Purification and characterization of a β-glucosidase capable of hydrolyzing soybean isoflavone glycosides from Pichia guilliermondii K123-1

A β-glucosidase, efficiently hydrolyzing isoflavone glycoside to isoflavone aglycone, was purified from Pichia guilliermondii K123-1, isolated from Korean soybean paste by ammonium sulfate precipitation, ion exchange column chromatography, gel filtration, and fast protein liquid chromatogram (FPLC). The molecular mass of purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The optimum temperature for enzyme activity was 45°c and it decreased dramatically above 50°c. The maximal activity was at pH 4.5 and more than 80% of the activity was retained for 24 hr in the pH range from 4.0 to 8.0 at 4°C. The N-terminal amino acid sequence of the enzyme was determined to be GLNWDYDNDK. Based on its substrate specificity and catalytic properties, the activity of the purified β-glucosidase was more effective when the sugar moiety of the glycoside was glucose and the size of the aglycone similar to that of the isoflavones. The purified β-glucosidase efficiently converts genistin and daidzin to genistein and daidzein 1.96 and 1.75 times more than almond meal β-glucosidase.     

Hydrolysis of grape monoterpenyl glycosides by Candida molischiana and Candida wickerhamii β-glucosidases

The exocellular β-glucosidases of Candida molischiana and Candida wickerhamii are able to hydrolyse geranyl, neryl, citronellyl, linalyl and α-terpinyl-β-D -glucopyranosides. The nature of the aglycone residue greatly affects the rate of hydrolysis. Glucosides with a tertiary alcohol as aglycone residue are more slowly hydrolysed than those with a primary alcohol. The β-glucosidase from C molischiana is also able to hydrolse α-L-arabinofuranosides. This results in the release of monoterpenols from a glycoside extract from grape juice.     

Characterization of the β-Glucosidase Activity Produced by Enological Strains of Non-Saccharomyces Yeasts

ABSTRACT: The β-glucosidase activities of 20 wine-related non- Saccharomyces yeasts were quantified, characterized, and assessed for their efficiency in releasing aroma-enhancing compounds during the winemaking process. Of these enzymatic activities, the β-glucosidase activity of Debaryomyces pseudopolymorphus revealed the most suitable combination of properties in terms of functionality at wine pH, resistance to wine-associated inhibitory compounds (glucose, ethanol, and sulfur dioxide), high substrate affinity, and large aglycone-substrate recognition. Its potential as a wine aroma-enhancing enzyme was confirmed by the significantly increasing concentrations of free volatiles (citronellol, nerol, and geraniol) during the fermentation of Chardonnay juice inoculated with both D. pseudopolymorphus and a widely used commercial starter culture strain of Saccharomyces cerevisiae , VIN13.     

Stability of β-glucosidase Activity Produced by Bifidobacterium and Lactobacillus spp. in Fermented Soymilk During Processing and Storage

ABSTRACT: Microorganisms possess endogenous enzymes, however the stability of these enzymes during storage in soymilk has not been studied. β-glucosidase is an important enzyme that could be used in the bioconversion of the predominant soy isoflavone glucosides to their bioactive aglycone forms. Fifteen probiotic microorganisms including bifidobacterium, Lactobacillus acidophilus , and Lactobacillus casei were screened for β-glucosidase activity using p-nitrophenyl-β-d-glucopyranoside as a substrate. Six strains were selected on the basis of β-glucosidase activity produced during fermentation of soymilk. The stability of the enzyme activity was assessed during incubation for up to 48 h and storage for 8 wk at frozen (-80°C), refrigerated (4°C), room (24.8°C), and incubation (37°C) temperatures. L. casei strains showed the highest β-glucosidase activity after 24 h of incubation followed by L. acidophilus strains, whereas bifidobacterium strains showedleast activity. However, p-glucosidase from Bifidobacterium animalis BB12 showed the best stability during the 48 h fermentation. Lower storage temperatures (-80°C and 4°C) showed significantly higher ( P < 0.05) β-glucosidase activity and better stability than that at higher temperatures (24.8°C and 37°C). The stability of β-glucosidase from these microorganisms should be considered for enzymic biotransformation during storage of isoflavone β-glucosides to bioactive isoflavone aglycone forms with potential health benefits.     

