用酸碱法从面包酵母中提取β-(1-3)-D葡聚糖

研究了用酸-碱法制备水不溶性葡聚糖的工艺.0.75 mol/L的NaOH在100℃下作用2 h,能有效地去除甘露聚糖,0.5 mol/L的酸使不溶性的葡聚糖的比粘度增加,除去糖元.分析表明,产品为β-D-葡聚糖,其中β-(1-3)-D-葡聚糖约为87%.     

用酸碱法从而包酵母中提取β——（1——3）——D葡聚糖

研究了用酸－碱法制备水不溶性葡聚糖的工艺。０．７５ｍｏｌ／Ｌ的ＮａＯＨ在１００℃作用２ｈ,能有效地去除甘露聚糖,０．５ｍｏｌ／Ｌ的酸使不溶性的葡聚糖的比粘度增加,除去糖元,分析表明,产品为β－Ｄ－葡聚糖,其中β－（１－３）－Ｄ－葡聚糖约为８７％。     

用废啤酒酵母制备碱不溶性葡聚糖的研究

研究了用碱法及酶－碱法从啤酒厂排弃的酵母泥中制备碱不溶性葡聚糖的工艺。对NaOH及蛋白酶用量的影响,NaOH作用时间、浓度、作用次数等因素的影响进行了较系统的探讨。先用蛋白酶处理酵母可减少NaOH用量从而减轻环境污染。对多糖产品进行了定性定量分析,得到的产品主要为β（1→3）-D-葡聚糖。     

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。     

灵芝提取物β-1,3-葡聚糖含量测定及多糖成分分析

采用酶法试剂盒检测法和苯胺蓝荧光检测法对不同结构多糖中β-葡聚糖含量进行测定,结果表明,两种方法对高纯度β-1,3/1,6-葡聚糖的含量检测均较为准确,但含有葡萄糖的杂多糖和多分支连接的β-葡聚糖均会对酶法试剂盒检测产生干扰,苯胺蓝荧光法检测β-1,3-葡聚糖专一性较好。通过比较不同灵芝提取物中β-1,3-葡聚糖的含量和得率可知,热水提取物和KOH提取物中β-1,3-葡聚糖的含量较高,分别为20.52%和45.86%,β-1,3-葡聚糖得率分别为0.27%和1.27%,占β-1,3-葡聚糖总提取得率的88%。HPSEC分析结果显示不同提取物中多糖的分子量分布不同,热水提取物中主要包含2个组分,分子量分别为2.835×10~6Da和9.587×10~4Da,KOH提取物中主要包含分子量分布在1×10~5–1×10~6Da范围内的3个组分。各提取物中多糖的单糖组成均以葡萄糖为主,水提多糖中含有少量半乳糖,酸碱提取多糖中含有少量木糖和甘露糖。本研究结果显示,分步提取中沸水和KOH碱溶液提取β-1,3-葡聚糖效率较高。     

外源物质对灵芝多糖和β-葡聚糖发酵的影响

研究了14种外源物质（化合物）对灵芝细胞生长和发酵合成多糖和β-葡聚糖的影响。结果表明,连翘水提物（3g/L）对灵芝细胞生长具有显著促进作用;薏苡仁酯（3g/L）对灵芝胞内多糖和β-葡聚糖的合成均具有促进作用;而桔梗水提物、硝酸铈铵、硝酸镨、茉莉酸甲酯和硝普钠对灵芝细胞生长和产物合成均具有抑制作用。进一步通过Box-Behnken试验设计和响应面法分析,建立了添加薏苡仁酯发酵产β-葡聚糖的二次多项式模型,经分析得到产β-葡聚糖的最优条件为：薏苡仁酯添加量10.5g/L、接种量16%、添加时间第88小时、发酵初始pH 7.00。在此条件下获得β-葡聚糖的产量可达(40.67±8.43)mg/L,与未添加薏苡仁酯的对照组相比,提高了41.86%;多糖产量为(0.99±0.21)g/L,与对照组相比,提高了31.99%。结果提示所得添加薏苡仁酯的优化条件可定向诱导灵芝β-葡聚糖的合成,同时也表明在灵芝液体发酵体系中添加薏苡仁酯发酵产多糖和β-葡聚糖具有一定的实用价值。     

