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。     

内切β-1,3-葡聚糖酶产生菌的分离筛选与鉴定

以茯苓多糖为碳源,以蓝色茯苓多糖为内切β－1,3－葡聚糖酶活力测定底物,从不同样品中筛选到4株产内切β－1,3－葡聚糖酶活力较高的菌株,经TLC分析酶解产物表明其中三株的酶解产物主要为二糖、三糖、四糖,另外一株的酶解产物主要为二糖。对其中一株内切酶活力最高（3．73U2/ml）的菌株经鉴定为生孢噬胞菌（Sporocytophaga)。     

微生物1,3-1,4-β-葡聚糖酶蛋白质改造及工业应用研究进展

1,3-1,4-β-葡聚糖酶(E.C.3.2.1.73)是一种重要的工业用酶,其可以通过特异性切割毗邻β-1,3-糖苷键的β-1,4-糖苷键将β-葡聚糖或地衣多糖降解为纤维三糖和纤维四糖。微生物β-葡聚糖酶属于糖苷水解酶家族16,其三维结构为卷心蛋糕状的逆向β-片层结构。文中综述了近些年来β-葡聚糖酶在工业上的应用情况及酶蛋白质工程改造的研究进展,并对其研究前景进行了展望。     

β-1,3-葡聚糖酶的研究和应用

β-1,3-葡聚精酶的生产菌种由冻土毛霉经紫外线诱变获得,据正交试验确定培养条件。经硫酸铵盐析和SephadexG-100柱层析,酶得到纯化,并对酶的性质进行了研究。酶对酵母自溶有明显促进作用,使自溶产物的游离氨基氮含量、还原糖含量、总蛋白质得率、干物质得率均得以提高。     

沙质微泡菌β-1,3(4)-葡聚糖酶的克隆表达及酶学性质

【背景】β-葡聚糖是自然界中广泛存在的非淀粉多糖,是谷类植物细胞壁的主要成分。β-葡聚糖酶能够水解β-葡聚糖生成低聚合度的寡糖,在食品、饲料、造纸等领域发挥着重要的作用。【目的】从海洋细菌沙质微泡菌(Microbulbifer arenaceous)中克隆到一个β-1,3(4)-葡聚糖酶基因,在大肠杆菌中可溶表达,研究其相关酶学性质。【方法】以沙质微泡菌(Microbulbifer arenaceous)基因组DNA为模板,克隆一个β-1,3(4)-葡聚糖酶基因(MaGlu16A),构建重组表达载体p ET-28a-MaGlu16A并在大肠杆菌BL21(DE3)中诱导表达,通过Ni-NTA亲和层析纯化后进行酶学性质研究。【结果】MaGlu16A的最适pH和最适温度分别为pH 6.0和40°C,在pH 5.0-10.5和35°C以下稳定。对EDTA具有较高的抵抗性,在1 mmol/L和10 mmol/L EDTA浓度下仍保持99.3%和82.5%的酶活力。该酶能够有效水解可得然多糖、昆布多糖、大麦葡聚糖、地衣多糖、燕麦葡聚糖和酵母葡聚糖,水解产物主要为葡萄糖、二糖、三糖和四糖。【结论】海洋细菌沙质微泡菌(Microbulbiferarenaceous)来源β-1,3(4)-葡聚糖酶的克隆表达及酶学性质的测定为β-葡聚糖酶的挖掘及β-葡寡糖的制备奠定了基础。     

番茄感染TMV诱导的β-1,3-葡聚糖酶的纯化和性质

番茄系统感染TMV诱导对胞外β—1,3—葡聚糖酶活性升高。番茄叶胞外提取液经冰冻干燥浓缩、-20℃丙酮沉淀、CM-Sephadex C-25离子交换层析和PBE 94聚焦层析纯化,获得PAGE和SDS-PAGE均一的β—1,3—葡聚糖酶。测得该酶的分子量为22kD;以昆布多糖为底物,该酶的最适pH5.4,最适温度30～40℃;K_m和V_(max)值分别为5.64mg/ml和 0.328nmol/s。在感染TMV的番茄叶中,β—1,3—葡聚糖酶活力大部分位于胞外,它是番茄叶胞外提取液中主要的病原相关蛋白。     

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。     

β—1,3—葡聚糖酶的研究和应用

β-1,3-葡聚精酶的生产菌种由冻土毛霉经紫外线诱变获得,据正交试验确定培养条件。经硫酸铵盐析和SephadexG-100柱层析,酶得到纯化,并对酶的性质进行了研究。酶对酵母自溶有明显促进作用,使自溶产物的游离氨基氮含量、还原糖含量、总蛋白质得率、干物质得率均得以提高。     