Influence of a Mixed Culture with Debaryomyces vanriji and Saccharomyces cerevisiae on the Volatiles of a Muscat Wine

ABSTRACT: :
A non- Saccharomyces yeast strain, Debaryomyces vanriji , isolated from grape berry flora was found to influence wine volatiles of the cv. Muscat of Frontignan when co-cultured with native or inoculated with a selected strain of Saccharomyces cerevisiae during fermentation. The concentrations of several volatiles were significantly different (p < 0.05) between the control and the 2 wines inoculated with D. vanriji. The latter were richer in fatty acids, esters, and terpenols. The increase in geraniol concentration in D. vanriji wines could be attributed to the hydrolysis of the corresponding glucosidic precursor by D. vanriji β-glucosidase. D. vanriji inoculated musts showed higher levels of β-glucosidase activity through fermentation compared to the control sample.     

利用β-葡萄糖苷酶提高葡萄酒香气的研究进展

葡萄酒中的萜类物质大多以结合态糖苷的形式存在,不易释放,而β-葡萄糖苷酶(β-glucosidase)是水解葡萄酒中结合态挥发性芳香化合物的关键酶,对葡萄酒香气的提升具有十分重要的作用。然而,由于葡萄醪或葡萄酒中高浓度的葡萄糖、乙醇以及低pH条件对该酶的活性及稳定性产生巨大的抑制或破坏作用,因此其在葡萄酒中的应用受到极大限制。近年来,针对如何提高β-葡萄糖苷酶在葡萄酒复杂生境中的活性及稳定性,研究者们做了大量的工作。本文在阅读大量文献的基础上,对通过β-葡萄糖苷酶产生菌的筛选、酶固定化技术、酿酒酵母细胞表面展示技术以及蛋白质的半理性化设计等不同策略来提高β-葡萄糖苷酶在葡萄酒复杂生境下的活性及稳定性方面的研究进行了综述,在此基础上,对利用β-葡萄糖苷酶来提高葡萄酒香气做了展望。     

β-Glucanases as a Tool for the Control of Wine Spoilage Yeasts

ABSTRACT:  In this study, the antimicrobial activity of a commercial β-glucanase preparation against wine spoilage yeasts such as  Cryptococcus albidus ,  Dekkera bruxellensis ,  Pichia membranifaciens ,  Saccharomyces cerevisiae ,  Zygosaccharomyces bailii , and  Zygosaccharomyces bisporus  has been evaluated. Yeast species tested showed different sensitivities to the enzyme preparation.  In vitro  assays in laboratory medium (GPY) showed inhibition by the β-glucanase preparation of  D. bruxellensis  and  Z. bailii  growth with IC₅₀ and MIC values approximately 3 to 4-fold greater than the recommended dose for improving wine filtration. Wine spoilage experiments showed antimicrobial action against  D. bruxellensis  and  Z. bailii  although with reduced effectiveness to that showed in laboratory medium. Under the conditions tested, the addition of β-glucanase did not affect wine enological parameters. Our data suggest the potential use of β-glucanases as antimicrobial agents in wine and indicate that the application of antimicrobial enzymes for yeast spoilage control deserves further investigation.     

非酿酒酵母产生的β-葡萄糖苷酶在发酵酒中的应用研究进展

β-葡萄糖苷酶对糖苷键的水解作用已被广泛应用于酿酒、茶增香、保健品开发等领域.发酵环境中非酿酒酵母产生的β-葡萄糖苷酶活力高于酿酒酵母,可在酿酒酵母酶活力不足时进行补充.本文对发酵过程中不同的影响因素如酵母合成和释放β-葡萄糖苷酶的能力、发酵环境中的温度、酸碱度和可发酵糖浓度等对β-葡萄糖苷酶活力的影响进行了综述,并对...     

