Feedback Control of Snf1 Protein and Its Phosphorylation Is Necessary for Adaptation to Environmental Stress.

Snf1, a member of the AMP-activated protein kinase family, plays a critical role in metabolic energy control in yeast cells. Snf1 activity is activated by phosphorylation of Thr-210 on the activation loop of its catalytic subunit; following activation, Snf1 regulates stress-responsive transcription factors. Here, we report that the level of Snf1 protein is dramatically decreased in a UBP8- and UBP10-deleted yeast mutant (ubp8Δ ubp10Δ), and this is independent of transcriptional regulation and proteasome-mediated degradation. Surprisingly, most Snf1-mediated functions, including glucose limitation regulation, utilization of alternative carbon sources, stress responses, and aging, are unaffected in this strain. Snf1 phosphorylation in ubp8Δ ubp10Δ cells is hyperactivated upon stress, which may compensate for the loss of the Snf1 protein and protect cells against stress and aging. Furthermore, artificial elevation of Snf1 phosphorylation (accomplished through deletion of REG1, which encodes a protein that regulates Snf1 dephosphorylation) restored Snf1 protein levels and the regulation of Snf1 activity in ubp8Δ ubp10Δ cells. Our results reveal the existence of a feedback loop that controls Snf1 protein level and its phosphorylation, which is masked by Ubp8 and Ubp10 through an unknown mechanism. We propose that this dynamic modulation of Snf1 phosphorylation and its protein level may be important for adaptation to environmental stress.

Sequential culture with aroma-producing yeast strains to improve the quality of Kyoho wine.

Despite many non-Saccharomyces yeasts being considered spoilage microorganisms, they can increase aroma and flavor diversity in alcoholic beverages. The purpose of this study was to investigate nontraditional inoculation strategies using aroma-producing yeast strains for Kyoho wine fermentation, followed by an instrumental analysis and sensory evaluation. The winemaking process was carried out using Saccharomyces cerevisiae Gr112, Hanseniaspora uvarum Pi235, and Pichia kluyveri Pe114. Multiple inoculation strategies were explored. In instrumental analysis results, mixed culture could promote the formation of esters (5.9-folds) and glycerol (1.3-folds) and reduce the content of ethanol (-0.5% [v/v]) in wine. The sensory analysis results suggested that the three yeast strains sequential inoculation treatment was associated with the aroma attributes "floral," "red fruity," and "tropical fruity." Co-cultivation contributed to an increase in complexity and aromatic intensity, with the three-strain inoculation treatment presenting a more distinctive appearance. PRACTICAL APPLICATION: The inoculation of S. cerevisiae improved the accumulation of volatile acids and esters by inhibiting the growth of non-Saccharomyces yeast strains. Inoculation of H. uvarum and P. kluyveri would effectively solve the defect of excessive content of higher alcohols in wines produced by S. cerevisiae. The suitable inoculation strategy between non-Saccharomyces yeasts could improve the overall quality of Kyoho wine whose starter might be widely used in fermentation industry.

Modulatory Effect of Fermented Black Soybean and Adlay on Gut Microbiota Contributes to Healthy Aging.

SCOPE: Aging is a natural process characterized by a multifactorial, physical decline, and functional disability. Nevertheless, healthy aging can be achieved by following a multidirectional strategy. The current study aims to investigate the anti-aging potential of fermented black soybean and adlay (FBA). METHODS AND RESULTS: FBA supplements are incorporated into a natural aging mouse model that is designed to evaluate anti-aging effects. Results show that FBA supplementation prevents muscle loss and visceral adipose tissue accumulation. FBA can also reduce aging biomarkers (including the expression of hepatic p16INK4A and galactosidase beta-1 (GLB1). Hepatic 8-hydoxy-2'-deoxyguanosine (8-oxodG) and pro-inflammatory cytokines have been significantly reduced. Lastly, FBA supplementation improves aging-related gut microbial dysbiosis by reshaping gut microbial composition and promoting the growth of beneficial microbes such as Alistipes, Anaeroplasma, Coriobacteriaceae UCG002, and Parvibacter members in both genders of aged mice. In the functional prediction of gut microbiota, correlations to metabolic, neurodegenerative, infectious, and immune system diseases have been reduced in supplemented mice compared to aged mice. Moreover, FBA supplementation can reverse the reduced ability of microbiota in aged mice for lipid metabolism and xenobiotics biodegradation. CONCLUSIONS: The results suggest that FBA exhibits noteworthy anti-aging effects and that it can potentially be developed into a functional food for healthy aging.

Cross-Sectional Study: New Approach for Diagnostic Identification of Non-Robust Older Adult.

