Assessment and Partial Characterization of Candidate Genes in Dihydrochalcone and Arbutin Biosynthesis in an Apple-Pear Hybrid by De Novo Transcriptome Assembly.

Apples (Malus × domestica Borkh.) and pears (Pyrus communis L.) are valuable crops closely related within the Rosaceae family with reported nutraceutical properties derived from secondary metabolites including phloridzin and arbutin, which are distinctive phenolic metabolites characterizing apples and pears, respectively. Here, we generated a de novo transcriptome assembly of an intergeneric hybrid between apple and pear, accumulating intermediate levels of phloridzin and arbutin. Combining RNA-seq, in silico functional annotation prediction, targeted gene expression analysis, and expression-metabolite correlations, we identified candidate genes for functional characterization, resulting in the identification of active arbutin synthases in the hybrid and parental genotypes. Despite exhibiting an active arbutin synthase in vitro, the natural lack of arbutin in apples is reasoned by the absence of the substrate and broad substrate specificity. Altogether, our study serves as the basis for future assessment of potential physiological roles of identified genes by genome editing of hybrids and pears.

Protective effects of arbutin against doxorubicin-induced cardiac damage.

BACKGROUND: Doxorubicin is an effective antineoplastic agent but has limited clinical application because of its cumulative toxicities, including cardiotoxicity. Cardiotoxicity causes lipid peroxidation, genetic impairment, oxidative stress, inhibition of autophagy, and disruption of calcium homeostasis. Doxorubicin-induced cardiotoxicity is frequently tried to be mitigated by phytochemicals, which are derived from plants and possess antioxidant, anti-inflammatory, and anti-apoptotic properties. Arbutin, a natural antioxidant found in the leaves of the bearberry plant, has numerous pharmacological benefits, including antioxidant, anti-bacterial, anti-hyperglycemic, anti-inflammatory, and anti-tumor activity. METHODS AND RESULTS: The study involved male Wistar rats divided into three groups: a control group, a group treated with doxorubicin (20 mg/kg) to induce cardiac toxicity, a group treated with arbutin (100 mg/kg) daily for two weeks before doxorubicin administration. After treatment, plasma and heart tissue samples were collected for analysis. The samples were evaluated for oxidative stress parameters, including superoxide dismutase, malondialdehyde, and catalase, as well as for cardiac biomarkers, including CK, CK-MB, and LDH. The heart tissues were also analyzed using molecular (TNF-α, IL-1ß and Caspase 3), histopathological and immunohistochemical methods (8-OHDG, 4 Hydroxynonenal, and dityrosine). The results showed that arbutin treatment was protective against doxorubicin-induced oxidative damage by increasing SOD and CAT activity and decreasing MDA level. Arbutin treatment was similarly able to reverse the inflammatory response caused by doxorubicin by reducing TNF-α and IL-1ß levels and also reverse the apoptosis by decreasing caspase-3 levels. It was able to prevent doxorubicin-induced cardiac damage by reducing cardiac biomarkers CK, CK-MB and LDH levels. In addition to all these results, histopathological analyzes also show that arbutin may be beneficial against the damage caused by doxorubicin on heart tissue. CONCLUSION: The study suggests that arbutin has the potential to be used to mitigate doxorubicin-induced cardiotoxicity in cancer patients.

Characterization of a novel sucrose phosphorylase from Paenibacillus elgii and its use in biosynthesis of α-arbutin.

