β-galactosidases (EC 3.2.1.23) catalyze the hydrolysis of β-galactosidic bonds in oligosaccharides and, under certain conditions, transfer a sugar moiety from a glycosyl donor to an acceptor. Cold-active β-galactosidases are identified in microorganisms endemic to permanently low-temperature environments. While mesophilic β-galactosidases are broadly studied and employed for biotechnological purposes, the cold-active enzymes are still scarcely explored, although they may prove very useful in biotechnological processes at low temperature. This review covers several issues related to cold-active β-galactosidases, including their classification, structure and molecular mechanisms of cold adaptation. Moreover, their applications are discussed, focusing on the production of lactose-free dairy products as well as on the valorization of cheese whey and the synthesis of glycosyl building blocks for the food, cosmetic and pharmaceutical industries.

Keywords: GH1; GH2; GH35; GH42; galacto-oligosaccharides; lactose hydrolysis; psychrophilic enzymes.

Background: β-mannanase can hydrolyze β-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, β-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. β-mannanase is widespread in various organisms, however, microorganisms are the main source of β-mannanases. Microbial β-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial β-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce β-mannanase, which shows more advantages than traditional microbial fermentation in various aspects.

Results: A β-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The β-mannanase activity was activated by low concentration of Co²⁺, Mn²⁺, Zn²⁺, Ba²⁺ and Ca²⁺. Man1E showed the highest affinity for Locust bean gum (LBG). The K_m and V_max values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 μmol/(mL min), respectively.

Conclusions: A new type of β-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported β-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.

Keywords: CBM; Enterobacter aerogenes; Enzymatic characterization; GH1 family; β-mannanase.

Dalcochinase and Abg are glycoside hydrolase family 1 β-glucosidases from Dalbergia cochinchinensis Pierre and Agrobacterium sp., respectively, with 35% sequence identity. However, Abg shows much higher catalytic efficiencies toward a broad range of glycone substrates than dalcochinase does, possibly due to the difference in amino acid residues around their glycone binding pockets. Site-directed mutagenesis was used to replace the amino acid residues of dalcochinase with the corresponding residues of Abg, generating three single mutants, F196H, S251V, and M369E, as well as the corresponding three double mutants and one triple mutant. Among these, the F196H mutant showed increases in catalytic efficiency toward almost all glycoside substrates tested, with the most improved catalytic efficiency being a 3-fold increase for hydrolysis of p-nitrophenyl β-D-mannoside, suggesting a preferred polar residue at this position and consistent with the presence of histidine at this position in two other GH1 glycosidases from barley and rice that prefer β-mannosides. In addition, the M369E mutation resulted in a small increase in catalytic efficiency for cleavage of p-nitrophenyl β-D-galactoside. By contrast, the multiple mutants were up to 8-fold less efficient than the recombinant wild-type dalcochinase, and displayed primarily antagonistic interactions between these residues. Thus, differences in catalytic efficiency between dalcochinase and Abg are therefore not primarily due to differences in the residues that directly contact the substrate, but derive largely from contributions from more remote residues and the overall architecture of the active site.

Phosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PI-dependent modulation remain largely elusive, the effects of disturbed PI metabolism could be employed to identify regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice (Oryza sativa). Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related proteins 2 and 3 (Arp2/3) complex-nucleated actin branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effects in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulting from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering cell development. These findings imply that the Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing insights into the relationship between PI homeostasis and plant growth and development.

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni2+ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a Km of 0.55 mM, a Vmax of 83.6 U/mg, a kcat of 74.3 s-1 and kcat/Km of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.