SCOPE: The aging biomarkers are alternatives and none of them can act as a strong predictor of frailty during the progression of aging. Several studies reveal the relationship between metabolites and frailty or gut microbiota and frailty. However, the connection between metabolites and gut microbiota in non-robust older adults has not been discussed yet. The study aims to combine the findings of serum metabolites and gut microbiota in non-robust subjects as a possible diagnostic biomarker. METHODS AND RESULTS: Frailty-related assessments are conducted to ensure the discrimination of non-robustness. The serum and fecal are collected for serum metabolomics and gut microbiota analysis. Robust and non-robust subjects show very different gut microbial compositions. Among the gut microbial differences, Escherichia/Shigella and its higher taxonomic ranks are found to have the most discriminative abundance among compared groups. More importantly, the abundance of Escherichia/Shigella is found to be positively correlated (p < 0.05) with the level of discriminant metabolites, such as serum oxoglutarate, glutamic acid, and 1-methyladenosine. CONCLUSION: These results indicate the obvious interrelation between gut microbiota and serum metabolites in non-robust older adults. Besides, the findings suggest that Escherichia/Shigella can be a potential biomarker candidate for robustness sub-phenotypic identification.

Ulva prolifera polysaccharide exerts anti-obesity effects via upregulation of adiponectin expression and gut microbiota modulation in high-fat diet-fed C57BL/6 mice.

Obesity is characterized by metabolic disorder and accompanying an altered and less diverse gut microbiota composition during a fat-enriched diet. Recent studies indicated that sulphated polysaccharide prevents high-fat diet (HFD) induced obesity, reduces metabolic disorder, and restores the gut microbiota. However, there are few studies about Ulva prolifera polysaccharide (UPP) may induce anti-obesogenic effects. Therefore, the present study investigates the enzymatic extracted UPP effects in HFD-fed mice. The results showed that UPP considerably slowed down the HFD-induced weight gain and improved metabolic disorders in HFD-fed mice. Notably, the effects were associated with lower body weight gain, reduced adipose tissue hypertrophy, triglyceride concentration in liver and systemic low-grade inflammation, and improved fasting blood glucose. Moreover, our result reveals that UPP may elevate the expression of AMPK via adiponectin activation. Interestingly, we found that UPP may induce PPARα agonist to enhance ß-oxidation since the elevation of CPT-1 and PPARα expression simultaneously. Meanwhile, gut microbiota analysis revealed UPP promoted the growth of Parasutterella, Feacalibaculum, and Bifidobacterium, and reduced the abundance of Acetatifactor, Tyzerella, Ruminococcus_1, and Desulfovibrio. The changes in microbiota may have a positively correlated effect on improving obesity and metabolic abnormalities. UPP may prevent HFD-induced obesity and associated metabolic diseases, as well as modulate the composition of gut microbiota to facilitate the growth of probiotics.

H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast.

BACKGROUND: The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. RESULTS: Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. CONCLUSION: These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

H3K4 Methylation in Aging and Metabolism.

During the process of aging, extensive epigenetic alterations are made in response to both exogenous and endogenous stimuli. Here, we summarize the current state of knowledge regarding one such alteration, H3K4 methylation (H3K4me), as it relates to aging in different species. We especially highlight emerging evidence that links this modification with metabolic pathways, which may provide a mechanistic link to explain its role in aging. H3K4me is a widely recognized marker of active transcription, and it appears to play an evolutionarily conserved role in determining organism longevity, though its influence is context specific and requires further clarification. Interestingly, the modulation of H3K4me dynamics may occur as a result of nutritional status, such as methionine restriction. Methionine status appears to influence H3K4me via changes in the level of S-adenosyl methionine (SAM, the universal methyl donor) or the regulation of H3K4-modifying enzyme activities. Since methionine restriction is widely known to extend lifespan, the mechanistic link between methionine metabolic flux, the sensing of methionine concentrations and H3K4me status may provide a cogent explanation for several seemingly disparate observations in aging organisms, including age-dependent H3K4me dynamics, gene expression changes, and physiological aberrations. These connections are not yet entirely understood, especially at a molecular level, and will require further elucidation. To conclude, we discuss some potential H3K4me-mediated molecular mechanisms that may link metabolic status to the aging process.

INO80-dependent chromatin remodeling regulates early and late stages of mitotic homologous recombination.