乙醇-酶体系预处理结合水提法提取灵芝中β-葡聚糖的研究

采用乙醇-酶预处理体系,结合水提法较系统地研究了灵芝子实体中β-葡聚糖的提取技术。结果表明,乙醇-酶体系预处理的关键参数为:乙醇质量分数60%(V/V),加酶量1.5%(M/V,g/m L)、酶解温度45℃、酶解p H8.0;在乙醇-酶体系预处理的基础上,进一步采用单因素试验和Box-Benhnken试验设计与响应面分析法对灵芝子实体中β-葡聚糖的热水提取工艺进行了优化。结果显示水提温度、提取时间、水料比3个因素及水提温度与水料比二者的交互作用对β-葡聚糖的提取有显著影响。经优化后获得3个核心因素的最佳水平为:提取温度80℃、提取时间2.5h、水料比35:1。在乙醇-酶预处理结合水提取条件下,灵芝子实体中β-葡聚糖的提取得率可达0.412mg/g,是传统无水乙醇回流预处理结合水提法提取β-葡聚糖得率的2.1倍。本研究为灵芝β-葡聚糖的进一步提取放大试验奠定了技术基础。     

不同处理方法对灵芝β-葡聚糖降解效果的比较研究

本研究采用酸法、碱法、酶法和微波法对灵芝β-葡聚糖进行降解,通过降解率、产物分子量变化、产物聚合度分布等指标比较了不同方法的降解效果。结果表明,微波法降解率高达94%,处理后产物的分子量明显降低,寡糖产物聚合度分布广。酶法降解率约为40%,寡糖产物中含有DP2-5的成分。酸法及碱法降解率低于20%,寡糖产物少。研究表明,与其他3种方法相比,微波法降解率高、产物丰富、操作条件易于控制,是一种简单、高效的降解灵芝β-葡聚糖、制备灵芝β-葡寡糖的方法。     

血浆(1-3)-β-D-葡聚糖诊断深部真菌感染的临床研究

目的探讨血浆(1-3)-β-D-葡聚糖对早期诊断深部真菌感染的临床价值。方法收集2009年3月～11月间在北京友谊医院感染科住院患者174例,根据侵袭性真菌感染诊断标准,将患者分为排除深部真菌组、确诊组、临床诊断组、拟诊组。应用MB-80微生物动态快速检测系统,检测各组患者血浆(1-3)-β-D-葡聚糖水平,分析比较深部真菌感染组和非深部真菌感染组血浆(1-3)-β-D-葡聚糖的水平。结果深部真菌感染组血清(1-3)-β-D-葡聚糖含量为(153.4±37.0)pg/mL,排除深部真菌感染组血清(1-3)-β-D-葡聚糖含量为(54.6±8.6)pg/mL,两组间有统计学差异(t=3.4,P<0.01);分析血浆(1-3)-β-D-葡聚糖诊断深部真菌感染,以20 pg/mL为诊断阈值,其准确率、敏感性、特异性、阳性预测值、阴性预测值分别为70.1%、87.5%、61.9%、52.1%、91.2%。确诊病例、临床诊断病例、拟诊病例,3组间血清(1-3)-β-D-葡聚糖含量无统计学差异(P>0.05)。结论深部真菌感染组的葡聚糖水平明显高于排除深部真菌感染组的葡聚糖水平,具有统计学意义。以20 pg/mL为诊断阈值,其准确率、敏感性、特异性、阳性预测值、阴性预测值分别为70.1%、87.5%、61.9%、52.1%、91.2%,可作为深部真菌感染的最佳诊断阈值。     