白菜型冬油菜β-1,3-葡聚糖酶基因在低温胁迫下的表达

为探明β-1,3-葡聚糖酶基因(β-1,3-glucanase)对油菜(Brassica campestris)抵御低温胁迫能力的作用,通过蛋白质谱分析得到β-1,3-葡聚糖酶蛋白,采用RT-PCR技术克隆白菜型冬油菜(B.rapa)陇油6号和天油4号β-1,3-葡聚糖酶的c DNA序列;并对该序列进行生物信息学分析;进而采用实时荧光定量PCR及半定量PCR检测β-1,3-葡聚糖酶基因在低温胁迫下的表达模式。结果获得长度为1 032 bp的陇油6号β-1,3-葡聚糖酶基因开放阅读框,编码343个氨基酸,相对分子量为38.102k Da,理论等电点为6.63,其与菜心(B.rapa subsp.chinensis)和甘蓝型油菜(B.napus)的蛋白质氨基酸序列同源性高达93.94%。该基因编码的酶是一个主要由α-螺旋组成的亲水性稳定蛋白,含有1个信号肽,存在2个跨膜结构域。该基因在进化上高度保守,其保守序列属于植物的糖基水解酶家族17特有的保守结构域。β-1,3-葡聚糖酶基因表达模式分析显示,4°C时该基因上调表达,继续低温(–4°C)胁迫处理,该基因上调表达至峰值,至–8°C时其表达下调。研究表明从白菜型冬油菜中克隆的β-1,3-glucanase在冬油菜品种陇油6号抗寒过程中发挥作用。     

解淀粉芽孢杆菌β-1,3-1,4-葡聚糖酶的高效表达

目的:克隆解淀粉芽孢杆菌β-1,3-1,4-葡聚糖酶基因(bglA)使其在解淀粉芽孢杆菌CICIM B4081中高效表达,并对重组酶进行酶学性质研究.方法:以解淀粉芽孢杆菌(CICIM B4801)染色体DNA为模板,经过PCR扩增得到了大小约为0.8kb的β-1,3-1,4-葡聚糖酶基因(bglA),构建了重组表达质粒pQ-bglA,通过电转化的方法将其转化人解淀粉芽孢杆菌(CICIM B4801)中.结果:得到了能高效表达β-1,3-1,4-葡聚糖酶的重组解淀粉芽孢杆菌.在250mL摇瓶条件下,重组菌分解地衣多糖的胞外最高酶活达到了1515.7U/mL,重组酶的最适作用温度为55℃,最适反应pH值为6.5.结论:重组菌的β-1,3-1,4-葡聚糖酶的酶活为原始菌株的11.84倍,实现了bglA基因在解淀粉芽孢杆菌中的高效表达.     

1,3-Beta-glucan synthase in cell-free extracts from mycelium and protoplasts of Sclerotium glucanicum

Conditions for obtaining stable protoplasts from Sclerotium glucanicum and their reversion to hyphal growth were determined. 1,3-beta-Glucan synthase activity was detected in particulate enzyme fractions from mycelium and protoplasts of Scl. glucanicum. UDP-[U-14C]glucose was linearly incorporated into a beta-glucan for about 20 min at 25 degrees C. Optimum pH and temperature values, as well as thermal stabilities of the 1,3-beta-glucan synthase activity, were determined. High concentrations of EDTA were inhibitory. Enzyme activity was stimulated by ATP and GTP. The apparent Km value for UDP-glucose was 0.54 mM. The reaction product was characterized as 1,3-beta-glucan by 13C NMR spectroscopy and hydrolysis products of an exo-1,3-beta-glucanase.     

Dilute-acid hydrolysis for fermentation of the Bolivian straw material Paja Brava

Hydrolysis of the straw material Paja Brava, a sturdy grass characteristic for the high plains of Bolivia, was studied in order to find suitable conditions for hydrolysis of the hemicellulose and cellulose parts. Dried Paja Brava material was pre-steamed, impregnated with dilute sulfuric acid (0.5% or 1.0% by wt), and subsequently hydrolyzed in a reactor at temperatures between 170 and 230 degrees C for a reaction time between 3 and 10 min. The highest yield of xylose (indicating efficient hydrolysis of hemicellulose) were found at a temperature of 190 degrees C, and a reaction time of 5-10 min, whereas considerably higher temperatures (230 degrees C) were needed for hydrolysis of cellulose. Fermentability of hemicellulose hydrolyzates was tested using the xylose-fermenting yeast species Pichia stipitis, Candida shehatae and Pachysolen tannophilus. The fermentability of hydrolyzates decreased strongly for hydrolyzates produced at temperatures higher than 200 degrees C.     