α-Arbutin, a naturally occurring glycosylated derivative of hydroquinone (HQ), effectively inhibits melanin biosynthesis in epidermal cells. It is widely recognized as a fourth-generation whitening agent within the cosmetic industry. Currently, enzymatic catalysis is universally deemed the safest and most efficient method for α-arbutin synthesis. Sucrose phosphorylase (SPase), one of the most frequently employed glycosyltransferases, has been extensively reported for α-arbutin synthesis. In this study, a previously reported SPase known for its effectiveness in synthesizing α-arbutin, was used as a probe sequence to identify a novel SPase from Paenibacillus elgii (PeSP) in the protein database. The sequence similarity between PeSP and the probe was 39.71%, indicating a degree of novelty. Subsequently, the gene encoding PeSP was coexpressed with the molecular chaperone pG-Tf2 in Escherichia coli, significantly improving PeSP's solubility. Following this, PeSP was characterized and employed for α-arbutin biosynthesis. The specific activity of co-expressed PeSP reached 169.72 U/mg, exhibited optimal activity at 35℃ and pH 7.0, with a half-life of 3.6 h under the condition of 35℃. PeSP demonstrated excellent stability at pH 6.5-8.5 and sensitivity to high concentrations of metal ions. The kinetic parameters Km and kcat/Km were determined to be 14.50 mM and 9.79 min- 1·mM- 1, respectively.The reaction conditions for α-arbutin biosynthesis using recombinant PeSP were optimized, resulting in a maximum α-arbutin concentration of 52.60 g/L and a HQ conversion rate of 60.9%. The optimal conditions were achieved at 30℃ and pH 7.0 with 200 U/mL of PeSP, and by combining sucrose and hydroquinone at a molar ratio of 5:1 for a duration of 25 h.

Arbutin inhibits androgen biosynthesis by rat immature Leydig cells in vitro.

Arbutin, a widely used skin lightening agent, has raised concerns regarding its potential side effects. In this study, we investigated the impact of arbutin on Leydig cell function using an in vitro model. We measured medium androgen levels, as well as the gene and protein expression related to Leydig cell steroidogenesis. Rat immature Leydig cells from age of 35 days were exposed to arbutin at concentrations ranging from 0.5 to 50 µM for a duration of 3 hrs. Following treatment, we observed a significant inhibition of androgen secretion by Leydig cells at both the 5 and 50 µM concentrations of arbutin. Furthermore, at a concentration of 50 µM, arbutin effectively blocked the stimulatory effects of luteinizing hormone (LH) and 8Br-cAMP on androgen secretion. Subsequent analysis revealed that arbutin downregulated the expression of crucial genes involved in androgen production, including Lhcgr, Hsd3b1, Cyp17a1, and Srd5a1. In silico computer program analysis predicted that arbutin exhibits good absorption, possesses a long elimination half-life, and may have other potential toxicity such as hepatoxicity. Taken together, our results demonstrate that arbutin negatively influences Leydig cell function and androgen production, potentially impacting male reproductive health.

Metabolomic analysis and bioactivities of Arbutus unedo leaves harvested across the seasons in different natural habitats of Sardinia (Italy).

BACKGROUND: Arbutus unedo L. is a wild tree of Mediterranean regions used as food and in traditional medicine and important for afforestation programs. There is no detailed information available on the variation of A. unedo leaves metabolome across the seasons. The leaves were analyzed by Proton nuclear magnetic resonance (1 H NMR)-based metabolomics, comparing samples harvested across the seasons and in ten different natural habitats of Sardinia (Italy). RESULTS: Multivariate analysis showed the impact of seasonal variation on the metabolome: glucose and quinic acid increased in summer, while in spring sucrose was accumulated. ß-Arbutin, the main known active principle of A. unedo, generally reached the highest concentration in autumn. In winter, O-ß-methylglucose, γ-aminobutyric acid (GABA), flavonols (quercetin-3-O-α-rhamnoside, myricetin-3-O-α-rhamnoside, kaempferol-3-O-α-rhamnoside), catechin, and gallocatechin increased. Characteristic metabolomic features were found also for samples collected in different locations. For instance, trees growing at the highest altitude and exposed to lower temperatures produced less flavonols and catechins. The only sample collected on trees growing on limestones, dolomites, and dolomitic limestones type of soil showed generally the highest content of arbutin. The highest phenolics content was found during spring, while samples collected on flowering branches in winter were the ones with the highest flavonoid content. The antioxidant activity was also variated, ranging from 1.3 to 10.1 mg of Trolox equivalents (TE)/mL of extract, and it was positively correlated to both total phenolics and flavonoid content. Winter samples showed the lowest antibacterial activity, while summer and autumn ones exhibited the highest activity (IC50 values ranging from 17.3 to 42.3 µg/mL against Staphylococcal species). CONCLUSION: This work provides 1 H-NMR fingerprinting of A. unedo leaves, elucidating the main metabolites and their variations during seasons. On the basis of arbutin content, autumn could be considered the balsamic period of this taxon. Samples collected in this season were also the most active ones as antibacterial. Moreover, an interesting metabolomic profile enriched in catechins and flavonols was observed in leaves collected in winter on flowering branches which were endowed with high antioxidant potential.

