, but their precise biological roles remain unclear. Here, we show that GH1-HMGA1 is required for efficient DNA damage repair and telomere integrity in Arabidopsis. GH1-HMGA1 mutants exhibit developmental and growth defects, accompanied by ploidy defects, increased telomere dysfunction-induced foci, mitotic anaphase bridges, and degraded telomeres. Furthermore, mutants have a higher sensitivity to genotoxic agents such as mitomycin C and γ-irradiation. Our work also suggests that GH1-HMGA1 is involved directly in the repair process by allowing the completion of homologous recombination.

Recessive mutations in GHRHR are associated with severe isolated growth hormone deficiency (IGHD), with a final height in untreated patients of 130 cm ± 10 cm (-7.2 ± 1.6 SDS; males) and 114 ± 0.7 cm (-8.3 ± 0.1 SDS; females).

We hypothesized that a consanguineous Pakistani family with IGHD in three siblings (two males, one female) would have mutations in GH1 or GHRHR.

Two novel homozygous missense variants [c.11G>A (p.R4Q), c.236C>T (p.P79L)] at conserved residues were identified in all three siblings. Both were absent from control databases, aside from pR4Q appearing once in heterozygous form in the Exome Aggregation Consortium Browser. The brothers were diagnosed with GH deficiency at 9.8 and 6.0 years (height SDS: -2.24 and -1.23, respectively), with a peak GH of 2.9 μg/liter with low IGF-1/IGF binding protein 3. Their sister presented at 16 years with classic GH deficiency (peak GH <0.1 μg/liter, IGF-1 <3.3 mmol/liter) and attained an untreated near-adult height of 144 cm (-3.0 SDS); the tallest untreated patient with GHRHR mutations reported. An unrelated Pakistani female IGHD patient was also compound homozygous. All patients had a small anterior pituitary on magnetic resonance imaging. Functional analysis revealed a 50% reduction in maximal cAMP response to stimulation with GHRH by the p.R4Q/p.P79L double mutant receptor, with a 100-fold increase in EC50.

We report the first coexistence of two novel compound homozygous GHRHR variants in two unrelated pedigrees associated with a partial loss of function. Surprisingly, the patients have a relatively mild IGHD phenotype. Analysis revealed that the pP79L mutation is associated with the compromise in function, with the residual partial activity explaining the mild phenotype.

Mutations affecting exon 3 splicing are the main cause of autosomal dominant Isolated GH Deficiency II (IGHDII) by increasing the level of exon 3-skipped mRNA encoding the functionally inactive dominant-negative 17.5-kDa isoform. The exons and introns of the gene encoding GH (GH1) were screened for the presence of mutations in 103 sporadic isolated GH deficiency cases. Four different variations within exon 3 were identified in 3 patients. One carried c.261C>T (p.Pro87Pro) and c.272A>T (p.Glu91Val), the second c.255G>A (p.Pro85Pro) and c.261 C>T, and the third c.246G>C (p.Glu82Asp). All the variants were likely generated by gene conversion from an homologous gene in the GH1 cluster. In silico analysis predicted that positions c.255 and c.272 were included within 2 putative novel exon splicing enhancers (ESEs). Their effect on splicing was confirmed in vitro. Constructs bearing these 2 variants induced consistently higher levels both of transcript and protein corresponding to the 17.5-kDa isoform. When c.255 and c.272 were combined in cis with the c.261 variant, as in our patients, their effect was weaker. In conclusion, we identified 2 variations, c.255G>A and c.272A>T, located in 2 novel putative exon splicing enhancers and affecting GH1 splicing in vitro by increasing the production of alternatively spliced isoforms. The amount of aberrant isoforms is further regulated by the presence in cis of the c.261 variant. Thus, our results evidenced novel putative splicing regulatory elements within exon 3, confirming the crucial role of this exon in mRNA processing.