Cell Wall Glucans of the Yeast and Mycelial Forms of Paracoccidioides brasiliensis

Glucans were isolated from the cell wall of the yeast (Y) and mycelial (M) forms of Paracoccidioides brasiliensis. The alkali-soluble glucan of the Y form had properties of alpha-1,3-glucan. The alkali-insoluble glucan of the M form was identified as a beta-glucan which contains a beta-(1 --> 3)-glycosidic linkage by infrared absorption spectrum, by effect of beta-1,3-glucanase, and by partial acid hydrolysis. The alkali-soluble glucans of the M form were a mixture of alpha- and beta-glucans and the ratio of alpha- to beta-glucan was variable, depending on the preparations.     

Sequencing of a 1,3-1,4-beta-D-glucanase (lichenase) from the anaerobic fungus Orpinomyces strain PC-2: properties of the enzyme expressed in Escherichia coli and evidence that the gene has a bacterial origin.

A 971-bp cDNA, designated licA, was obtained from a library of Orpinomyces sp. strain PC-2 constructed in Escherichia coli. It had an open reading frame of 738 nucleotides encoding LicA (1,3-1,4-beta-D-glucanase; lichenase) (EC 3.2.1.73) of 245 amino acids with a calculated molecular mass of 27,929 Da. The deduced amino acid sequence had high homology with bacterial beta-glucanases, particularly in the central regions and toward the C-terminal halves of bacterial enzymes. LicA had no homology with plant beta-glucanases. The genomic DNA region coding for LicA was devoid of introns. More than 95% of the recombinant beta-glucanase produced in E. coli cells was found in the culture medium and periplasmic space. A N-terminal signal peptide of 29 amino residues was cleaved from the enzyme secreted from Orpinomyces, whereas 21 amino acid residues of the signal peptide were removed when the enzyme was produced by E. coli. The beta-glucanase produced by E. coli was purified from the culture medium. It had a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gels. The Km and Vmax values with lichenin as the substrate at pH 6.0 and 40 degrees C were 0.75 mg/ml and 3,790 micromol/min/mg, respectively. With barley beta-glucan as the substrate, the corresponding values were 0.91 mg/ml and 5,320 micromol/min/mg. This enzyme did not hydrolyze laminarin, carboxymethylcellulose, pustulan, or xylan. The main products of lichenin and barley beta-glucan hydrolysis were triose and tetraose. LicA represented the first 1,3-1,4-beta-D-glucanase reported from fungi. The results presented suggest that licA of Orpinomyces had a bacterial origin.     

Effect of beta-glucanases on Penicillium oxalicum cell wall fractions

The polysaccharidic effect of a purified 1,3-beta-glucanase, a purified beta-glucosidase, and of partially purified endo-1,3-beta-glucanase from autolysed Penicillium oxalicum cultures on cell wall isolate fractions from the same fungus were studied. Fractionation of 5-day-old cell wall gave rise to a series of fractions that were identified using infrared spectrophotometry. The fractions used were: F1, an alpha-glucan; F3, a beta-glucan; F4, a chitin-glucan; and F4b, a beta-glucan. The fractions were incubated with each of the enzymes and with a mixture of equal parts of the three enzymes and the products of the enzymatic hydrolysis were analyzed after 96 h incubation. The enzymes were found to degrade fraction F4b (beta-glucan); the greatest degree of hydrolysis was reached when the three enzymes were used together, suggesting the need for synergic action by these enzymes in the cell wall degradation process.     

Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

The pH and temperature dependence of the activity of the high Km cyclic nucleotide phosphodiesterase of bakers' yeast

The hydrolysis of adenosine 3':5'-monophosphate by the high Km cyclic nucleotide phosphodiesterase of bakers' yeast was studied over a range of temperature and pH at I = 0.17. The effects of ionic strength and MgCl2 concentration were studied at pH 7.7 and 30 degrees C. Km and Vmax were insensitive to changes in the MgCl2 concentration between 1 and 30 mM, implying that this enzyme (which does not require free divalent metal ions) does not discriminate between free cyclic AMP- and the Mg-cyclic AMP+ complex. Vmax decreased below pH 6.8 because of protonation of a group required in the basic form in the enzyme x substrate complex. On the basis of its pK (5.46 at 30 degrees C) and delta H (23 kJ/mol) this group was tentatively identified as imidazole. Vmax/Km decreased above pH 6.8 because of ionization of a group required in the acid form in the free enzyme, with a pK of 7.88 at 30 degrees C and a delta H of about 13 kJ/mol. Several possibilities exist for the identity of this group, the most likely being a second imidazole, sulfhydryl, or a water molecule bonded to tightly bound zinc. At pH 7.90, log Vmax and log Km both changed linearly with 1/T (between 12 degrees C and 37 degrees C) with enthalpies of 47 and 55 kJ/mol, respectively. Consequently, at low enough cyclic AMP concentration, the rate of reaction at pH 7.90 decreases slightly when the temperature is increased. This is also true at higher pH, but in the physiological pH range (6.4 to 7.5) Vmax/Km and, therefore, the rate of reaction at very low cyclic AMP concentration were nearly independent of temperature. Under physiological conditions, the Km approaches the upper limit of in vivo cyclic AMP concentrations in yeast, and at normal in vivo cyclic AMP concentrations the pH optimum is within or below the physiological range of pH in yeast.     

Value-added subcritical water hydrolysate from rice bran and soybean meal

New value-added product was derived from agricultural by-products: rice bran and soybean meal by means of subcritical water (SW) hydrolysis. The effect of temperature (200-220 degrees C), reaction time (10-30 min), raw material-to-water weight ratio (1:5 and 2:5), was determined on the yields of protein, total amino acids, and reducing sugars in the soluble products. The suitable hydrolysis time was 30 min and the proper weight ratio of the raw material-to-water was 1:5. The reaction temperature suitable for the production of protein and amino acids was 220 degrees C for raw and deoiled rice bran, 210 degrees C for raw soybean meal, and 200 degrees C for deoiled soybean meal. The products were also found to have antioxidant activity as tested by ABTS(.)(+) scavenging assay. In addition, sensory evaluation of milk added with the hydrolysis product of deoiled rice bran indicated the potential use of the product as a nutritious drink.     

Effects of temperature on allosteric and catalytic properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

We have investigated effects of temperature on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Vmax for cAMP and cGMP increased as assay temperature increased from 5 to 45 degrees C. At substrate concentrations below Kmapp, however, hydrolysis increased as temperature decreased from 45 to 5 degrees C and was much greater at 5 degrees C than at 45 degrees C. As assay temperature decreased, Kmapp for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were approximately 1.9 at 45 degrees C and 1.2-1.0 at 5 degrees C. cGMP stimulated hydrolysis of 0.5 microM [3H]cAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like Vmax, was lowest at 5 degrees C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (K alpha app) for cGMP stimulation was lower at 5 degrees C than at 45 degrees C. Thus, affinity for both substrate and effector was increased at 5 degrees C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity" state. That cGMP stimulated cAMP hydrolysis at 5 degrees C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 degrees C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 microM [3H] cAMP) concentrations. At higher substrate concentrations (50 microM [3H]cAMP), with the enzyme in the "activated" state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 degrees C, as at 30 degrees C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 microM [3H]cAMP and reduced hydrolysis of 50 microM [3H]cAMP. At 5 degrees C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 microM [3H]cAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 degrees C than at 45 degrees C. Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.     

Extraction of protein and amino acids from deoiled rice bran by subcritical water hydrolysis

This study investigated the production of value-added protein and amino acids from deoiled rice bran by hydrolysis in subcritical water (SW) in the temperature range between 100 and 220 degrees C for 0-30 min. The results suggested that SW could effectively be used to hydrolyze deoiled rice bran to produce useful protein and amino acids. The amount of protein and amino acids produced are higher than those obtained by conventional alkali hydrolysis. The yields generally increased with increased temperature and hydrolysis time. However, thermal degradation of the product was observed when hydrolysis was carried out at higher temperature for extended period of time. The highest yield of protein and amino acids were 219 +/- 26 and 8.0 +/- 1.6 mg/g of dry bran, and were obtained at 200 degrees C at hydrolysis time of 30 min. Moreover, the product obtained at 200 degrees C after 30 min of hydrolysis exhibited high antioxidant activity and was shown to be suitable for use as culture medium for yeast growth.