Tea (Camellia sinensis) aroma is an important factor affecting tea quality. Many tea aroma compounds are present as glycosidically conjugated forms in tea leaves, and can be hydrolyzed by β-glucosidase (β-Glu) and β-primeverosidase to release free tea aromas. β-Primeverosidase has been identified and functionally characterized, while β-Glu has not been identified in tea leaves. In the present study, we established a yeast expression system to recombine CsGH1BG1, CsGH3BG1, and CsGH5BG1, which belonged to GH1, GH3, and GH5 families in plants, respectively. These three recombinant Csβ-Glus hydrolyzed the β-glucopyranosidically conjugated aromas to form free aromas, suggesting that there was no specific Csβ-Glus for the hydrolysis of β-glucopyranosidically conjugated aromas in vitro. Furthermore, subcellular localization of the Csβ-Glus indicated that CsGH1BG1 and CsGH3BG1 were located in the cytosol and vacuole, respectively, while CsGH5BG1 was located in the cell wall. This suggested that CsGH1BG1 and CsGH3BG1 might be responsible for the hydrolysis of β-glucopyranosidically conjugated aromas in tea leaves during the tea manufacturing process. This study provides the first evidence of Csβ-Glus in tea leaves, and will advance understanding of tea aroma formation.

BACKGROUND

In the search for biomarkers that allow the prediction of neonatal growth and development, placental growth hormone(PGH), pituitary growth hormone (GH1), insulin-like growth factor 1 (IGF-1), and ghrelin concentrations were assessed in the amniotic fluid and in the umbilical cord blood of 92 neonates.

METHODS

The proteins were assayed by the ELISA method. Their concentration values were compared in 57 full-term neonates and 35 prematurely born neonates, as well as in both large >> 4,000 g) and small neonates (< 2,500 g). Also, body mass and placenta mass were compared.

RESULTS

Statistically significant differences both between prematurely born neonates and full-term neonates and between large and small neonates were obtained only in terms of the body mass of neonates and placenta mass. The concentration values of the hormones studied did not show statistically significant differences. A distinct tendency was noticed towards an increase in PGH concentration in both prematurely born and small neonates. In large neonates, statistically significantly higher IGF-1 concentrations were found compared to the prematurely born neonates.

CONCLUSIONS

Our studies indicate an important role for PGH in maintaining a proper IGF-1 pool and demonstrate the existence of a direct influence on the function of the placenta in prematurely born neonates through the activation of compensation mechanisms,which stimulate IGF-1 synthesis.

β-Glucosidases primarily catalyze removal of terminal glucosyl residues from a variety of glucoconjugates and also perform transglycosylation and reverse hydrolysis. These catalytic properties can be readily exploited for degradation of lignocellulosic biomass as well as for pharmaceutical, food and flavor industries. β-Glucosidases have been either isolated in the native form from the producer organism or recombinantly expressed and gaged for their biochemical properties and substrate specificities. Although almond and Aspergillus niger have been instantly recognizable sources of β-glucosidases utilized for various applications, an intricate pool of novel β-glucosidases from different sources can provide their potent replacements. Moreover, one can envisage the better efficacy of these novel candidates in biofuel and biorefinery industries facilitating efficient degradation of biomass. This article reviews properties of the novel β-glucosidases such as glucose tolerance and activation, substrate specificity, and thermostability which can be useful for their applications in lignocellulose degradation, food industry, and pharmaceutical industry in comparison with the β-glucosidases from the conventional sources. Such β-glucosidases have potential for encouraging white biotechnology.

Keywords: Cold active; Flavor enhancement; GH1; Glucose tolerant; Lignocellulose; β-glucosidase.

A novel GH1 β-glucosidase gene (bgla) from marine bacterium was sequenced and expressed in Escherichia coli. After purification by Ni²⁺ affinity chromatography, the recombinant protein was characterized. The purified recombinant enzyme showed maximum activity at 40 °C, pH 7.5 and was stable between temperatures that range from 4 to 30 °C and over the pH range of 6-10. The enzyme displayed a high tolerance to glucose and maximum stimulation at the presence of 100 mM glucose. To improve glucose tolerance of the enzyme, a site-directed mutation (f171w) was introduced into β-glucosidase. The recombinant F171W showed a higher glucose tolerance than the wild type and maintained more than 40% residual activity at the presence of 4 M glucose. Additionally, the recombinant enzymes showed notable tolerance to ethanol. These properties suggest the enzymes may have potential applications for the fermentation of lignocellulosic sugars and the production of biofuels.