灵芝菌丝体中β-葡聚糖的提取

比较了热水回流法、热水-酶回流法、超声波法以及超声波-酶水解法等4种提取方法对灵芝菌丝体中β-葡聚糖提取得率的影响。结果表明,采用超声波-酶水解法所得β-葡聚糖的得率最大,影响提取的关键因素为超声波功率(A),水料比(B),p H(C)和加酶量(D)。进一步采用Box-Behnken设计及响应面分析法对这4个因素进行了优化。结果显示,因素A、B、C对β-葡聚糖提取得率的影响达到极显著水平(P0.01),但因素D和所有因素之间的交互作用并不显著(P0.05)。通过回归拟合,建立了预测灵芝β-葡聚糖提取的多项式模型。经响应面最优分析,获得4个因素的最佳水平为:超声波功率275.54W,水料比25.62:1(V/M,m L/g),p H 7.17,加酶量1.05%。经实际试验验证,在该条件下,灵芝菌丝体β-葡聚糖得率可达5.50mg/g。     

Analysis of wall glucans from yeast, hyphal and germ-tube forming cells of Candida albicans

Acid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germ-tube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained beta(1----3)- and beta(1----6)-chains but no mixed intra-chain beta(1----3)/(1----6) linkages. Methylation and 13C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (67.0% to 76.6%) of beta(1----6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29.6% to 38.9% beta(1----3) and 43.3% to 53.2% beta(1----6) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67.0% beta(1----3) and 14% beta(1----6) linkages. Branch points accounted for 6.7%, 12.3% and 17.4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively.     

浓盐法与稀碱法在啤酒废酵母中提取RNA的研究

通过从啤酒废水生产的酵母中提取核糖核酸的实验,对稀碱法和浓盐法从操作步骤、产品产率等几方面进行比较,从而得出浓盐法优于稀碱法的结论。     

Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.     

Comparison of beta-glucan structures in a cell wall mutant of Saccharomyces cerevisiae and the wild type

A mutant of Saccharomyces cerevisiae defective in the cell wall beta-glucan structure was obtained. The mutant cells are extremely sensitive to (beta 1-3)-glucanase digestion and mild alkali treatment. Structural analysis revealed that the alkali-insoluble, skeletal glucan from wild type cells contains two components, a (beta 1-3) linked glucan with a laminated structure, and a highly branched glucan containing predominantly (beta 1-6) linkages. The mutant cells lack the latter component.     

Evidence for the periplasmic location of glycogen in Saccharomyces

Treatment of yeast cells with hot alkali fails to solubilize a significant amount of glycogen. The insoluble glycogen is readily hydrolyzed by insolubilized α-amylase indicating that the apparent insolubility of this glycogen fraction does not result from its physical entrapment within an insoluble glucan membrane. The alkali-insoluble glycogen fraction of glutaraldehyde treated-cells is rapidly degraded by a mixture of snail gut enzymes during the formation of spheroplasts but the alkali-soluble glycogen fraction is unaffected. These results indicate that a major fraction of yeast glycogen is located outside the cellular membrane.     

Natural Abundance 13C Nuclear Magnetic Resonance Spectra of Intact Fungal Mycelia

We examined the change of the composition of the cell wall polysaccharides prepared from cells of the salt-tolerant yeast Zygosaccharomyces rouxii grown in two media containing 20% NaCl and 0% NaCl. Comparative analysis of their walls showed that the wall obtained from salt-free medium had greater quantities of alkali-insoluble fraction and smaller quantities of mannan than the walls obtained from 20% NaCl medium. The alkali-insoluble fractions from the cell walls obtained from salt-free medium contained a large amount of glucosamine and a smaller amount of linear β-1,3-glucosidic linkage than the fractions from the cell walls obtained from 20% NaCl medium. Structural analyses showed that the mannans from each cell wall contained an α-1,6-mannbsidic linked backbone to which single mannose and mannobiose units were connected as side chains by α-1,2-mannosidic linkages. However, when cells were grown in the presence of 20% NaCl, the side chains of the mannans consisting of a mannobiose unit were largely reduced.

These results indicated that the structure of alkali-insoluble glucan and mannan were greatly affected by the presence of NaCl in the final medium.     