Human (h) growth hormone (GH) production studies are largely limited to effects on secretion. How pituitary hGH gene (hGH-N/GH1) expression is regulated is important in our understanding of the role hGH plays in physiology and disease. Here we assess for the first time the effect of sleep deprivation (SD) and high-fat diet (HFD) on hGH-N expression in vivo using partially humanized 171hGH/CS transgenic (TG) mice, and attempted to elucidate a role for DNA methylation. Activation of hGH-N expression requires interactions between promoter and upstream locus control region (LCR) sequences including pituitary-specific hypersensitive site (HS) I/II. Both SD and diet affect hGH secretion, but the effect of SD on hGH-N expression is unknown. Mice fed a HFD or regular chow diet for three days underwent SD (or no SD) for 6 hours at zeitgeber time (ZT) 3. Serum and pituitaries were assessed over 24 hours at 6-hour intervals beginning at ZT 14. SD and HFD caused significant changes in serum corticosterone and insulin, as well as hGH and circadian clock-related gene RNA levels. No clear association between DNA methylation and the negative effects of SD or diet on hGH RNA levels was observed. However, a correlation with increased methylation at a CpG (cytosine paired with a guanine) in a putative E-box within the hGH LCR HS II was suggested in situ. Methylation at this site also increased BMAL1/CLOCK-related nuclear protein binding in vitro. These observations support an effect of SD on hGH synthesis at the level of gene expression.

Adaptation to a bamboo diet is an essential process for giant panda growth, and gut microbes play an important role in the digestion of the polysaccharides in bamboo. The dietary transition in giant panda cubs is particularly complex, but it is an ideal period in which to study the effects of gut microbes on polysaccharide use because their main food changes from milk to bamboo (together with some bamboo shoot and coarse pastry). Here, we used 16S rDNA and internal transcribed spacer 1 (ITS1) DNA sequencing and metagenomic sequencing analysis to investigate the succession of the gut microbial structure in feces sampled from twin giant panda cubs during the completely dietary transition and determine the abundances of polysaccharide-metabolizing genes and their corresponding microbes to better understand the degradation of bamboo polysaccharides. Successive changes in the gut microbial diversity and structure were apparent in the growth of pandas during dietary shift process. Microbial diversity increased after the introduction of supplementary foods and then varied in a complex way for 1.5-2 years as bamboo and complex food components were introduced. They then stabilized after 2 years, when the cubs consumed a specialized bamboo diet. The microbes had more potential to metabolize the cellulose in bamboo than the hemicellulose, providing genes encoding cellulase systems corresponding to glycoside hydrolases (GHs; such as GH1, GH3, GH5, GH8, GH9, GH74, and GH94). The cellulose-metabolizing species (or genes) of gut bacteria was more abundant than that of gut fungi. Although cellulose-metabolizing species did not predominate in the gut bacterial community, microbial interactions allowed the giant pandas to achieve the necessary dietary shift and ultimately adapt to a bamboo diet.

Keywords: Clostridium; GH5; cellulose degradation; functional gene prediction; giant panda cub; gut microbes; succession.

Human cytomegalovirus (HCMV) is a common pathogen that causes persistent infections in immune deficient patients and results in significant morbidity and mortality, particularly among transplant recipients and children. Different HCMV glycoprotein H (gH) genotypes may cause different diseases and affect the severity of these diseases. To develop a sensitive quantitative real-time PCR assay that could rapidly distinguish between two HCMV gH genotypes, primers were designed to target the conserved region of the gH gene. gH1 and gH2 probes were designed to target the two variable regions. Standard HCMV strains (AD169 and TOWNE) and 203 clinical urine samples from HCMV infected children were used for the present study. Based on the primer-probe set used to detect the target gH gene segment of HCMV, our quantitative real-time PCR assay specifically discriminated between HCMV gH1 and gH2 with a detection limit of approximately 10(2) viral copies/ml. Among the 203 clinical urine samples tested, 145 were gH1 positive, 56 were gH2 positive, and 2 were positive for both. Thus, we developed a gH gene-based real time-PCR method that could rapidly, stably, and specifically distinguish between two HCMV gH genotypes. We found HCMV gH1 to be common among children examined in Zhejiang, China.