Release of glycosidically bound flavour compounds of Chardonnay by Oenococcus oeni during malolactic fermentation

Glycosidases, produced by Oenococcus oeni strain Lalvin EQ54 during malolactic fermentation (MLF) performed in a chemically defined wine (CDW) medium, contributed to the release of volatile aglycons from their glycosylated precursors, present in a Chardonnay wine glycosidic extract. The liberation of wine volatiles during MLF was limited by the low activity of these enzymes in this strain. Six different aglycons examined were 3-hydroxydamascone, alpha-terpineol, vanillin, methyl vanillate, 4-hydroxybenzoic acid and tyrosol. Using p-nitrophenyl synthetic substrates, it was shown that O. oeni Lalvin EQ54 has β-glucosidase, and limited α-l-rhamnopyranosidase and α-l-arabinofuranosidase activities. Release of aglycons from the Chardonnay wine glycosidic extract was increased when purified α-l-rhamnopyranosidase and α-l-arabinofuranosidase were added to the CDW medium together with the malolactic bacteria culture. The results obtained confirmed that O. oeni has the necessary glycosidases for the sequential liberation of the disaccharide sugars from wine glycosides. The rate of MLF proceeded much faster in the CDW supplemented with the Chardonnay wine glycoside extract (8 days compared to 20 days), even though the bacterial growth was unaffected.     

Screening of non-Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes

Twenty-six strains of yeasts, belonging to the genera Candida, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces and Torulaspora previously isolated from wines, were screened for the production of extracellular pectinases, amylases, lipases, proteases and β-glucosidases. Some strains of Candida species and Hanseniaspora uvarum/Kloeckera apiculata produced extracellular proteolytic or lipolytic activities. Most yeasts exhibited β-glucosidase activity, but particularly high activity was observed in strains of Pichia anomala/Candida pelliculosa (formerly Hansenula anomala ) and Hanseniaspora uvarum/Kloeckera apiculata . The potential impact of these enzymes on wine quality is discussed.     

酒类酒球菌β-葡萄糖苷酶研究进展

酒类酒球菌（Oenococcus oeni）是葡萄酒苹果酸乳酸发酵（MLF）中的主要微生物,糖苷物质是葡萄酒中的重要香气前体物,β-葡萄糖苷酶是降解糖苷物质的关键酶。酒类酒球菌β-葡萄糖苷酶对增加葡萄酒香气,提升葡萄酒整体品质具有重要作用。该文介绍了β-葡萄糖苷酶的定义、分类、作用机制和测定方法,阐述了酒类酒球菌β-葡萄糖苷酶活,探讨了pH值、发酵温度、乙醇浓度、糖含量和二氧化硫含量对酶活的影响,在分子生物学水平上研究了酒类酒球菌β-葡萄糖苷酶基因,并对酒类酒球菌葡萄糖苷酶未来的研究热点和研究方向进行了展望。这对深入认识葡萄酒生物增香机理和提高葡萄酒整体品质具有重要意义。     

A Novel Extracellular β-Glucosidase from Trichosporon asahii: Yield Prediction, Evaluation and Application for Aroma Enhancement of Cabernet Sauvignon

The production and application of novel β-glucosidase from Trichosporon asahii were studied. The β-glucosidase yield was improved by response surface methodology, and the optimal media constituents were determined to be dextrin 4.67% (w/v), yeast extract 2.99% (w/v), MgSO(4) 0.01% (w/v), and K(2) HPO(4) 0.02% (w/v). As a result, β-glucosidase production was enhanced from 123.72 to 215.66 U/L. The effects of different enological factors on the activity of β-glucosidases from T. asahii were investigated in comparison to commercial enzymes. β-Glucosidase from T. asahii was activated in the presence of sugars in the range from 10% to 40% (w/v), with the exception of glucose (slight inhibition), and retained higher relative activities than commercial enzymes under the same conditions. In addition, ethanol, in concentrations between 5% and 20% (v/v), also increased the β-glucosidase activity. Although the β-glucosidase activity decreased with decreasing pH, the residual activity of T. asahii was still above 50% at the average wine pH (pH 3.5). Due to these properties, extracellular β-glucosidase from T. asahii exhibited a better ability than commercial enzymes in hydrolyzing aromatic precursors that remained in young finished wine. The excellent performs of this β-glucosidase in wine aroma enhancement and sensory evaluation indicated that the β-glucosidase has a potential application to individuate suitable preparations that can complement and optimize grape or wine quality during the winemaking process or in the final wine. Practical Application: The present study demonstrated the usefulness of response surface methodology based on the central composite design for yield enhancement of β-glucosidase from T. asahii. The investigation of the primary characteristics of the enzyme and its application in young red wine suggested that the β-glucosidase from T. asahii can provide more impetus for aroma improvement in the future.     