Key secondary metabolite markers for Wuchang Daohuaxiang rice discrimination in China.

This study aimed to comprehensively elucidate the vital secondary metabolites of Wuchang Daohuaxiang (DHX) rice through widely targeted metabolomics analysis. Among the secondary metabolites detected, a total of 30 differential ones were screened out and categorized into 4 different classes, including 6 alkaloids (20%), 15 flavonoids (50%), 6 phenolic acids (20%), and 3 terpenoids (10%) between DHX and control groups. Of these, compounds as zarzissine, fagomine, arbutin, p-Hydroxypheny-ß-D-allopyranoside, pimaric acid, kaurenoic acid, and isopimaric acid were more abundant in DHX than control group, with the possibility in serve as key secondary metabolites of DHX rice. Furthermore, arbutin, trigonelline and 6'-O-Feruloyl-D-sucrose were optimized as potential biomarkers for DHX rice discrimination. This study would supply data support for DHX rice authenticity and quality improvement.

Protective effect of arbutin against cyclophosphamide-induced oxidative stress, inflammation, and hepatotoxicity via Nrf2/HO-1 pathway in rats.

Cyclophosphamide (CP) is a potent anticancer drug widely employed in chemotherapy against various types of cancer. However, CP leads to toxicity to non-targeted organs, including the liver and this limits its clinical use. This study explored the role of arbutin (ARB) against CP-mediated oxidative and inflammatory reactions and hepatotoxicity. Rats were administered ARB (25 and 50 mg/kg) for 14 days and CP (150 mg/kg). CP triggered liver tissue injury with marked increase in serum AST, ALT, ALP, and bilirubin, and hepatic malondialdehyde (MDA) and nitric oxide (NO) coupled with diminution of GSH, SOD, catalase, and GPx. Liver NF-kB p65, NOS, IL-6, TNF-α, Bax and caspase-3 were upregulated by CP injection and IL-10 and Bcl-2 were decreased. ARB prevented liver injury, suppressed MDA, NO, NF-kB p65, inflammatory markers, Bax and caspase-3 in CP-treated rats. ARB restored antioxidants, IL-10 and Bcl-2, and enhanced Nrf2 and hemeoxygenase-1 (HO) both gene and protein in the liver of rats. In conclusion, these results pinpointed the protective role of ARB on oxidative and inflammatory reactions, apoptosis, and hepatotoxicity in rats. This hepatoprotective activity was linked to the ability of ARB to modulate Nrf2/HO-1 pathway.

Arbutin abrogates testicular ischemia/reperfusion injury in rats through repression of inflammation and ER stress.

The aim of this study was to investigate the effects of arbutin (ARB) administration on oxidative stress, inflammation, endoplasmic reticulum (ER) stress and apoptosis in an experimental testicular torsion/detorsion (T/D)-induced testicular injury model for the first time. A total of 24 male Sprague-Dawley rats were divided into four groups with six rats in each group: sham control, T/D, T/D+ARB (50 mg/kg) and T/D+ARB (100 mg/kg). Torsion and detorsion times were applied as 4 h and 2 h, respectively. The levels of lipid peroxidation [malondialdehyde (MDA)] and oxidative stress [total oxidant status (TOS) and total antioxidant status (TAS)] in testicular tissues were determined using colorimetric methods. The levels of DNA damage [8-hydroxy-2'-deoxyguanosine (8-OHdG)], antioxidant system [superoxide dismutase (SOD) and catalase (CAT)], pro-inflammatory cytokines [high mobility group box 1 (HMGB1), nuclear factor kappa B protein 65 (NF-κB p65), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and myeloperoxidase (MPO)], ER stress [78-kDa glucose-regulated protein (GRP78), activating transcription factor 6 (ATF6) and CCAAT-enhancer-binding protein homologous protein (CHOP)] and apoptosis (caspase-3) markers in testicular tissues were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits. Johnsen's testicle scoring system was used for histological evaluation. In the T/D group, it was determined that statistically significant increasing in the levels of oxidative stress, inflammation, ER stress and apoptosis compared with sham control group (p < 0.05). ARB administrations statistically significantly restored testicular I/R damage in a dose dependent manner (p < 0.05). In addition, it was determined that the data of histological examinations supported the biochemical results. Our findings support the hypothesis that ARB may be used as a protective agent against T/D-induced testicular damage.