Keywords: Ethanol tolerance; Glucose tolerance; Marine bacterium; Site-directed mutation; β-Glucosidase.

Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, β-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast.

Background: The advent of genomic information and the reduction in the cost of genotyping have led to the use of genomic information to estimate genomic inbreeding as an alternative to pedigree inbreeding. Using genomic measures, effects of genomic inbreeding on production and fertility traits have been observed. However, there have been limited studies on the specific genomic regions causing the observed negative association with the trait of interest. Our aim was to identify unique run of homozygosity (ROH) genotypes present within a given genomic window that display negative associations with production and fertility traits and to quantify the effects of these identified ROH genotypes.

Methods: In total, 50,575 genotypes based on a 50K single nucleotide polymorphism (SNP) array and 259,871 pedigree records were available. Of these 50,575 genotypes, 46,430 cows with phenotypic records for production and fertility traits and having a first calving date between 2008 and 2018 were available. Unique ROH genotypes identified using a sliding-window approach were fitted into an animal mixed model as fixed effects to determine their effect on production and fertility traits.

Results: In total, 133 and 34 unique ROH genotypes with unfavorable effects were identified for production and fertility traits, respectively, at a 1% genome-wise false discovery rate. Most of these ROH regions were located on bovine chromosomes 8, 13, 14 and 19 for both production and fertility traits. For production traits, the average of all the unfavorably identified unique ROH genotypes effects were estimated to decrease milk yield by 247.30 kg, fat yield by 11.46 kg and protein yield by 8.11 kg. Similarly, for fertility traits, an average 4.81-day extension in first service to conception, a 0.16 increase in number of services, and a - 0.07 incidence in 56-day non-return rate were observed. Furthermore, a ROH region located on bovine chromosome 19 was identified that, when homozygous, had a negative effect on production traits. Signatures of selection proximate to this region have implicated GH1 as a potential candidate gene, which encodes the growth hormone that binds the growth hormone receptor. This observed negative effect could be a consequence of unfavorable alleles in linkage disequilibrium with favorable alleles.

Conclusions: ROH genotypes with unfavorable effects on production and fertility traits were identified within and across multiple traits on most chromosomes. These identified ROH genotypes could be included in mate selection programs to minimize their frequency in future generations.

Electrophysiologic studies in patients with autosomal dominant myotonia congenita (ADMC) have implicated defects of both muscle membrane sodium and chloride channels. An adult skeletal muscle sodium channel (ASkM1) gene maps to chromosome 17q23-25, and defects in this gene are almost certainly responsible for at least three variants of hyperkalemic periodic paralysis (HPP)--myotonic HPP, nonmyotonic HPP, and paramyotonia congenita. A gene for a muscle chloride channel has not yet been mapped in humans, but has been identified in the mouse. The gene for the cystic fibrosis transmembrane regulator (CFTR), which has chloride channel properties, is located on chromosome 7q31. This region is syntenic with the area of mouse chromosome 6 that contains the muscle chloride channel gene, a defect in which is responsible for the ADR phenotype, a murine model of myotonia. We performed linkage analysis using chromosome 17q polymorphisms at D17S74, SCN4A, and GH1, two chromosome 7q31 restriction fragment length polymorphisms, and a dinucleotide repeat polymorphism within the CFTR gene (CFTR-DNR), in three pedigrees with ADMC. The lod scores obtained show that the locus for ADMC is not at ASkM1 and is excluded from a region of at least 24 cM on either side of the CFTR gene.

