差示扫描量热法直接测定抗冻蛋白质溶液的热滞效应

文献上一直用显微镜观察法等测定抗冻蛋白的热滞效应,其冰晶量是靠观察晶核体积估算得得的,人为性很大,用并示扫描量热法直接测定沙冬青抗冻蛋白质溶液的热滞效应,通过熔融焓和冻结焓值准确地测定了冰晶含有量和热滞温度。同文献比较,沙冬青ＡＦＰ具有极高的抗冻活性,而且开创了一个新的有效的测量抗冻蛋白抗冻活性的途径。     

热变性前后水合醛缩酶中水状态的热力学研究

 用差示扫描量热计,测定了不同水合态(水含量h从0.08g/g至2.50g /g)冻结的水合醛缩酶样品中冰的熔化温度和熔化焓,并计算出熔化熵。实验结果表明,变性前,水合醛缩酶中的水转化成四种状状:(1)不冻结水,(2)熔化焓和熔化温度均低了普通水的可冻结水,(3)熔化焓与普通水相同而熔化温度低于普通水的可冻结水,(4)熔化焓和熔化温度均与普通水相同的容积水。变性后,水合醛缩酶中的水可处于五种状态,即不冻结水,熔化焓低于普通水而熔化温度与普通水相同的可冻结水,以及熔化温度均低于普通水且彼此不相同的三种状态的可冻结水。实验还观察到,水合醛缩酶在热变性前后,水在这些态中的量是不同的。     

嗜热古菌S-腺苷甲硫氨酸合成酶的序列分析、基因克隆与异源表达

将Methanopyrus sp.SNP6进行功能基因组测序,获得S-腺苷甲硫氨酸合成酶基因(sam)序列,并进行序列分析。将sam基因扩增后连接至表达质粒p ET-28b(+),转化E.coli BL21(DE3),获得重组菌后进行诱导表达。生物信息学分析表明,sam基因编码蛋白的理论分子量为44 086.4Da,三级结构为同源四聚体,与其他相关古菌来源的S-腺苷甲硫氨酸合成酶的蛋白序列较保守。实验结果显示,构建的重组表达质粒p ET28b(+)-sam可在E.coli BL21(DE3)宿主菌中高水平表达。重组蛋白的分子量与预期值基本一致,部分为胞内可溶性表达,另一部分以包涵体形式存在。本研究首次实现了Methanopyrus sp.SNP6菌株S-腺苷甲硫氨酸合成酶的异源表达,为后期的蛋白纯化、酶学性质研究和酶促转化法生产S-腺苷甲硫氨酸奠定了理论基础。     

第一类鱼抗冻蛋白对冰晶生长界面层结构的影响

根据冰晶在水溶液中生长的基本热力学性质,应用多层界面模型,分别得到了冰晶在纯水及抗冻蛋白溶液中生长界面层的吉布斯自由能.由冰晶生长界面层的吉布斯自由能,分析了冰晶在三种不同第一类鱼抗冻蛋白分子溶液中,热平衡状态下生长界面层的微观平衡结构,发现冰晶在抗冻蛋白溶液中生长与其在纯水中生长相比,界面层结构有明显变化,结合抗冻蛋...     

明绿豆活性蛋白的分离纯化及抗菌性鉴定

利用25%及75%饱和度的硫酸铵沉淀、阳离子交换层析CM-52和葡聚糖凝胶Sephadex G-75对明绿豆蛋白进行了分离纯化。实验结果表明:分离纯化出的该蛋白对两种真菌:棉花枯萎病菌(Fusarium oxysporum f.sp.vasinfectum)、玉米纹枯病菌(Rhizoctonia solani Kuha)的生长具有一定的抑制作用;对金黄色葡萄球菌(Staphylococcus Aureus)的细菌生长虽有一定的抑制作用,但较其抗真菌活性相对偏弱。利用高效液相色谱法(HPLC)对分离出的抗菌蛋白的分子量进行了测定,其分子量在29.7~33.8 k Da之间。     

