Characterization of a β-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to β-glucan-binding proteins

Spodoptera frugiperda β-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of β-1,3-glucanases and β-glucan-binding protein supports the assumption that the β-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived β-1,3-glucanases by the loss of an extended N-terminal region and the β-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.     

啤酒废酵母中β-1，3-葡聚糖的提取工艺

研究采用酶-碱法从经超声波处理的废酵母残渣中提取β-1,3-葡聚糖的工艺,通过正交试验得出理想的酶处理工艺条件：酶添加量208U／g,温度50℃、pH6,酶解8h,蛋白质去除率为62．82％,每L废酵母液中可回收0．348g多肽、氨基酸的蛋白水解液;碱处理工艺条件：用30mL质量分数为2％ NaOH溶液在70℃处理酶解后的沉淀物5h。所得β-1,3-葡聚糖纯度为90．50％,得率为11．00％,经紫外光谱、薄层层析和性质分析为高纯度的β-1,3-葡聚糖。     

The influence of β-glucan on the growth and cell wall architecture of Aspergillus spp

β-1,3-glucan is a major component of fungal cell walls with various biological activities, including effects on the production of inflammatory mediators in vivo and in vitro. However, few reports have examined its influence on the fungal cell itself. In this study, the influences of β-1,3-glucan on the growth and cell wall structure of fungi was examined. Aspergillus fumigatus was cultured with a synthetic medium, C-limiting medium, in the presence or absence of β-1,3-glucan. Hyphal growth was promoted in liquid and solid-cultures by adding β-1,3-glucan. Glucose and dextran did not induce growth. The influence on cell wall structure of the β-glucan-added cultures was examined by enzymolysis and NMR spectroscopy and the amount of β-1,3-glucan found to be changed. β-1,3-glucan has been widely detected in the environment. In this study, it was demonstrated that β-1,3-glucan causes promotion of the growth, and a change in the cell wall architecture, of Aspergillus. Unregulated distribution of β-1,3-glucan would be strongly related to the incidence of infectious diseases and allergy caused by Aspergillus spp.     

Purification,characterization and sequencing of the major β-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major β-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes β-1,3-glucans as laminarin and yeast β-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched β-1,3-glucans or mixed β-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.     

The yeast-phase virulence requirement for α-glucan synthase differs among Histoplasma capsulatum chemotypes

Histoplasma capsulatum strains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype II Histoplasma virulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucan in vitro, yet they remain fully virulent in vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishes AGS1 expression. Nonetheless, AGS1 mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced during in vivo growth despite its absence in vitro. To directly test whether AGS1 contributes to chemotype I strain virulence, we prevented AGS1 function by RNA interference and by insertional mutation. Loss of AGS1 function in chemotype I does not impair the cytotoxicity of ags1(-) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, the ags1(-) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate that AGS1 expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a unique Histoplasma chemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.     

食药用菌功能性β-葡聚糖荧光法测定研究

以酵母功能性β-1,3/1,6-葡聚糖为对照品,利用苯胺蓝和β-1,3/1,6-葡聚糖特异结合荧光特性,研究了葡聚糖荧光法测定时的各影响因素,建立了荧光法测定食药用菌功能性β-葡聚糖的方法。pH9.6缓冲液,80℃条件下避光反应15min,室温30min冷却后,398nm激发波长,508nm发射波长,20℃下进行荧光测定。在测定浓度2-20μg/mL范围内,荧光强度与浓度具有良好的线性关系(R2=0.9977),其中检出限为45μg/L,测定精密度和加样回收率良好,相对标准偏差(RSD)分别为1.86%和3.40%,并与酶法进行了比对验证,一致性良好,且荧光法更为节约时间和成本,并对灰树花菌、巴氏蘑菇、香菇和鲍氏针层孔菌四种食药用菌β-葡聚糖提取样品进行了葡聚糖纯度和提取率测定。     

Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay

BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.     