Our aim was to investigate the long-term effects of intramuscular injection of rAAV2/1-CMV-GH1 viral particles on GH1 expression in normal adult male rats. We found that specific and sustained GH1 expression did not improve muscle exercise performance despite inducing local muscle hypertrophy. Injection of rAAV2/1-CMV-GH1 had some systemic effects on the liver and heart and on lipid metabolism in the healthy rats. Serum levels of hGH (human growth hormone), insulin, glucose and leptin increased significantly, which might induce insulin resistance. The serum concentration of IGF-1 (insulin-like growth factor 1), IGF-BP3 (insulin-like growth factor binding protein 3) and PIIINP (N-terminal propeptide of type III procollagen) markedly increased at 24 weeks after injection of GH1. In conclusion, GH1 expression driven by AAV2/1 in normal animals did not improve muscle strength but did increase muscle mass and may have systemic effects in healthy animals.

The cytopathicity and susceptibility of prototype HIV-1 [HTLVIIIB] and Ghanaian HIV isolates [GH1, GH2, GH3] to inhibition by ddCyd and ddIno were determined by the tetrazolium-based colorimetric method. HIV-1 [HTLVIIIB] caused the most cytopathic effect followed by HIV-2 [GH2]. At low MOI, HIV-2 [GH1] was more cytopathic than HIV-1 [GH3] but the reverse was true at high MOI. Using EC90 concentrations for comparison at similar cytopathicities, both HIV-1 [HTLVIIIB] and HIV-1 [GH3] which belong to prototype HIV-1 group, were effectively inhibited by one or both drugs. In contrast, HIV-2 [GH1] which belongs to prototype HIV-2 group, and especially, the highly divergent HIV-2 [GH2] which belongs to HIV-2b group were relatively resistant to inhibition by ddCyd and ddIno.

Comparison of coding nucleotide sequences of the paralogous GH1 and GH2 genes, as well as of the growth hormone amino acid sequences, in the species of closely related salmonid genera Salvelinus, Oncorhynchus, and Salmo was performed. It was demonstrated that, in different groups of salmonids, the amino acid substitution rates were considerably different. In some cases, an obvious discrepancy between the divergence of growth hormone genes and phylogenetic schemes based on other methods and approaches was revealed. These findings suggest that the reason may be multidirectional selection at duplicated genes at different stages of evolution.

Isolated growth hormone deficiency type II (IGHD2) is mainly caused by heterozygous splice-site mutations in intron 3 of the GH1 gene. A dominant negative effect of the mutant GH lacking exon 3 on wild-type GH secretion has been proposed; however, the molecular mechanisms involved are elusive. To uncover the molecular systems underlying GH deficiency in IGHD2, we established IGHD2 model mice, which carry both wild-type and mutant copies of the human GH1 gene, replacing each of the endogenous mouse Gh loci. Our IGHD2 model mice exhibited growth retardation along with intact cellular architecture and mildly activated ER stress in the pituitary gland, caused by decreased GH releasing hormone receptor (Ghrhr) and Gh gene promoter activities. Decreased Ghrhr and Gh promoter activities were likely caused by reduced levels of nuclear CREB3L2, which was demonstrated to stimulate Ghrhr and Gh promoter activity. This is the first in vivo study to reveal a novel molecular mechanism of GH deficiency in IGHD2, representing a new paradigm that differs from widely accepted models.

Since growth hormone deficiency (GHD) causes short stature and metabolic derangements, the processes which control its release are important physiologically. These processes can be illuminated by an understanding of genetically determined GHD. In 2 Indian Moslem cousins from a consanguineous family, GHD resistant to growth hormone releasing hormone (GHRH) stimulation was found. No mutations were found in the growth hormone gene (GH1) (J. Phillips). The receptor for GHRH (GHRHR), implicated in the dwarfism of the little mouse, thus becomes a candidate gene to explain their GHD. Amplification and sequencing a region of GHRHR homologous to that mutated in the little mouse showed a mutation (265G*T) leading to a stop codon at position 72 which would completely prevent GHRHR expression. Subsequently, Maheshwari et al. found an identical mutation in a multiplex kindred from Sindh, Pakistan, about 800 km from the place of origin of our patients. GHD is more commonly caused by recessive or dominant mutations of GH1. The latter are of great interest in understanding the mechanism of GH secretion. In a large kindred with dominant GHD we found a heterozygous 666G*A mutation replacing of Arg with His at amino acid 183. We speculate that the introduced histidine interferes with interactions necessary for correct GH secretion.