Human cytomegalovirus (HCMV) is the leading cause of congenital infection and an opportunistic pathogen capable of establishing lifelong latency. In the present study, we aimed to investigate the distribution of glycoprotein B, H, and N in infants of Shanghai and correlate the genotype with active and latent HCMV infection. A total of 129 urine samples were collected between August 2014 and December 2015 from infants under 3 years with HCMV infection. Nested PCR was used to amplify the regions of UL55 (gB), UL75 (gH), and UL73 (gN). Gene sequencing and phylogenetic analyses were used to classify the genotypes. Overall, regarding gB, gB1 (57.27%) was predominant, followed by gB3 (41.82%) and gB4 (0.91%). gH1 (54.33%) was the most prevalent genotype of gH, followed by gH2 (45.67%). Concerning gN, we detected gN1 (17.44%), gN2 (2.33%), gN3a (29.07%), gN3b (8.14%), gN4a (13.95%), gN4b (15.12%), and gN4c (13.95%), among which gN3a was the dominant genotype. All the expected genotypes were present except gB2 in children with active infection: gB1 (56.25%), gB3 (42.5%), gB4 (1.25%), gH1 (58.70%), gH2 (41.30%), gN1 (19.05%), gN2 (3.17%), gN3a (25.40%), gN3b (6.35%), gN4a (15.87%), gN4b (17.46%), and gN4c (12.70%). However, among latent cases, we detected gB1 (60%), gB3 (40%), gH1 (42.86%), gH2 (57.14%), gN1 (13.04%), gN3a (39.13%), gN3b (13.04%), gN4a (8.70%), gN4b (8.70%), and gN4c (17.39%), respectively. gB2, gB4, and gN2 were absent in this group. The results revealed that gB1, gH1, and gN3a were predominant in the infants of Shanghai. gH showed different trends among children with active and latent infection.

Keywords: gene polymorphisms; glycoprotein B; glycoprotein H; glycoprotein N; human cytomegalovirus.

UNASSIGNED

To determine the prevalence of glycoprotein B (gB), glycoprotein N (gN), and glycoprotein H (gH) genotypes of human cytomegalovirus (HCMV) superimposed on ulcerative colitis (UC) patients in Japan.

UNASSIGNED

Four archived stool samples and 7-archived extracted DNA from stool samples of 11 UC patients with positive multiplex polymerase chain reaction (PCR) results for HCMV were used UL55 gene encoding gB, UL73 gene encoding gN, and UL75 gene encoding gH were identified by PCR. Genotypes of gB and glycoprotein N were determined by sequencing.

UNASSIGNED

Among 11 samples, 8 samples were amplified through PCR. gB, gN, and gH genotypes were successfully detected in 3 of 8 (37.5%), 4 of 8 (50%), and 8 of 8 (100%), respectively. The distribution of gB and gN genotypes analyzed through phylogenetic analysis were as follows: gB1 (2/3, 66.7%), gB3 (1/3, 33.3%), gN3a (2/4, 50%), and gN3b (2/4, 50%). Other gB genotypes (gB2 and gB4) and gN genotypes (gN1, gN2, and gN4) were not detected in this study. Out of successfully amplified 8 samples of gH genotype, gH1 and gH2 were distributed in 12.5% and 75% samples, respectively. Only 1 sample revealed mixed infection of gH genotype. The distribution of gH1 and gH2 differed significantly (1:6, P<0.05) in UC patients. The distribution of single gH genotype also revealed significant difference in UC patients who were treated with immunosuppressive drug (P<0.05).

UNASSIGNED

In this study, gB1, gN3, and gH2 gene were determined as the most frequently observed genotypes in UC patients, which suggest that there might be an association between these genotypes of HCMV and UC.