Cell wall composition of the yeast and mycelial forms of Paracoccidioides brasiliensis

Isolation and chemical analyses of the cell walls of the yeast (Y form) and mycelial forms (M form) of Paracoccidioides brasiliensis and Blastomyces dermatitidis revealed that their chemical composition is similar and depends on the form. Lipids, chitin, glucans, and proteins are the main constituents of the cell walls of both forms of these fungi. There is no significant difference in the amount of lipids (5 to 10%) and glucans (36 to 47%) contained by the two forms. In both fungi, the Y form has a larger amount of chitin (37 to 48%) than the M form (7 to 18%), whereas the M form has a larger amount of proteins (24 to 41%) than the Y form (7 to 14%). Several properties of the glucan of P. brasiliensis were studied. Almost all of the glucan in the Y form was soluble in 1 n NaOH, was weakly positive in the periodic acid-Schiff reaction, was not hydrolyzed by snail digestive juice, and had alpha-glycosidic linkage. Glucans of the M form were divided into alkali-soluble (60 to 65%) and alkali-insoluble (35 to 40%) types. The alkali-soluble glucan was similar to that of the Y form; the alkali-insoluble glucan was positive in the periodic acid-Schiff reaction and was hydrolyzed by snail digestive juice.     

(1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin

The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430). The (1-->6)-beta-D-glucan was purified from cell walls of C. boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor. Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min. Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin.     

Presence of a large β(1-3)glucan linked to chitin at the Saccharomyces cerevisiae mother-bud neck suggests involvement in localized growth control

Previous results suggested that the chitin ring present at the yeast mother-bud neck, which is linked specifically to the nonreducing ends of β(1-3)glucan, may help to suppress cell wall growth at the neck by competing with β(1-6)glucan and thereby with mannoproteins for their attachment to the same sites. Here we explored whether the linkage of chitin to β(1-3)glucan may also prevent the remodeling of this polysaccharide that would be necessary for cell wall growth. By a novel mild procedure, β(1-3)glucan was isolated from cell walls, solubilized by carboxymethylation, and fractionated by size exclusion chromatography, giving rise to a very high-molecular-weight peak and to highly polydisperse material. The latter material, soluble in alkali, may correspond to glucan being remodeled, whereas the large-size fraction would be the final cross-linked structural product. In fact, the β(1-3)glucan of buds, where growth occurs, is solubilized by alkali. A gas1 mutant with an expected defect in glucan elongation showed a large increase in the polydisperse fraction. By a procedure involving sodium hydroxide treatment, carboxymethylation, fractionation by affinity chromatography on wheat germ agglutinin-agarose, and fractionation by size chromatography on Sephacryl columns, it was shown that the β(1-3)glucan attached to chitin consists mostly of high-molecular-weight material. Therefore, it appears that linkage to chitin results in a polysaccharide that cannot be further remodeled and does not contribute to growth at the neck. In the course of these experiments, the new finding was made that part of the chitin forms a noncovalent complex with β(1-3)glucan.     

Cellulose synthesis by extracts of Acanthamoeba castellanii during encystment. Stimulation of the incorporation of radioactivity from UDP-(14C)glucose into alkali-soluble and insoluble beta-glucans by glucose 6-phosphate and related compounds.

1. The activity of a particulate enzyme prepared from encysting cells of Acanthamoeba castellanii (Neff), previously shown to catalyze the incorporation of glucose from UDP-[14C]glucose into both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans, was stimulated several fold by glucose-6-phosphate and several related compounds. 2. Incorporation was observed when [14C]glucose-6-P was incubated with the particles in the presence of UDP-glucose. The results of product analysis by partial acid hydrolysis indicated that glucose-6-P stimulates the formation of both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans from UDP-[14C]glucose and was itself incorporated into an alkali-insoluble beta-(1 leads to 4)glucan. 3. When particles incubated with UDP-[14C]glucose and glucose-6-P were reisolated and then reincubated with unlabeled UDP-glucose and glucose-6-P, a loss of counts from the alkali-soluble fraction was detected along with a corresponding rise in the radioactivity of the alkali-insoluble fraction. This suggests that the alkali-soluble beta-glucan was converted to an alkali-insoluble product and possibly may be an intermediate stage in cellulose synthesis.