Purification and characterization of a novel alkaline β-1,3-1,4-glucanase (lichenase) from thermophilic fungus Malbranchea cinnamomea

A novel alkaline β-1,3-1,4-glucanase (McLic1) from a thermophilic fungus, Malbranchea cinnamomea, was purified and biochemically characterized. McLic1 was purified to homogeneity with a purification fold of 3.1 and a recovery yield of 3.7 %. The purified enzyme was most active at pH 10.0 and 55 °C, and exhibited a wide range of pH stability (pH 4.0–10.0). McLic1 displayed strict substrate specificity for barley β-glucan, oat β-glucan and lichenan, but did not show activity towards other tested polysaccharides and synthetic p-nitrophenyl derivates, suggesting that it is a specific β-1,3-1,4-glucanase. The K _m values for barley β-glucan, oat β-glucan and lichenan were determined to be 0.69, 1.11 and 0.63 mg mL^?1, respectively. Moreover, the enzyme was stable in various non ionic surfactants, oxidizing agents and several commercial detergents. Thus, the alkaline β-1,3-1,4-glucanase may have potential in industrial applications, such as detergent, paper and pulp industries.     

Analysis of the expression of some stress induced genes in several commercial wine yeast strains at the beginning of vinification

AIMS: During fermentation yeast cells should cope with stress conditions. We pursue a better understanding of the stress response in wine yeasts at the beginning of vinification. METHODS AND RESULTS: We analyse by means of quantitative PCR the expression of several stress induced genes in 24 efficient commercial wine yeast strains at the beginning of vinifications performed under standard conditions or with small variations in pH and temperature. In all cases, high levels (with differences among strains) of GPD1 mRNA but quite low expression of other stress genes (TRX2, HSP104 and SSA3) were found. For all these genes, mRNA levels increase as temperature decreases or pH increases. CONCLUSIONS: Important levels of expression of GPD1 (but not of other stress genes) are required during the first hours of vinification, because of the need for glycerol production to counteract the hyperosmotic stress at this point. The differences among strains suggest that certain level of expression is enough to ensure the continuity of the process. Variations in the pH and temperature of the vinification can affect gene expression. SIGNIFICANCE AND IMPACT OF THE STUDY: A common pattern of stress response between efficient wine strains exists, which could be used as a criterion for selection. Studies of this kind can allow the establishment of connections between gene expression and physiological traits.     

Characterisation of a novel Bacillus sp. SJ-10 β-1,3–1,4-glucanase isolated from jeotgal, a traditional Korean fermented fish

A novel β-1,3–1,4-glucanase gene was identified in Bacillus sp. SJ-10 (KCCM 90078) isolated from jeotgal, a traditional Korean fermented fish. We analysed the β-1,3–1,4-glucanase gene sequence and examined the recombinant enzyme. The open reading frame of the gene encoded 244 amino acids. The sequence was not identical to any β-glucanases deposited in GenBank. The gene was cloned into pET22b(+) and expressed in Escherichia coli BL21. Purification of recombinant β-1,3–1,4-glucanase was conducted by affinity chromatography using a Ni-NTA column. Enzyme specificity of β-1,3–1,4-glucanase was confirmed based on substrate specificity. The optimal temperature and pH of the purified enzyme towards barley β-glucan were 50 °C and pH 6, respectively. More than 80 % of activity was retained at temperatures of 30–70 °C and pH values of 4–9, which differed from all other bacterial β-1,3–1,4-glucanases. The degradation products of barley β-glucan by β-1,3–1,4-glucanase were analysed using thin-layer chromatography, and ultimately glucose was produced by treatment with cellobiase.     