Silencing FHL2 inhibits bleomycin-induced pulmonary fibrosis through the TGF-ß1/Smad signaling pathway.

OBJECTIVE: This study aimed to investigate the inhibiting effects of FHL2 and Arbutin on cell fibrosis and their possible mechanisms. METHODS: The mRNA expression of FHL2 in pulmonary fibrosis tissues was analyzed by bioinformatics. TGF⁃ß1 induced fibrosis of mouse lung fibroblast (Mlg) and mouse primary pulmonary fibroblast (PPF) in rat's lung fibroblasts. FHL2 siRNA was transfected into Mlg and mouse PPF cells to inhibit FHL2. FHL2, α-smooth muscle actin (α-SMA), collagen 1 (Col I), and Fibronectin (Fn) were detected by qRT-PCR. Western blot expression levels of Smad3, p-Smad3, Smad2, and p-Smad2 proteins in cells. High-throughput drug screening for FHL2 inhibitors and the inhibitory effect of Arbutin on pulmonary fibrosis were validated in cellular and animal models of pulmonary fibrosis. RESULTS: The mRNA expression of FHL2 in lung fiber tissue was increased. Meanwhile, the decrease of FHL2 expression significantly inhibited the cellular fibrosis morphological changes of rat's lung fibroblasts (Mlgs) and primary lung fibroblasts (PPFs). The expression levels of α⁃SMA, Col I, and Fn were decreased. High-throughput screening showed that Arbutin targeted FHL2. Arbutin alleviated bleomycin (BLM)-induced pulmonary fibrosis in rats by inhibiting FHL2 and then the TGF-ß1/Smad signaling pathway. CONCLUSION: Inhibition of FHL2 can effectively reduce the fibrosis process induced by TGF⁃ß1 and bleomycin, and then inhibit the fibrosis.

Effects of arbutin on glucose uptake by glucose transporter 4 (GLUT4) and its cytoprotective properties in L6 skeletal muscle cell line.

It has been well known that oxidative stress and increased intracellular reactive oxygen species (ROS) have a pivotal role in disrupting the insulin signaling pathways leading to cellular insulin resistance. In this study, we evaluated arbutin's effects on glucose uptake by GLUT4 and cytoprotective properties in the L6 skeletal muscle cell line. The effect of arbutin and tertiary butyl hydrogen peroxide (t-BHP) on glucose uptake in cultured L6 cells was investigated by flow cytometry. We also evaluated gene expression levels of GLUT1 and GLUT4 in the L6 cells by quantitative real-time polymerase chain reaction analysis. The results from the study demonstrated that the optimum ROS generation occurred 3 h after 100 µM t-BHP treatment and pretreatment with arbutin (500 and 1000 µM) significantly inhibited the t-BHP induced ROS generation (p < .05). Our result indicated that 3 h pretreatment of L6 cells with 1000 µM of arbutin before 50 µM t-BHP significantly increased glucose uptake than the 50 µM t-BHP alone group (p < .05). Our findings may suggest that an increase in the uptake of 2-NBDG by L6 cells with arbutin pretreatment can be associated with increased expression of GLUT4 and GLUT1 under oxidative stress.

Arbutin attenuates monosodium L-glutamate induced neurotoxicity and cognitive dysfunction in rats.