粘质沙雷氏菌中果糖-1,6-/景天庚酮糖-1,7-二磷酸两个双功能酶的研究

[目的]明确粘质沙雷氏菌中两个FBPases是否同时具有催化果糖-1,6-二磷酸(F-1,6-BP)与景天庚酮糖-1,7-二磷酸(S-1,7-BP)的功能。[方法]PCR构建p ET28a-Sm-FBPase和p ET28a-Sm-Glp X,IPTG诱导表达于大肠杆菌并应用镍柱纯化靶蛋白。应用体外酶学分析Sm-FBPase与Sm-Glp X酶学特性。[结果]SDS-PAGE结果表明Sm-FBPase、Sm-Glp X的分子量是45 k Da、38 k Da。体外酶学分析表明两个酶均可催化F-1,6-BP和S-1,7-BP。Sm-Glp X是Mn2+或Mg2+依赖型酶,而Sm-FBPase的激活需要Mn2+。根据被测试的底物,Sm-FBPase最适p H 7. 0～7. 5、最适温度40℃; Sm-Glp X最适p H 8. 0、最适温度40～45℃。当F-1,6-BP作底物时,Sm-FBPase与Sm-Glp X的Kcat/Km分别为6. 88、7. 55 S-1mmol-1L。然而,当S-1,7-BP作底物时,Sm-FBPase与Sm-Glp X的Kcat/Km分别是3. 17、8. 54 S-1mmol-1L。[结论]Sm-FBPase和Sm-Glp X均可催化F-1,6-BP与S-1,7-BP,Sm-Glp X催化S-1,7-BP的效率是Sm-FBPase的2. 69倍。     

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。     

一种水溶性党参多糖的分离纯化及结构分析

采用水提醇沉法得到党参粗多糖(COP),采用Sevag法除去蛋白成份,接着通过Sephacry1 S-200HR及Sephadex G-25凝胶柱色谱分离得到均一多糖COP-1。凝胶渗透色谱法(HPGPC)测定其纯度和平均分子量。高效液相色谱法(HPLC)确定其单糖的组成。红外光谱及核磁推测COP-1结构。结果表明COP-1平均分子量约为2.1×10~3 Da,且均由阶D-(2→1)呋喃果糖组成。     

水稻条纹病毒蛋白与核酸的特性

提纯的水稻条纹病毒(云南宜良分离物)在电镜下的形态为多型性,但主要是宽8-10 nm,长80-250的分枝丝状体,有些为直径3 nm或8 nm的开环环状体,有些为13 nm宽,130-190 nm长的丝状体,但其基本结构应是直径3 nm、长度不等的丝状体.经聚丙烯酰胺凝胶电泳分析,vRNA4编码的病害特异蛋白(SP)分子量为19.9 kDa,而vcRNA3编码的外壳蛋白(CP)约为33.6 kDa.在非变性条件下,RSV的4条ssRNAs大小分别为3.0×106(ssRNA1)、1.2×106(ssRNA2)、0.9×106(ssRNA3)和0.8×106 Da(ssRNA4),有时出现一条大小为0.58×106 Da的单链RNA(ssRNA5);而4条dsRNAs的分子量分别为4.9×106(dsRNA1)、2.8×106(dsRNA2)、2.0×106(dsRNA3)和1.7×106 Da(dsRNA4).利用制备电泳分离提纯的外壳蛋白免疫家兔,得到了高特异性的抗血清.A蛋白夹心ELISA检测结果表明,RSV-CP与水稻草状矮化病毒(RGSV)CP抗血清有微弱的反应,但与RSV、RGSV的SP抗血清没有反应,而RSV-CP抗血清与RSV-SP及RGSV的SP、CP都无血清学关系,这个结果表明RGSV与RSV之间在进化上具有一定的亲缘关系.     

海豚链球菌诱变发酵法制备透明质酸及其在动物皮肤修复中的应用

透明质酸(HA)是一种非常重要的生物材料,是体内广泛存在的细胞外基质成分之一。为了获得产量、分子量及纯度较高的透明质酸,并研究透明质酸水凝胶在动物皮肤修复中的潜在作用。通过紫外诱变的方法对海豚链球菌进行诱变,并对此突变菌发酵后产物的蛋白含量及HA分子量进行了测定,通过CTAB法对发酵产物进行提纯,运用物理冻融法将透明质酸制成水凝胶后,用于兔背部全层皮肤修复的初探。结果表明通过诱变海豚链球菌产透明质酸的能力从(82.3±3.3)mg/L增加到(120±10.6)mg/L,增加了46.4%;产物经纯化后蛋白含量从(0.178±0.011)mg/L减少到(0.032±0.017)mg/L,减少了82.02%;所制得透明质酸的分子量约为3.0×105 Da;透明质酸水凝胶对兔全层皮肤缺损的修复有较明显的促进作用,能减轻炎症和伤口瘢痕的形成。     