Degradative GH5 β-1,3-1,4-glucanase PpBglu5A for glucan in Paenibacillus polymyxa KF-1

A novel β-1,3-1,4-glucanase in the glycoside hydrolase family 5 (GH5) has been identified in the secretome of Paenibacillus polymyxa KF-1. The recombinant GH5 enzyme PpBglu5A shows broad substrate specificity, with strong lichenase activity, medium β-1,3-glucanase activity, and minimal cellulase activity. Barley β-glucan, lichenan, curdlan, and carboxymethyl cellulose are hydrolyzed to varying degrees by PpBglu5A, with the highest catalytic activity being observed with barley β-glucan. Hydrolysates from barley β-glucan or lichenan are primarily glucan oligosaccharides with degrees of polymerization from 2 to 4. PpBglu5A also hydrolyzes oat bran into oligosaccharides mainly consisted of di-, tri-, and tetra- oligosaccharides that are useful in the preparation of gluco-oligosaccharides. In addition to hydrolytic activity, transglycosylation was also observed with PpBglu5A and cellotriose as substrate. An in vitro assay indicated that the recombinant PpBglu5A has antifungal activity and can inhibit the growth of Canidia albicans. These results suggest that PpBglu5A exhibits unique properties and may be useful as an antifungal agent.     

Effect of the addition of inert cellulose substrates to grape must on Saccharomyces cerevisiae diversity and the evolution of alcoholic fermentation

AIMS: To study the addition of cellulose-based adjuvant as a resource to offset the negative effects produced by grape juice clarification during alcoholic fermentations. METHODS AND RESULTS: The effect of the addition of two kinds of inert cellulose substrates in white wine vinification was investigated in two different musts. In one of these musts, stuck fermentations were detected. One of the types of cellulose examined had a fining effect, which caused a decrease in the number of viable yeasts in the medium and altered the distribution and frequency of the clones, which performed the fermentation. The other cellulose substrate made the medium cloudier but did not alter the distribution of yeasts in comparison with the control. CONCLUSIONS: The behaviour of the inert cellulose substrates on vinification depends on its physical characteristics and its capacity for making the must cloudy. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of inert cellulose substrates in white wine vinification improves the fermentation process and the quality of wines obtained. This effect is more noticeable in difficult fermentations. One variety of cellulose showed an inhibitory effect on Torulaspora delbrueckii yeasts.     

Proteomic characterization of a wild-type wine strain of Saccharomyces cerevisiae

Saccharomyces cerevisiae is the optimal eukaryotic model system to study mammalian biological responses. At the same time Saccharomyces cerevisiae is also widely utilized as a biotechnological tool in the food industry. Enological Saccharomyces cerevisiae strains have been so far routinely analyzed for their microbiological aspects. Nevertheless, wine yeasts are gaining an increasing interest in the last years since they strongly affect both the vinification process and the organoleptic properties of the final product wine. The protein repertoire is responsible of such features and, consequently, 2D-PAGE can be an useful tool to evaluate and select optimal wine yeast strains. We present here the first proteomic map of a wild-type wine Saccharomyces cerevisiae strain selected for the guided fermentation of very high quality wines.     

Yeast strain effect on the concentration of major volatile compounds and sensory profile of wines from Vitis vinifera var. Treixadura

The fermentative ability of five Saccharomyces cerevisiae strains and their influence on the aroma and sensory properties of wine from Treixadura were evaluated to determine the most suitable yeast that produces a high quality wine from this grapevine variety. The results indicated that all strains, except T2, were able to lead the vinification process and have good fermentative powers. The chemical composition of wines obtained with resident cellar yeasts consisted of a significant amount of glycerol, a compound that contributes to the structure and smoothness in taste of the wine. In addition, these strains were good producers of acetates, ethyl esters and fatty acids, compounds that positively influence wine aroma. Compounds with a direct contribution to the aroma of Treixadura wines (Odour Activity Value >1) included 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol, isoamyl and ethyl acetate and the ethyl esters, ethyl butyrate, ethyl hexanoate, ethyl octanoate and ethyl decanoate. Sensory analysis supported the fact that cellar strains produced more fruity and floral wines than commercial or spontaneous fermentations. We conclude that resident cellar yeasts enhanced sensory quality of wines from Treixadura.     