Β-glucosidases are key enzymes used in second-generation biofuel production. They act in the last step of the lignocellulose saccharification, converting cellobiose in glucose. However, most of the β-glucosidases are inhibited by high glucose concentrations, which turns it a limiting step for industrial production. Thus, β-glucosidases have been targeted by several studies aiming to understand the mechanism of glucose tolerance, pH and thermal resistance for constructing more efficient enzymes. In this paper, we present a database of β-glucosidase structures, called Glutantβase. Our database includes 3842 GH1 β-glucosidase sequences collected from UniProt. We modeled the sequences by comparison and predicted important features in the 3D-structure of each enzyme. Glutantβase provides information about catalytic and conserved amino acids, residues of the coevolution network, protein secondary structure, and residues located in the channel that guides to the active site. We also analyzed the impact of beneficial mutations reported in the literature, predicted in analogous positions, for similar enzymes. We suggested these mutations based on six previously described mutants that showed high catalytic activity, glucose tolerance, or thermostability (A404V, E96K, H184F, H228T, L441F, and V174C). Then, we used molecular docking to verify the impact of the suggested mutations in the affinity of protein and ligands (substrate and product). Our results suggest that only mutations based on the H228T mutant can reduce the affinity for glucose (product) and increase affinity for cellobiose (substrate), which indicates an increment in the resistance to product inhibition and agrees with computational and experimental results previously reported in the literature. More resistant β-glucosidases are essential to saccharification in industrial applications. However, thermostable and glucose-tolerant β-glucosidases are rare, and their glucose tolerance mechanisms appear to be related to multiple and complex factors. We gather here, a set of information, and made predictions aiming to provide a tool for supporting the rational design of more efficient β-glucosidases. We hope that Glutantβase can help improve second-generation biofuel production. Glutantβase is available at http://bioinfo.dcc.ufmg.br/glutantbase .

Keywords: Biofuel; GH1; Glucose-tolerant; Rational design of enzymes; Thermostability; β-Glucosidases.

β-glucosidase hydrolyzes the β-1,4 linkage of cellobiose, a product generated from the action of endoglucanase and cellobiohydrolase on cellulose, and generates glucose. Accumulated glucose during saccharification leads to product inhibition of β-glucosidase, which in turn cause an accumulation of cellobiose and inhibition of other cellulolytic enzymes. Thus, glucose tolerant and active β-glucosidase is required for the efficient saccharification of biomass. O08324 is a glucose tolerant β-glucosidase isolated from archaeon Thermococcus sp. which shows no loss in enzyme specific activity in the presence of up to 4 M glucose and is active at 78 °C. Since O08324 has such high glucose tolerance, knowing the rationale for glucose tolerance will be helpful in engineering glucose tolerant β-glucosidase. In the present study, we designed mutations at eleven sites across the gatekeeper, aglycone, and glycone region. Based on the kinetic studies of O08324 mutants, the gatekeeper residues at positions 160, 166, 167, 168, and the aglycone binding residue 156 were identified to play a role in glucose inhibition. However, only residues at the tunnel entrance, and not all gatekeeper residues contribute to glucose tolerance. This study sheds some light on the unusual glucose tolerance of O08321 archaeal GH1 β-glucosidase.

A GH1 β-glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4'-galactosyllactose, a tri-galactooligosaccharide, at 3.0 å and 2.1 å resolutions, respectively. These structures reveal details of the structural elements that define the catalytic activity and substrate binding of HsBglA, and provide a possible interpretation for its high catalytic potency for transgalactosylation reaction. This article is protected by copyright. All rights reserved.

Keywords: Hamamotoa singularis; crystal structure; galactooligosaccharide; transgalactosylation; β-galactosidase; β-glucosidase.

Marine microalgae are promising sources of novel glycoside hydrolases (GHs), which have great value in biotechnical and industrial applications. Although many GH1 family β-glucosidases have been extensively studied, studies on β-glucosidases from microalgae are rare, and no structure of algal GH1 β-glucosidase has been reported. Here, we report the biochemical and structural study of a GH1 β-glucosidase BGLN1 from Nannochloropsis oceanica, an oleaginous microalga. Phylogenetic analysis of BGLN1, together with the known structures of GH1 β-glucosidases, has indicated that BGLN1 is branched at the root of the eukaryotic part of the phylogenetic tree. BGLN1 showed higher activity against laminaribiose compared to cello-oligosaccharides. Unlike most of the other GH1 β-glucosidases, BGLN1 is partially inhibited by metal ions. The crystal structure of BGLN1 revealed that BGLN1 adopts a typical (α/β)₈-barrel fold with variations in loops and N-terminal regions. BGLN1 contains extra residues at the N-terminus, which are essential for maintaining protein stability. BGLN1 has a more acidic substrate-binding pocket than other β-glucosidases, and the variations beyond the conserved -1 site determine the substrate specificity. These results indicate that GH enzymes from microalgae may have unique structural and functional features, which will provide new insight into carbohydrate synthesis and metabolism in marine microalgae.