Chromatin remodeling is emerging as a critical regulator of DNA repair factor access to DNA damage, and optimum accessibility of these factors is a major determinant of DNA repair outcome. Hence, chromatin remodeling is likely to play a key role in genome stabilization and tumor suppression. We previously showed that nucleosome eviction near double-strand breaks (DSBs) in yeast is regulated by the INO80 nucleosome remodeling complex and is defective in mutants lacking the Arp8 subunit of INO80. In the absence of homologous donor sequences, RPA recruitment to a DSB appeared normal in arp8Delta, but Rad51 recruitment was defective. We now show that the early strand invasion step of homologous recombination (HR) is markedly delayed in an arp8Delta haploid, but there is only a minor defect in haploid HR efficiency (MAT switching). In an arp8Delta diploid, interhomolog DSB repair by HR shows a modest defect that is partially suppressed by overexpression of Rad51 or its mediator, Rad52. In wild type cells, DSB repair typically results in gene conversion, and most gene conversion tracts are continuous, reflecting efficient mismatch repair of heteroduplex DNA. In contrast, arp8Delta gene conversion tracts are longer and frequently discontinuous, indicating defects in late stages of HR. Interestingly, when a homologous donor sequence is present, Rad51 is recruited normally to a DSB in arp8Delta, but its transfer to the donor is delayed, and this correlates with defective displacement of donor nucleosomes. We propose that retained nucleosomes at donors destabilize heteroduplex DNA or impair mismatch recognition, reflected in delayed strand invasion and altered conversion tracts.

Intracranial brain abscess preceded by orbital cellulitis and sinusitis.

A 17-year-old boy with pyrexia, headache, and frequent drop attacks reported an acute onset of periorbital pain and swelling 1 month previously. Coronal computed tomography (CT) identified an ethmoid sinusitis, which was treated with functional endoscopic sinus surgery and intravenous gentamicin, prostaphylline, and metronidazone. Because of persistent symptoms, the patient returned 1 month later. The CT identified accumulation of debris in both frontal sinuses and a multilobulated lesion over the right frontal lobe. Bicoronal craniotomy was performed, and a mass located in the right frontal lobe was excised; the mass comprised chronic inflammatory tissues without evidence of malignancy. A postoperative brain CT confirmed the absence of a residual mass, and no recurrence or neurologic deficits were noted during the 3-month follow-up period. Intracranial complications cannot be prevented entirely even with the judicious use of antibiotics. Early application of the appropriate imaging modality and institution of aggressive therapy in any patient, not just pediatric patients, to prevent potential long-term disabilities and death are essential.

Biotransformation of mogrosides from Siraitia grosvenorii by Ganoderma lucidum mycelium and the purification of mogroside III E by macroporous resins.

Mogrosides are the major triterpenoidal saponins found in swingle, the fruit of Siraitia grosvenorii, which have recently been widely used throughout the world as natural food sweeteners. Among this class of compounds, mogroside III E (MG III E) exhibits the most intense sweetness, and it was also found to effectively regulate blood glucose levels. However, the relative abundance of naturally occurring MG III E is low compared to other mogrosides. Therefore, the purpose of this study was to enrich MG III E through biotransformation of fruit extracts and to develop a reliable method for its purification. We used HPLC coupled with mass spectrometry and nuclear magnetic resonance spectroscopy for metabolite analysis and identified MG III E as a major metabolite of Ganoderma lucidum mycelium. This organism converts the most abundant mogroside, mogroside V, to MG III E via a deglycosylation reaction; high levels of ß-glucosidase activities were also detected. In addition, we established an efficient purification method for MG III E using HP-20 macroporous resin. Optimization of the method was accomplished by kinetic model fitting, dynamic adsorption studies, and desorption experiments. The purity of MG III E was increased from 11.71% to 54.19%, with a 70%-76% recovery rate, and the scaled-up purification process allowed us to harvest 17.38 g of MG III E with 55.14% purity and a 74.71% of recovery rate. Therefore, our low cost, time-saving, easy to scale-up procedure for isolating MG III E could be applicable in industrial processes.

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress.

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.

Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity.

RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3alpha, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Delta. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a "random walk," and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Delta. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.

Green tea extract promotes DNA repair in a yeast model.

Green tea polyphenols may protect cells from UV damage through antioxidant activities and by stimulating the removal of damaged or cross-linked DNA. Recently, DNA repair pathways have been predicted as possible targets of epigallocatechin gallate (EGCG)-initiated signaling. However, whether and how green tea polyphenols can promote nucleotide excision repair and homologous recombination in diverse organisms requires further investigation. In this report, we used the budding yeast, Saccharomyces cerevisiae, as a model to investigate the effects of green tea extract on DNA repair pathways. We first showed that green tea extract increased the survival rate and decreased the frequency of mutations in yeast exposed to UVB-irradiation. Furthermore, green tea extract increased the expression of homologous recombination genes, RFA1, RAD51 and RAD52, and nucleotide excision repair genes, RAD4 and RAD14. Importantly, we further used a specific strand invasion assay to show that green tea extract promotes homologous recombination at double-strand breaks. Thus, green tea extract acts to preserve genome stability by activating DNA repair pathways in yeast. Because homologous recombination repair is highly conserved in yeast and humans, this study demonstrates yeast may be a useful platform for future research to investigate the underlying mechanisms of the bioactive compounds in DNA repair.