Excitotoxicity, oxidative stress, and neuro-inflammation underlie the pathogenesis of neurodegenerative brain disorders. Although L-glutamate is the prime excitatory neurotransmitter involved in diverse brain functions, however, overabundance at synapse can activate cell death mechanisms. Previous studies indicate that arbutin affords relief in metabolic, cardiovascular, and gastrointestinal disorders. Recently, arbutin showed benefits in animal models of epilepsy, Parkinson's disease, and Alzheimer's disease that further expanded its therapeutic potential against brain disorders. In the present study, we aimed to evaluate the potential of arbutin against monosodium L-glutamate (MSG) neurotoxicity in rats. Wistar rats (male, 180-200 g) were administered MSG (4 mg/kg) and arbutin (50 and 100 mg/kg) intraperitoneally for 21 days. Cognitive functions were assessed using elevated plus maze and novel object recognition task. Biochemical parameters of oxidative stress, tumour necrosis factor-α (TNF-α), γ-amino butyric acid (GABA), acetylcholinesterase (AChE) activity, lactate dehydrogenase (LDH), and intracellular cation-levels (Na+, Ca2+, K+) were determined using whole brain. Administration of MSG augmented cation-levels, oxidative stress, inflammation, AChE, and LDH activities, and decreased GABA levels in the brain. Arbutin (50 and 100 mg/kg, i.p.) significantly decreased these biochemical disturbances in the brain of MSG administered rats. Behavioural results showed that MSG triggered cognitive deficits in rats that were significantly attenuated by arbutin. Histopathological findings in hippocampus and cortex revealed neuroprotective outcome of arbutin treatments against MSG. MK-801 and N(G)-nitro-L-arginine methyl ester (L-NAME) enhanced memory and neuroprotective effects in rats treated with arbutin and MSG. Arbutin may afford therapeutic advantages in neurodegenerative brain disorders by suppressing the excitotoxic pathways.

Identification of Sitogluside as a Potential Skin-Pigmentation-Reducing Agent through Network Pharmacology.

Many traditional Chinese medicines (TCMs) with skin-whitening properties have been recorded in the Ben-Cao-Gang-Mu and in folk prescriptions, and some literature confirms that their extracts do have the potential to inhibit pigmentation. However, no systematic studies have identified the specific regulatory mechanisms of the potential active ingredients. The aim of this study was to screen the ingredients in TCMs that inhibit skin pigmentation through a network pharmacology system and to explore underlying mechanisms. We identified 148 potential active ingredients from 14 TCMs, and based on the average "degree" of the topological parameters, the top five TCMs (Fructus Ligustri Lucidi, Hedysarum multijugum Maxim., Ampelopsis japonica, Pseudobulbus Cremastrae Seu Pleiones, and Paeoniae Radix Alba) that were most likely to cause skin-whitening through anti-inflammatory processes were selected. Sitogluside, the most common ingredient in the top five TCMs, inhibits melanogenesis in human melanoma cells (MNT1) and murine melanoma cells (B16F0) and decreases skin pigmentation in zebrafish. Furthermore, mechanistic research revealed that sitogluside is capable of downregulating tyrosinase (TYR) expression by inhibiting the ERK and p38 pathways and inhibiting TYR activity. These results demonstrate that network pharmacology is an effective tool for the discovery of natural compounds with skin-whitening properties and determination of their possible mechanisms. Sitogluside is a novel skin-whitening active ingredient with dual regulatory effects that inhibit TYR expression and activity.

Arbutin prevents alterations in mitochondrial and lysosomal enzymes in isoproterenol-induced myocardial infarction: An in vivo study.

The present study demonstrated the protective effects of arbutin (ARB) on hyperlipidemia, mitochondrial, and lysosomal membrane damage and on the DNA damage in rats with isoproterenol (ISO)-induced myocardial infarction (MI). Rats were pretreated with ARB (25 and 50 mg/kg body weight (bw)) for 21 days. After pretreatment with ARB, MI was induced by subcutaneous injection of ISO (60 mg/kg bw) for two consecutive days at an interval of 24 h. The levels of TC, TG, and FFA were increased and decreased the level of PL in the heart tissue of ISO-induced MI rats. Very-low-density lipoprotein cholesterol and low-density lipoprotein cholesterol were increased while high-density lipoprotein cholesterol was decreased in the plasma of ISO-administered rats. A heart mitochondrial fraction of the ISO rats showed a significant decrease in the activities of mitochondrial enzymes isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. The activities of lysosomal enzymes (ß-glucosidase, ß-glucuronidase, α-galactosidase, ß-galactosidase, cathepsin-B, and cathepsin-D) were increased significantly in the heart tissue homogenate of disease control rats. In ISO-induced MI, rat's significant increase in the percentage of tail DNA and tail length, and a decrease in the level of head DNA were also observed. ARB administration to MI rats brought all these parameters to near normality, showing the protective effect of ARB against MI in rats. The results of this study demonstrated that the 50 mg/kg bw of ARB shows higher protection than 25 mg/kg bw against ISO-induced damage.