Synthesis of β-1, 3-Glucan and β-1, 3 and β-1, 4-Mixed Glucan from UDP-α-d-Glucose

A particulate enzyme preparation from Phaseolus aureus (mung bean) seedlings catalyzed the synthesis of a water insoluble β-1,3-glucan from UDP-α-d-glucose (UDPG) at high concentrations (0.4~20 mm) and an alkaline insoluble β-1,3 and β-1,4-mixed glucan from UDPG at a low concentration (8.5 µm).

Furthermore, the two kinds of β-glucan synthetases which were investigated with two reaction systems at high and low concentrations of UDPG had different properties in optimal pH, stability of enzyme activity, and metallic ion requirement.     

Production and partial characterization of β-1,3-glucanase obtained from Rhodotorula oryzicola

The current study aims to assess the kinetics of population growth of Rhodotorula oryzicola and the production of β-1,3-glucanase (EC 3.2.1.39) enzyme by this yeast. It also aims to obtain the optimum conditions of β-1,3-glucanase enzymatic activity by varying the pH as well as to study the enzyme thermostability. R. oryzicola population doubled within 12?hr. During this period, 9.26 generations were obtained, with 1?hr and 29?min of interval from one generation to the other, with specific growth rate (µ) of 0.15 (hr^?1). The entire microorganism growth process was monitored during β-1,3-glucanases production, and the maximum value was obtained in the stationary phase in the 48-hr fermentation period. pH and temperature optimum values were 4.7 and 96°C, respectively. The enzyme maintained 88% of its activity when submitted to the temperature of 90°C for an incubation period of 1?hr. The results show that the enzyme can be used in industrial processes that require high temperatures and acidic pH.     

Absence of fks1p in lager brewing yeast results in aberrant cell wall composition and improved beer flavor stability

The flavor stability during storage is very important to the freshness and shelf life of beer. However, beer fermented with a yeast strain which is prone to autolyze will significantly affect the flavor of product. In this study, the gene encoding β-1,3-glucan synthetase catalytic subunit (fks1) of the lager yeast was destroyed via self-clone strategy. β-1,3-glucan is the principle cell wall component, so fks1 disruption caused a decrease in β-1,3-glucan level and increase in chitin level in cell wall, resulting in the increased cell wall thickness. Comparing with wild-type strain, the mutant strain had 39.9 and 63.41 % less leakage of octanoic acid and decanoic acid which would significantly affect the flavor of beer during storage. Moreover, the results of European Brewery Convention tube fermentation test showed that the genetic manipulation to the industrial brewing yeast helped with the anti-staling ability, rather than affecting the fermentation ability. The thiobarbituric acid value reduced by 65.59 %, and the resistant staling value increased by 26.56 %. Moreover, the anti-staling index of the beer fermented with mutant strain increased by 2.64-fold than that from wild-type strain respectively. China has the most production and consumption of beer around the world, so the quality of beer has a significant impact on Chinese beer industry. The result of this study could help with the improvement of the quality of beer in China as well as around the world.     

Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts

β-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of β-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or β-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for β-1,3-1,4 glucans, showing maximum activity on barley β-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley β-glucan and lichean were 3-O-β-cellotriosyl-D-glucose and 3-O-β-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for β-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase.     

Production of extracellular β-1,3-/β-1,6-glucan by mono- and dikaryons ofSchizophyllum commune

From the dikaryotic mycelium ofSchizophyllum commune ATCC 38548 several monokaryotic strains were obtained by isolating the two types of monokaryotic protoplasts and their reversion to hyphal growth. The dikaryoticS. commune ATCC 38548 produced about 10 g/liter of extracellular β-1,3-/β-1,6-glucan (schizophyllan) after 96 h of growth, while the monokaryons excreted much less of this polysaccharide. During growth of strains of both types of monokaryons indigo and β-1,3-glucanase activities were excreted. Two selected monokaryons were mated with other monokaryoticS. commune strains and some of the dikaryotic mycelia obtained produced about 12 g/liter of extracellular β-1,3-/β-1,6-glucan after 120 h of cultivation.