Keywords: Laminaribiose; Microalgae; β-Glucosidase.

Lignocellulosic waste has offered a cost-effective and food security-wise substrate for the generation of biofuels and value-added products. Here, whole-genome sequencing and comparative genomic analyses were performed for Serratia sp. AXJ-M. The results showed that strain AXJ-M contained a high proportion of strain-specific genes related to carbohydrate metabolism. Furthermore, the genetic basis of strain AXJ-M for efficient degradation of cellulose was identified. Cellulase activity tests revealed strong cellulose degradation ability and cellulase activities in strain AXJ-M. mRNA expression indicated that GH1, GH3 and GH8 might determine the strain's cellulose degradation ability. The SWISS-MODEL and Ramachandran Plot were used to predict and evaluate the 3D structure, respectively. High performance liquid chromatography (HPLC) and gas chromatography-mass spectrometer (GC-MS) were used to analyze the cellulose degradation products. Further research is needed to elucidate the cellulose degradation mechanism and to develop industrial applications for lignocellulosic biomass degradation and waste management.

Keywords: Biodegradation; Cellulose; Genomic and comparative genomic analysis; Glycoside hydrolase family; Lignocellulosic wastes.

β-glucosidase causes hydrolysis of β-1,4-glycosidic bond in glycosides and oligosaccharides. It is an industrially important enzyme owing to its potential in biomass processing applications. In this study, computational screening of an extreme temperature aquatic habitat metagenomic resource was done, leading to the identification of a novel gene, bgl_M, encoding a β-glucosidase. The comparative protein sequence and homology structure analyses designated it as a GH1 family β-glucosidase. The bgl_M gene was expressed in a heterologous host, Escherichia coli. The purified protein, Bgl_M, was biochemically characterized for β-glucosidase activity. Bgl_M exhibited noteworthy hydrolytic potential towards cellobiose and lactose. Bgl_M, showed substantial catalytic activity in the pH range of 5.0-7.0 and at the temperature 40 °C-70 °C. The enzyme was found quite stable at 50 °C with a loss of hardly 20% after 40 h of heat exposure. Furthermore, any drastically negative effect was not observed on the enzyme's activity in the presence of metal ions, non-ionic surfactants, metal chelating, and denaturing agents. A significantly high glucose tolerance, retaining 80% relative activity at 1 M, and 40% at 5 M glucose, and ethanol tolerance, exhibiting 80% relative activity in 10% ethanol, enrolled Bgl_M as a promising enzyme for cellulose saccharification. Furthermore, its ability to catalyze the hydrolysis of daidzin and polydatin ascertained it as an admirably suited biocatalyst for enhancement of nutritional values in soya and wine industries.

Keywords: Bioflavoring; Cellobiose; Ethanol-tolerant; Glucose-tolerant; Lactose; β-glucosidase.

β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent K_i,_Glc = 686 mM). We report our steady-state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose-protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

CircRNAs have been identified to be expressed differently and stably in numerous species and tissues, but their functions in growth hormone (GH) secretion are still largely unknown. In summary, we have revealed a circRNA-miRNA-mRNA network that may play a biological role in the rat pituitary gland. First, we verified the chromosome location information of circAgtpbp1 according to sequencing analysis. The circAgtpbp1 characteristics were authenticated through PCR, qRT-PCR, treating with RNase and fluorescent in situ hybridization (FISH). Second, we detected the expression pattern of circAgtpbp1 in the rat anterior pituitary by qRT-PCR. We also designed circAgtpbp1 siRNA and constructed overexpression plasmid to evaluate the effect of circAgtpbp1 function on GH secretion by qRT-PCR, ELISA and Western blot. CircAgtpbp1 is a stable, truly circular molecule. We found that circAgtpbp1 interacted with miR-543-5p and can regulate GH secretion in pituitary cells through a circAgtpbp1-miR-543-5p-GH axis. Overall, the evidence generated by our study suggests that circAgtpbp1 can act as a sponge of miR-543-5p to reduce the inhibitory effect of miR-543-5p on Gh1 and further promote GH secretion. These findings expand our existing knowledge on the mechanisms of hormone regulation in the pituitary gland.

Keywords: GH; animal growth; circRNA; miRNA sponge; pituitary.