The effects of strawberry tree (Arbutus unedo L.) water leaf extract and arbutin upon kidney function and primary DNA damage in renal cells of rats.

Although strawberry tree (Arbutus unedo L.) leaves have long been used as a herbal remedy, insufficient information is available on their nephrotoxicity. We assessed the safety of strawberry tree water leaf extract and its key component arbutin, administered per os to Lewis rats of both genders at 200 mg/kg b.w./day for 14 and 28 days. The effects of the tested compounds on DNA integrity in renal cells was evaluated using alkaline comet assay, while kidney function was studied using serum creatinine and urea levels. Strawberry tree water leaf extract showed high biocompatibility with kidney tissue. It did not impair DNA integrity of renal cells and kidney function, either in male or female rats. However, exposure to single arbutin affected the levels of primary DNA damage in renal cells which could be related to metabolic conversion of arbutin into hydroquinone, whose nephrotoxicity has previously been proven.

Recent progress on biological production of α-arbutin.

Arbutin, a glucoside of hydroquinone, is used as a powerful skin lightening agent in the cosmeceutical industry because of its strong inhibitory effect on the human tyrosinase activity. It is a natural compound occurring in a number of plants, with a ß-anomeric form of the glycoside bond between glucose and hydroquinone. α-Arbutin, which glycoside bond is generated with α-anomeric form, is the isomer of natural arbutin. α-Arbutin is generally produced by transglucosylation of hydroquinone by microbial glycosyltransferases. It is interesting that α-arbutin is found to be over 10 times more effective than arbutin, and thus biological production of α-arbutin attracts increasing attention. Seven different microbial enzymes have been identified to be able to produce α-arbutin, including α-amylase, sucrose phosphorlase, cyclodextrin glycosyltransferase, α-glucosidase, dextransucrase, amylosucrase, and sucrose isomerase. In this work, enzymatic and microbial production of α-arbutin is reviewed in detail.

Qualitative and quantitative screening of the ß-glucosidase activity in Saccharomyces cerevisiae and Saccharomyces uvarum strains isolated from refrigerated must.

The aim of the present work was to screen a pool of 75 yeasts belonging to the species Saccharomyces cerevisiae and Saccharomyces uvarum in order to select the strains endowed with ß-glucosidase activity. The first screening was a qualitative assay based on chromogenic substrates (arbutin and esculin). The second screening was the quantitative evaluation of the ß-glucosidase activity via a p-nitrophenyl-ß-d-glucopyranoside assay. The measurement was performed on three different cell preparations, including the extracellular compartment, the cell lysates and the whole cells. This study pointed out the high frequency of ß-glucosidase activity in S. uvarum strains. In particular, we retrieved three promising S. uvarum strains, CRY14, VA42 and GRAS14, featuring a high enzymatic activity, exploitable for winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: In yeasts, ß-glucosidase activity has been extensively described, especially in non-Saccharomyces species, while there is only little evidence of this activity in strains belonging to the Saccharomyces species. In winemaking, ß-glucosidase plays essential roles in the hydrolysis of glyco-conjugated precursors and the release of active aromatic compounds. This study provides new insights into the ß-glucosidase activity in strains belonging to Saccharomyces cerevisiae and Saccharomyces uvarum species, which are the most important strains in wine industry. Our results point out a marked enzymatic activity for the tested S. uvarum strains. These strains could be exploited for their potential ability to enhance the aroma profiles of wine. In addition, they could be potential sources for the commercial production of enzymes to be applied in winemaking.

Diversity of Streptococcus equi subsp. zooepidemicus strains isolated from the Spanish sheep and goat population and the identification, function and prevalence of a novel arbutin utilisation system.

The zoonotic bacterium Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a diverse, opportunistic pathogen that can cause mastitis in dairy sheep and goats. We used multilocus sequence typing (MLST) to define the genetic diversity of 60 isolates of S. zooepidemicus, which were recovered from sheep and goats in Spain between 2003 and 2010. We identify a novel clonal complex based on sequence type (ST), ST-236, which accounted for 39 of the 60 isolates. A representative ST-236 strain, S. zooepidemicus strain C7 (SzC7), was sequenced and interrogated for the presence of novel nutritional uptake or utilisation systems, the acquisition of which have previously been shown to be important for environmental adaptation in other streptococcal pathogens. A novel phosphoenolpyruvate sugar phosphotransferase system (PTS), which enabled the utilisation of arbutin, was identified. Functionality of the PTS was confirmed following deletion of the PTS from SzC7. Arbutin is found in multiple animal foodstuffs and we propose that the ability to utilise arbutin may have conferred a selective advantage to strains infecting animals, the diet of which contains this sugar.