EFFECTS OF β-GLUCOSIDASE ACTIVITY ON THE CHARACTERISTICS OF AROMATIC RED RICE WINE

The pigment in the bran layer of aromatic red rice (Oryza sativa var. Indica, Tapol) is rather stable, has a characteristic red color just like that of grape wine and has a peak of absorbance at 530 nm at an acidic pH. A commercial saccharifying agent, glucoamylase AN-2, produced by Aspergillus niger was fractionated to separate glucoamylase and β-glucosidase activities by column chromatography on CM Sephadex C-50. The red rice wine made from uncooked, unpolished aromatic red rice using the fractionated, β-glucosidase-free preparation of glucoamylase had a characteristic red color. By contrast, red rice wine made with glucoamylase AN-2, which contained β-glucosidase activity, was inferior in color. The red pigment of aromatic red rice wine was decolorized and glucose originating from the red pigment was released by enzymatic digestion with the fractionated preparation of β-glucosidase. The partial decolorization of aromatic red rice wine was ascribed to the enzymatic action of β-glucosidase that was present in glucoamylase AN-2. Thus β-glucosidase activity has an undesirable effect on the brewing of aromatic red rice wine.     

Microbial degradation of cellulose polymers used in cosmetics and toiletries

Cellulose polymers which are commonly found in cosmetics can act as potential targets for microbial attack and sometimes support extensive growth under suitable conditions. As a result, cellulosic substances can be converted from a stiff gel into a running liquid, thereby rendering the cosmetic unfit for use.
To provide an understanding of the biodeterioration of cellulose in cosmetics, this paper reviews the structure of cellulose, mechanism of enzyme degradation and effects of structural properties of cellulose on the rate of hydrolysis. At least three different types of enzymes (exo-β-1, 4-glucanase, endo-β-1,4-glucanase and β-glucosidase) are involved in the degradation of crystalline cellulose. Enzyme production by three fungi belonging to the genera Aspergillus, Fusarium and Penicillium is described.
Dégradation microbienne des polymers de cellulose utilisés dans les produits cosmétiques et de toilette     

Improvement on enzymatic hydrolysis of resveratrol glucosides in wine

An enzymatic hydrolysis method, useful for the analysis of total wine resveratrol, was optimised. The wine resveratrol O-glycosides were totally hydrolysed in ∼9 h after incubation with β-glucosidase at 50 °C; then the trans- and cis-aglycones were measured by high-performance liquid chromatography after solid phase extraction (SPE). Kinetic study of enzymatic hydrolysis at different temperatures showed that hydrolysis times were reduced with increasing temperature, up to 50 °C, because the enzyme retains its activity even at this temperature.     

Stability of naturally occurring 2,5-dimethyl-4-hydroxy-3 [2H]-furanone derivatives

The stability, in aqueous buffer solutions at different pH values (pH 2.0–8.0, interval: 1.5 pH units), of 2,5-dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, DMHF, 1), its methoxy derivative 2,5-dimethyl-4methoxy-3[2H]-furanone (methoxyfuraneol, mesifurane, DMMF, 2 and the glycosidically bound forms DMHF β-D-glucopyranoside 3 and DMHF 6′-O-malonyl-β-Dglucopyranoside 4 was investigated over a period of 32 days at 23 °C. Only slight decomposition of 2 and 3 was observed, whereas 1 and 4 were found to be unstable at all pH values. In addition, 3 and 4 were subjected to enzymatic hydrolysis. In contrast to the rapid hydrolysis of 3, the malonylated glycoside, 4, remained unaffected by enzymatic treatment with β-glucosidase (Emulsin). Using a pectinolytic enzyme preparation (Rohapect D5L; R?hm, Darmstadt, Germany) with esterase activities, hydrolysis of 4 was achieved. Received: 25 September 1996     