Dekkera bruxellensis, a beer yeast that specifically bioconverts mogroside extracts into the intense natural sweetener siamenoside I.

In response to growing concerns about the consumption of artificial sweeteners, the demand for natural sweeteners has recently increased. Mogroside V is a common natural sweetener extracted from the fruit of Siraitia grosvenorii, but its taste should be improved for marketability. Here, we screened various microbes for the ability to perform selective hydrolysis of glycosidic bonds in mogroside V, converting it to siamenoside I, which has a higher sweetening power and better taste than other mogrosides. Dekkera bruxellensis showed the most promising results in the screen, and the Exg1 gene (coding for a ß-glucosidase) of D. bruxellensis was cloned and purified. We then used HPLC-MS/MS to assess the ß-glucosidase activity of purified enzymes on p-nitrophenyl ß-glucoside and mogroside V. The results demonstrated that D. bruxellensis had a unique enzyme that can selectively hydrolyze mogrol glycosides and promote the conversion of the natural sweetener mogroside V to siamenoside I.

Inferring transcriptional compensation interactions in yeast via stepwise structure equation modeling.

BACKGROUND: With the abundant information produced by microarray technology, various approaches have been proposed to infer transcriptional regulatory networks. However, few approaches have studied subtle and indirect interaction such as genetic compensation, the existence of which is widely recognized although its mechanism has yet to be clarified. Furthermore, when inferring gene networks most models include only observed variables whereas latent factors, such as proteins and mRNA degradation that are not measured by microarrays, do participate in networks in reality. RESULTS: Motivated by inferring transcriptional compensation (TC) interactions in yeast, a stepwise structural equation modeling algorithm (SSEM) is developed. In addition to observed variables, SSEM also incorporates hidden variables to capture interactions (or regulations) from latent factors. Simulated gene networks are used to determine with which of six possible model selection criteria (MSC) SSEM works best. SSEM with Bayesian information criterion (BIC) results in the highest true positive rates, the largest percentage of correctly predicted interactions from all existing interactions, and the highest true negative (non-existing interactions) rates. Next, we apply SSEM using real microarray data to infer TC interactions among (1) small groups of genes that are synthetic sick or lethal (SSL) to SGS1, and (2) a group of SSL pairs of 51 yeast genes involved in DNA synthesis and repair that are of interest. For (1), SSEM with BIC is shown to outperform three Bayesian network algorithms and a multivariate autoregressive model, checked against the results of qRT-PCR experiments. The predictions for (2) are shown to coincide with several known pathways of Sgs1 and its partners that are involved in DNA replication, recombination and repair. In addition, experimentally testable interactions of Rad27 are predicted. CONCLUSION: SSEM is a useful tool for inferring genetic networks, and the results reinforce the possibility of predicting pathways of protein complexes via genetic interactions.

Mre11 and Ku regulation of double-strand break repair by gene conversion and break-induced replication.

The yeast Mre11-Rad50-Xrs2 (MRX) and Ku complexes regulate single-strand resection at DNA double-strand breaks (DSB), a key early step in homologous recombination (HR). A prior plasmid gap repair study showed that mre11 mutations, which slow single-strand resection, reduce gene conversion tract lengths and the frequency of associated crossovers. Here we tested whether mre11Delta or nuclease-defective mre11 mutations reduced gene conversion tract lengths during HR between homologous chromosomes in diploid yeast. We found that mre11 mutations reduced the efficiency of HR but did not reduce tract lengths or crossovers, despite substantially reduced end-resection at the test (ura3) locus. End-resection is increased in yku70Delta, but this change also had no effect on tract lengths. Thus, heteroduplex formation and tract lengths are not regulated by the extent of end-resection during DSB repair in a chromosomal context. In a plasmid-chromosome DSB repair assay, tract lengths were again similar in wild-type and mre11Delta, but they were reduced in mre11Delta in a gap repair assay. These results indicate that tract lengths are not affected by the extent of end processing when broken ends can invade nearby sites, perhaps because MRX coordination of the two broken ends is dispensable when ends invade nearby sites. Although HR outcome was largely unaffected in mre11 mutants, break-induced replication (BIR) and chromosome loss increased, suggesting that Mre11 function in mitotic HR is limited to early HR stages. Interestingly, yku70Delta suppressed BIR in mre11 mutants. BIR is also elevated in rad51 mutants, but yku70Delta did not suppress BIR in a rad51 background. These results indicate that Mre11 functions in Rad51-independent BIR, and that Ku functions in Rad51-dependent BIR.

