重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究

将产热稳定性过氧化氢酶的重组大肠杆菌培养后菌体破碎得到的粗酶液经热处理、硫酸铵分级沉淀、DEAE\|Sephadex A\|50离子交换层析、HiPrep16/10 Phenyl疏水作用层析、Superdex200 HR 10/30凝胶层析提纯后得到电泳纯的酶,比酶活达到15629U/mg。此酶的最适温度为70℃,最适pH70,在60℃保温60min酶活力基本不变,在pH3～8的范围内比较稳定。此酶的Km和Vmax分别为775mmol/L和278mmol\5min\+\{-1\}·mg-1。1mmol/L的Zn2+、Ba2+、Mn2+可使该酶完全失活,KCN、NaN\-3、Na\-2S\-2O\-4、巯基乙醇对酶活力有抑制作用,50mmol/L的EDTA不影响酶活性。     

Natural cellulose fibers from soybean straw

This paper reports the development of natural cellulose technical fibers from soybean straw with properties similar to the natural cellulose fibers in current use. About 220 million tons of soybean straw available in the world every year could complement the byproducts of other major food crops as inexpensive, abundant and annually renewable sources for natural cellulose fibers. Using the agricultural byproducts as sources for fibers could help to address the concerns on the future price and availability of both the natural and synthetic fibers in current use and also help to add value to the food crops. A simple alkaline extraction was used to obtain technical fibers from soybean straw and the composition, structure and properties of the fibers was studied. Technical fibers obtained from soybean straw have high cellulose content (85%) but low% crystallinity (47%). The technical fibers have breaking tenacity (2.7 g/den) and breaking elongation (3.9%) higher than those of fibers obtained from wheat straw and sorghum stalk and leaves but lower than that of cotton. Overall, the structure and properties of the technical fibers obtained from soybean straw indicates that the fibers could be suitable for use in textile, composite and other industrial applications.     

Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, and sphingolipid-enriched lipid rafts

The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.     

温特曲霉转富马酸为L-苹果酸的初步研究*

从大量霉菌中选育到一株具有较高富马酸酶活性的温特曲霉(Aspergillus wentii) A5-61。在摇瓶培养条件下,32℃ 96小时,产L-苹果酸达10.49g/100ml,对富马酸的转化率达90.80%。利用菌体细胞,进行酶转化试验,结果表明：1.6g湿菌体接入25ml含富马酸10.0%（用NaOH中和至pH7.0）的转化液中,35℃16～24小时,连续转化三次,分别产生L—苹果酸9.61g/100ml、9.73g/100ml、6.93g/100ml。对菌体整体细胞酶学性质的研究表明,其最适反应温度35℃,最适反应pH7.0,Cu2＋对该酶有明显的抑制作用,该酶的Km＝0.154mol/L,Vmax＝0.0571mol/L·h。     

链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２株脂肪酶产生菌中,链霉菌Ｚ９４－２产脂肪酶活力为５９６ｕ／ｍＬ,其最适培养基（ｇ／Ｌ）为：糊精１０、黄豆饼粉３０、尿素１０、Ｋ２ＨＰＯ４０．５、ＭｇＳＯ４０．５、ＮａＣｌ１和ＡＥＯ９０．５,产酶的最适条件为：初始ｐＨ９．５～１０．０,在２６℃培养４８ｈ。用ＰＶＡ橄榄油乳化系统测定该酶的最适ｐＨ９．８,最适温度３７℃,在ｐＨ８．６～１０．２于５℃存放２４ｈ,酶活力不变。０．１４ｍｏｌ／Ｌ的氯     

Time course changes to structural,mechanical and material properties of bone in rats after complete spinal cord injury

Objective:Characterise the spatiotemporal trabecular and cortical bone responses to complete spinal cord injury (SCI) in young rats.Methods:8-week-old male Wistar rats received T9-transection SCI and were euthanised 2-, 6-, 10- or 16-weeks post-surgery. Outcome measures were assessed using micro-computed tomography, mechanical testing, serum markers and Fourier-transform infrared spectroscopy.Results:The trabecular and cortical bone responses to SCI are site-specific. Metaphyseal trabecular BV/TV was 59% lower, characterised by fewer and thinner trabeculae at 2-weeks post-SCI, while epiphyseal BV/TV was 23% lower with maintained connectivity. At later-time points, metaphyseal BV/TV remained unchanged, while epiphyseal BV/TV increased. The total area of metaphyseal and mid-diaphyseal cortical bone were lower from 2-weeks and between 6- and 10-weeks post-SCI, respectively. This suggested that SCI-induced bone changes observed in the rat model were not solely attributable to bone loss, but also to suppressed bone growth. No tissue mineral density differences were observed at any time-point, suggesting that decreased whole-bone mechanical properties were primarily the result of changes to the spatial distribution of bone.Conclusion:Young SCI rat trabecular bone changes resemble those observed clinically in adult and paediatric SCI, while cortical bone changes resemble paediatric SCI only.     

干酪乳杆菌L-乳酸脱氢酶在大肠杆菌中的表达、纯化及酶学性质

L-乳酸脱氢酶(L-lactate dehydrogenase,L-LDH)是发酵生产L-乳酸中催化丙酮酸转化成L-乳酸的关键酶。以干酪乳杆菌G-02(Lactobacillus casei G-02)基因组DNA为模板,克隆得到L-LDH基因(ldhL),经序列分析后将其连接到表达载体pET-28a(+)上,构建成重组质粒pET-ldhL转化到大肠杆菌BL21(DE3)中,实现ldhL基因的表达。30°C加入IPTG诱导表达后,经镍柱亲和层析纯化的重组蛋白样品通过SDS-PAGE分析,约在40 kD处出现显著的特异性条带。对表达的L-LDH生物学特异性研究显示:重组L-LDH的比酶活为1 722 U/mg,最适反应温度为40°C-45°C;果糖-1,6-二磷酸(FBP)为别构激活剂,使最适pH向中性方向偏移(pH为6.6-6.8),Mn2+可拓宽最适酶活pH范围;Mn2+、Ca2+和Mg2+对L-LDH有激活作用,而Zn2+对L-LDH有抑制作用。     

土壤水解酶类催化动力学研究进展

土壤水解酶是存在于土壤中的一种重要的酶类,参与了土壤中为数众多的重要生物化学反应,与土壤中多种营养元素转化密切相关其催化反应的动力学研究常用来阐明其催化过程的特性、酶的本质属性及其对环境变化的响应等,研究土壤水解酶动力学特征对探讨其来源、性质及影响因素,对进一步调控多种营养元素参与的反应过程有着重要意义。文中概述了土壤水解酶的种类及其参与的生物化学反应;探讨了土壤水解酶动力学研究的理论基础;综述了土壤水解酶催化动力学研究的进展和影响因素,在此基础上,对今后研究提出了几点建议。