Degradation of cyanogenic glycosides of bitter apricot seeds (Prunus armeniaca) by endogenous and added enzymes as affected by heat treatments and particle size

Bitter apricot (Prunus armeniaca) seeds (kernels) are by-products of the apricot processing industry. They contain approximately 50–150 μMol/g (dry weight basis) of potentially toxic cyanogenic glycosides, mainly amygdalin and prunasin. The present paper deals with the degradation of these glycosides by endogenous and added enzymes in raw and blanched seeds of different particle sizes. A hot water blanching treatment of 20 min at 100 °C was adequate to inactivate endogenous β-glucosidase activity in raw bitter apricot seeds. In addition to raw seeds, such blanched seeds were used as an experimental model to investigate the effect of particle size and added individual enzyme preparations on the degradation of cyanogenic glycosides. Finely ground (< 2 mm) fractions showed increased glycoside degradation, supporting the hypothesis that particle size is a limiting factor for enzymic degradation. Our hypothesis that added pectinase activity would enhance degradation of glycosides by improving enzyme-substrate contact could not be affirmed. Furthermore, it was observed that substantial enzyme addition (β-glucosidase) is required to fully degrade residual glycoside levels in raw and/or blanched seeds.     

Immobilization of β-Glucosidase and Its Application for Enhancement of Aroma Precursors in Muscat Wine

Enzyme immobilization is becoming more widely practised in biotechnology because of the advantages that this method brings. In this study, commercial β-glucosidase for aroma released in winemaking was immobilized on diverse supports (alginate–chitin, chitosan–chitin) by using different methods. It was found that the most appropriate matrix was chitosan by adsorption and reticulation. The optimal immobilization conditions were pH 3.5, immobilization time 120 min, and concentration of cross-linker glutaraldehyde 0.25 %. Stability of the immobilized enzymes was assessed, which revealed a number of advantages, such as a lower enzyme dose required for immobilization (367 times lower than the free enzyme dose recommended by the manufacturer), high stability over time, and reusability. In vitro studies of cellobiose and in vivo studies of wine and aroma precursors isolated from grape must yielded similar outcomes with respect to enzyme hydrolysis of free and immobilized proteins.     

新型碱性低分子量β-葡萄糖苷酶基因的分离与酶学性质研究

目的:获得新型β-葡萄糖苷酶编码基因,并对其表达产物进行酶学性质研究。方法:采用宏基因组学方法提取兔盲肠内微生物总DNA,构建DNA文库,通过筛选培养基获得产β-葡萄糖苷酶的克隆,测序获得其插入基因序列,氨基酸多重序列比对分析其序列特征,并用DNS显色法测定表达产物在不同条件下的酶活力。结果:在文库中获得一个基因片段nglu07,能编码一个低分子量的β-葡萄糖苷酶。nglu07基因片段长180bp,编码60个氨基酸残基。与已提交的糖苷水解酶Ⅰ家族成员进行氨基酸多重序列比对表明nglu07具有预测的活性位点Glu4与底物结合位点Tyr47,其最适反应温度为50℃,最适pH为10.0,粗酶活为8.12U/mL。结论:成功获得一新型低分子量的碱性β-葡萄糖苷酶编码基因,其蛋白温度稳定性高,在pH4.0～11.0的环境中均能保持较高的酶活力,具有作为食品工业用酶以及碱性酶的潜力,尤其在食品饮料加工、棉织品生物整理、洗涤及废纸脱墨等工业方面具有一定的应用价值。     

β-葡萄糖苷酶在酒类酿造中研究进展

β-葡萄糖苷酶EC3.2.1.21是一类可水解含β-D-葡萄糖苷键底物,释放具有香气特性的游离糖苷配体的水解酶,广泛应用于食品、药品、保健品等领域。该文基于国内科研人员在β-葡萄糖苷酶方面取得的研究成果,综述了近年来我国在产β-葡萄糖苷酶酿酒微生物选育方面以及β-葡萄糖苷酶在酒类酿造领域中的应用研究进展,以期为进一步扩大β-葡萄糖苷酶菌种的选育和应用提供理论参考。