Characterization of an extracellular ß-glucosidase from Dekkera bruxellensis for resveratrol production.

Polygonum cuspidatum is a widely grown crop with a rich source of polydatin (also called piceid) for resveratrol production. Resveratrol is produced from piceid via enzymatic cleavage of the sugar moiety of piceid. In this study, Dekkera bruxellensis mutants were selected based on their high p-nitrophenyl-ß-d-glucopyranoside and piceid conversion activities. The enzyme responsible for piceid conversion was a heterodimeric protein complex that was predominantly secreted to the extracellular medium and consisted of two subunits at an equal ratio with molecular masses of 30.5 kDa and 48.3 kDa. The two subunits were identified as SCW4p and glucan-ß-glucosidase precursor in D. bruxellensis. Both proteins were individually expressed in Saccharomyces cerevisiae exg1Δ mutants, which lack extracellular ß-glucosidase activity, to confirm each protein's enzymatic activities. Only the glucan-ß-glucosidase precursor was shown to be a secretory protein with piceid deglycosylation activity. Our pilot experiments of piceid bioconversion demonstrate the possible industrial applications for this glucan-ß-glucosidase precursor in the future.

Association between radioiodine treatment for thyroid cancer and risk of stroke.

BACKGROUND: The purpose of this study was to evaluate the association between radioiodine (I-131) therapy for thyroid cancer and the risk of stroke in Taiwan. METHODS: A total of 10 104 of the patients aged 20 years or older, who were newly diagnosed with thyroid cancer during 2000-2010, were recruited and classified into 2 cohorts according to whether they received I-131 therapy through 1:1 propensity score matching. The cumulative Kaplan-Meier curves for the incidence of stroke in the 2 cohorts were compared using the log-rank test. RESULTS: After adjustment for age, sex, and comorbidities, the I-131 therapy group showed no significantly higher risk of ischemic stroke (adjusted HR [aHR] = 1.05; 95% confidence interval [CI] = 0.82-1.34) or hemorrhagic stroke (aHR = 1.06; 95% CI = 0.58-1.93) than did the non-I-131 therapy group. CONCLUSION: The I-131 treatment for thyroid cancer did not increase the risk of stroke during 10-year follow-up.

Pilot scale repeated fed-batch fermentation processes of the wine yeast Dekkera bruxellensis for mass production of resveratrol from Polygonum cuspidatum.

Resveratrol has long been used as an ingredient in functional foods. Currently, Polygonum cuspidatum extract is the greatest natural source for resveratrol because of high concentrations of glycosidic-linked resveratrol. Thus, developing a cost-effective procedure to hydrolyze glucoside could substantially enhance resveratrol production from P. cuspidatum. This study selected Dekkera bruxellensis from several microorganisms based on its bioconversion and enzyme-specific activities. We demonstrated that the cells could be reused at least nine times while maintaining an average of 180.67U/L ß-glucosidase activity. The average resveratrol bioconversion efficiency within five rounds of repeated usage was 108.77±0.88%. This process worked effectively when the volume was increased to 1200L, a volume at which approximately 35mgL-1h-1 resveratrol per round was produced. This repeated fed-batch bioconversion process for resveratrol production is comparable to enzyme or cell immobilization strategies in terms of reusing cycles, but without incurring additional costs for immobilization.

Hydrolysis of isoflavone in black soy milk using cellulose bead as enzyme immobilizer.

The establishment of a catalytic system to enrich isoflavone aglycones in black soybean milk was investigated in this study. Beta-glucosidase, which was covalently immobilized onto cellulose beads, exhibited a significant efficiency for the conversion of 4-nitrophenyl ß-d-glucuronide to p-nitrophenol over the sol-gel method. The Michaelis constant (Km) of the cellulose bead enzymatic system was determined to be 1.50±0.10 mM. Operational reusability of the cellulose bead enzymatic system was justified for more than 10 batch reactions in black soy milk. Moreover, the storage stability verification indicated that the cellulose bead catalytic system was able to sustain its highest catalytic activity for 10 days. High-performance liquid chromatography results demonstrated that this enzymatic system required only 30 minutes to achieve complete isoflavone deglycosylation, and the aglycone content in the total isoflavones in black soy milk was enriched by 67% within 30 minutes by the cellulose bead enzymatic system.