The influence of acclimation of the euryhaline gilthead sea bream (Sparus aurata) larvae/post-larvae to brackish water on growth, energetic contents, and mRNA levels of selected hormones and growth-regulating hypothalamic neurohormones was assessed. Specimens from 49 days post-hatching were acclimated during 28 days to two different environmental salinities: 38 and 20 psu (as brackish water). Both groups were then transferred to 38 psu and acclimated for an additional week. Early juveniles were sampled after 28 days of acclimation to both salinities and one week after transfer to 38 psu. Pituitary adenylate cyclase-activating peptide (adcyap1; pacap), somatostatin-I (sst1), growth hormone (gh1), insulin-like growth factor-I (igf1), and prolactin (prl) mRNA expression were all studied by QPCR. Post-larvae acclimated to 20 psu showed better growth performance and body energetic content than post-larvae maintained at 38 psu. prl, adcyap1, and igf1 mRNA expression levels increased in 20-psu-acclimated post-larvae but decreased upon transfer to 38 psu. GH1 expression did not show significant changes under both experimental conditions. Our results suggested an enhanced general performance for post-larvae in brackish water, supported by the actions of adcyap1, igf1, and prl.

This study was performed to clarify the pathophysiology of familial short stature with moderate GH deficiency.The siblings showed moderate GH deficiency with short stature. Pedigree analysis revealed an accumulation of the history of short stature in father's relatives, although there was no consanguinity.We performed sequencing analysis of GH1 and GHSR gene in the siblings.We detected SNPs in the GH1 gene in the combination of the - 278G, - 57T, +1169T, and +2103C in one allele from the father and the - 278T, - 57G, +1169 A, and +2103T in the other allele from the mother in the siblings. In the previous report, the -278G and - 57T allele are associated with low serum IGF-I levels in patients with isolated GH deficiency and the haplotype of the - 278T, - 57G, +1169 A, and +2103T allele exhibited an impaired GH secretion in vitro.It is suggested that these haplotypes were responsible at least in part for the GH deficiency and short stature in these siblings.

This article is referred to the research article entitled "Development of a novel method for specific detection of genetically modified Atlantic salmon, AquAdvantage, using real-time polymerase chain reaction" by Soga et al. (2020). Applicability of the developed <i>growth hormone 1</i> (<em>GH1</em>) and <i>18S ribosomal DNA</i> (18S rDNA) detection methods using real-time polymerase chain reaction (PCR) for detecting Atlantic salmon (<i>Salmo salar</i>) to processed food commodities was examined. DNAs extracted and purified from 24 commodities labelled to include salmon as an ingredient were used as template. Yield and purity of DNAs obtained and Cq values from real-time PCR analyses were provided.

The effects of natural variation in the number of copies of the growth hormone (GH) gene on growth parameters, plasma GH profiles, and the response to GHRH challenge were compared in Coopworth ram lambs from selection lines differing in body composition and GH levels. Different genotypes at the GH locus carried two, three, or four copies of the GH gene and GH secretion was studied under ad libitum feeding conditions and in the fasted state. There were no significant effects of GH genotype on any parameters of growth or body composition. Basal serum GH concentration, GH pulse frequency, and GH pulse amplitude differed significantly with selection line and fasting, but did not differ significantly between the GH genotypes. Significant differences of subtle nature were found between the GH genotypes in their responsiveness to GHRH. For the ad libitum-fed Lean selection line animals, the first GHRH challenge resulted in a higher mean maximum response for GH1/GH1 than GH2/GH2 (P < 0.05). Between the first and the second challenges there was a decrease in maximum response for the GH1/GH1 genotype and an increase for the GH2/GH2 genotype (P < 0.05 for GH genotype main effect). The differences between GH genotypes in response to GHRH challenge suggest that polymorphism in the number of GH gene copies in sheep may have physiological implications for the function of the GH axis, which may be manifested in growing lambs only under specific genotype-environment combinations.