Effects of structural modifications on some physical characteristics of exopolysaccharides from Lactococcus lactis

The relationship between the primary structure and the chain stiffness of exopolysaccharides (EPSs) and modified EPSs produced by two strains of Lactococcus lactis subsp. cremoris was investigated. The molar mass and radius of gyration of these exopolysaccharides were analyzed by multiangle static light scattering after size-exclusion chromatography. From these results and the chemical structure of the repeating units of the investigated EPSs, the Kuhn lengths could be calculated. We found that the initial Kuhn lengths of the two native EPSs are similar. Modification of the EPSs by removing parts of the side groups resulted in a decrease in both the absolute value and the normalized value of the Kuhn length. It is therefore concluded that partial removal of the side groups of these polysaccharides could make them less efficient as thickeners if no specific interaction with other components occurs.     

壳聚糖辐照降解产物涂膜保鲜草莓研究

壳聚糖具有抑菌性与成膜性。将壳聚糖辐照降解得到的一系列不同粘均分子量产物进行涂膜草莓保鲜,研究涂膜液中壳聚糖粘均分子量、浓度、pH值、有机酸、明胶含量对草莓保鲜效果的影响;并设计四因素三水平正交试验。实验结果表明：1％（w／v）7．0×10^4Da壳聚糖、1％（v／v）醋酸、pH5、添加明胶0．5％的涂膜配方具有最好的保鲜效果;在常温（20℃、湿度80％～90％）下可以延长贮藏期2d;低温（3℃-4℃、湿度80％-90％）下可以延长贮藏期3d。     

Production and molecular characteristics of four groups of exopolysaccharides from submerged culture of Phellinus gilvus

AIMS: The objective of the present study was to determine the optimal culture conditions for the production of four groups of exopolysaccharides (EPSs) in Phellinus gilvus by submerged culture and to investigate their molecular properties by multi-angle laser-light scattering (MALLS) analysis. METHODS AND RESULTS: The optimal temperature and initial pH for the production of both mycelial biomass and EPSs in P. gilvus by submerged flask cultures were found to be 30 degrees C and pH 9.0, respectively. Glucose and corn steep powder were the most suitable carbon and nitrogen source for both mycelial biomass and EPS production. Optimal medium composition was determined to be glucose 30 g l-1, corn steep powder 5 g l-1, MgSO4 1.23 g l-1, KH2PO4 0.68 g l-1, and K2HPO4 0.87 g l-1. Four groups of EPSs (Fr-I, II, III, and IV) were obtained from the culture filtrates by gel filtration chromatography on Sepharose CL-4B and characterized by size exclusion chromatography (SEC) coupled with MALLS. The weight average molar mass (Mw) of Fr-I, Fr-II, Fr-III and Fr-IV were determined to be 8.628 x 106 (+/-129 420), 1.045 x 106 (+/-19 855), 61.09 x 104 (+/-1244), and 33.55 x 104 (+/-134) g mol-1, respectively. CONCLUSIONS: Under optimal culture conditions, the maximum EPS production in a 5-l stirred fermenter indicated 5.3 g l-1 after 11 days of fermentation. The SEC/MALLS analysis revealed that Fr-I, which has extremely high molecular weight, was presumably an aggregate of complex polysaccharides forming a compact globular shape; whereas Fr-II was nearly spherical, Fr-III and Fr-IV were rod-like chains in an aqueous solution. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the production of high amounts of EPSs from liquid-culture of the basidiomycete, P. gilvus. The SEC/MALLS approach used in this study could be useful in providing greater insight into the characterization of the mushroom polysaccharides without carrying out elaborate fractionation procedures prior to analysis.     

Characterization of potential stress responses in ancient Siberian permafrost psychroactive bacteria

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.     

Structural characterization of the exocellular polysaccharides produced by Streptococcus thermophilus SFi39 and SFi12.