A comparative study of the evolution of cellobiose utilization in Escherichia coli and Shigella sonnei.

The chb operon of Escherichia coli is involved in the utilization of chitooligosaccharides. While acquisition of two classes of mutations leading to altered regulation of the chb operon is necessary to confer the ability to utilize the glucose disaccharide cellobiose to wild-type strains of E. coli, in the closely related organism Shigella sonnei, Cel+ mutants arise relatively faster, requiring only a single mutational event. In Type I mutants, the insertion of IS600 at -21 leads to ChbR regulator-independent, constitutive expression of the operon. In Type II mutants, the insertion of IS2/600 within the distal binding site of the negative regulator NagC leads to ChbR-dependent cellobiose-inducible expression of the operon. These studies underscore the significance of strain background, specifically the diversity of transposable elements, in the evolution of novel metabolic functions. Constitutive expression of the chb operon also enables utilization of the aromatic ß-glucosides arbutin and salicin, implying that the chb structural genes are inherently promiscuous.

Fermentation scale up for α-arbutin production by Xanthomonas BT-112.

α-Arbutin is a glycosylated hydroquinone that has an inhibitory function against tyrosinase. The aim of the present study is to develop an efficient and inexpensive method for large-scale production of α-arbutin by using Xanthomonas BT-112 as biocatalyst. To accomplish this goal, various surfactants were tested to enhance the α-arbutin production, and the optimal operational conditions for 30L jar fermenter were scaled up for a production level of 3000L with using a constant volumetric oxygen transfer coefficient (KLa) and the volumetric aeration rate per volume unit (Q/V) as scale-up criteria. Under the optimized conditions, the α-arbutin produced in the presence of 0.4% (w/v) Tween-80 was 124.8% higher than that of the control, and the yield of α-arbutin in 3000L fermenter was 38.2g/L with a molar conversion ratio of 93.7% based on the amount of hydroquinone supplied. This result is comparable to the results from laboratory-scale fermenter. Hence, 100-fold scale-up was successfully achieved.

Purification and enzymatic characterization of secretory glycoside hydrolase family 3 (GH3) aryl ß-glucosidases screened from Aspergillus oryzae genome.

By a global search of the genome database of Aspergillus oryzae, we found 23 genes encoding putative ß-glucosidases, among which 10 genes with a signal peptide belonging to glycoside hydrolase family 3 (GH3) were overexpressed in A. oryzae using the improved glaA gene promoter. Consequently, crude enzyme preparations from three strains, each harboring the genes AO090038000223 (bglA), AO090103000127 (bglF), and AO090003001511 (bglJ), showed a substrate preference toward p-nitrophenyl-ß-d-glucopyranoside (pNPGlc) and thus were purified to homogeneity and enzymatically characterized. All the purified enzymes (BglA, BglF, and BglJ) preferentially hydrolyzed aryl ß-glycosides, including pNPGlc, rather than cellobiose, and these enzymes were proven to be aryl ß-glucosidases. Although the specific activity of BglF toward all the substrates tested was significantly low, BglA and BglJ showed appreciably high activities toward pNPGlc and arbutin. The kinetic parameters of BglA and BglJ for pNPGlc suggested that both the enzymes had relatively higher hydrolytic activity toward pNPGlc among the fungal ß-glucosidases reported. The thermal and pH stabilities of BglA were higher than those of BglJ, and BglA was particularly stable in a wide pH range (pH 4.5-10). In contrast, BglJ was the most heat- and alkaline-labile among the three ß-glucosidases. Furthermore, BglA was more tolerant to ethanol than BglJ; as a result, it showed much higher hydrolytic activity toward isoflavone glycosides in the presence of ethanol than BglJ. This study suggested that the mining of novel ß-glucosidases exhibiting higher activity from microbial genome sequences is of great use for the production of beneficial compounds such as isoflavone aglycones.