We investigated the structures of the exopolysaccharides (EPSs) produced by Streptococcus thermophilus SFi39 and SFi12. Both polymers were found to have molecular masses of greater than 2 x 10(6) Da. The SFi39 EPS consisted of D-glucose and D-galactose in a molar ratio of 1:1, whereas the SFi12 EPS was composed of D-galactose, L-rhamnose, and D-glucose in a molar ratio of 3:2:1. Methylation analysis of and nuclear magnetic resonance spectra recorded from the native polysaccharide, as well as oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of the SFi39 EPS, which consists of the following tetrasaccharide repeating unit: [formula: see text] Similar spectra recorded only from the native polysaccharide were sufficient to allow the structural determination of the SFi12 EPS, which consists of the following hexasaccharide repeating unit: [formula: see text] This study shows that the texturizing properties of different S. thermophilus ropy strains are based on the production of EPSs exhibiting chemical similarities but structural differences.     

生淀粉酶生产菌株Paenibacillus sp.的筛选和酶的纯化及酶学性质

分离得到1株产生淀粉酶的菌株,通过扩增和测定16S rDNA序列并进行比对,发现是Paenibacillus属的细菌。液体摇瓶发酵结束后,其产生的生淀粉酶比酶活达108.5U/mL。通过饱和(NH4)2SO4沉淀、Sephacryl S-300层析的方法对其所产的生淀粉酶进行分离纯化,得到纯化的酶蛋白比酶活为5112.04U/mg,纯化倍数为14.1,相对分子质量约为1.0×105。该酶以木薯生淀粉为底物时,最适pH5.6,最适温度50℃。金属离子Ca2+、Mg2+对该酶具有激活作用,Zn2+、Cu2+、Fe2+、Ni2+、Mn2+、Co2+和EDTA2-对该酶均具有抑制作用。     

Physicochemical properties of the exopolysaccharides produced by marine bacterium Zoogloea sp. KCCM10036

The physicochemical properties of the exopolysaccharide (EPS) produced by marine bacterium Zoogloea sp. KCCM10036 were investigated. Two types of isolated EPSs were shown to have average relative molecular masses (Mr) of 4.07 x 10(6) of CBP (cell-bound polysaccharide) and 3.43 x 10(6) of WSP (water-soluble polysaccharide), respectively. When the CBP was utilized as an emulsifier, it stabilized the emulsion for up to 148 h. Compared with other commercially available hydrocolloids such as xanthan gum, the Tween series, and Triton, the CBP showed much better emulsifying capability on a water-in-oil system. Phase separation occurred in the Tween series after 24 h, whereas the emulsion was better stabilized by the CBP. The CBP thus has potential as an emulsifying agent in commercial emulsions. The flocculating activity was also greatest at 0.01% (w/v) and decreased at higher concentrations than the optimized concentration of the WSP and CBP. The results also showed that both types of exopolysaccharides from Zoogloea sp. had excellent flocculating activity.     

Freezing and melting water in lamellar structures.

The manner in which ice forms in lamellar suspensions of dielaidoylphosphatidylethanolamine, dielaidoylphosphatidylcholine, and dioleoylphosphatidylcholine in water depends strongly on the water fraction. For weight fractions between 15 and 9%, the freezing and melting temperatures are significantly depressed below 0 degree C. The ice exhibits a continuous melting transition spanning as much as 20 degrees C. When the water weight fraction is below 9%, ice never forms at temperatures as low as -40 degrees C. We show that when water contained in a lamellar lipid suspension freezes, the ice is not found between the bilayers; it exists as pools of crystalline ice in equilibrium with the bound water associated with the polar lipid headgroups. We have used this effect, together with the known chemical potential of ice, to measure hydration forces between lipid bilayers. We find exponentially decaying hydration repulsion when the bilayers are less than about 7 A apart. For larger separations, we find significant deviations from single exponential decay.     

Enhancing the Production and Monosaccharide Composition of Exopolysaccharides of Lactobacillus plantarum VAL6 by Applying Thermal Stress and Increased Carbon Dioxide Concentration

Microbiology - Lactobacilli are able to produce exopolysaccharides (EPSs) with a wide diversity in structure and composition. However, changes in EPS production under environmental challenges are...     

Cultivation technology development of Rhodothermus marinus DSM 16675

Extremophiles - This work presents an evaluation of batch, fed-batch, and sequential batch cultivation techniques for production of R. marinus DSM 16675 and its exopolysaccharides (EPSs) and...