碱性普鲁兰酶产生菌的原生质体制备与诱变选育

研究报道了Bacillus sp SX—12原生质体制备与再生最佳条件。实验表明,在液体完全培养基中加入2％的甘氨酸培养10h,原生质体制备最佳条件为：溶菌酶浓度0．5mg／mL,酶解温度37℃,酶解时间1．5h时原生质体形成率为93．8％。原生质体形成最佳高渗稳定剂为甘露醇,再生率26．4％。在原生质体制备的最佳条件下,用紫外线诱变技术选育产碱性普鲁兰酶的高产菌株。筛选到1株高产菌株SX—12C87,酶活由出发菌株的2．42μ／mL提高到6．87μ／mL,提高了约1．8倍。     

耐酸耐热普鲁兰酶菌株的筛选及发酵条件的研究

从土壤中分离出一株产普鲁兰酶的菌株 ,初步鉴定为芽孢杆菌 ,通过紫外及 EMS多次诱变及筛选 ,酶活从 1.6 u/ m L提高到 5.4 u/ m L。在此基础上对产酶条件进行了优化 ,酶活有了较大提高 ,最高产酶水平达 8.8u/ m L。初步研究了酶的性质 ,酶反应的最适温度和 p H分别为 75℃和 4 .6 ,在 55℃反应条件下 ,酶在p H4 .0～ 8.0范围内活性稳定。     

茁芽短梗霉原生质体激光诱变及高产菌株筛选

目的：通过普鲁兰(pullulan)产生菌-茁芽短梗霉(Aureobasidium Pullulans)原生质体的激光诱变,以得到普鲁兰高产菌株。方法：利用正交实验研究了茁芽短梗霉原生质体的制备与再生并确定了其最佳条件为：菌体以1％的甘氨酸预处理;在0．1mol／L pH 6．0柠檬酸一柠檬酸钠缓冲液,含0．7mol／L NaCl的高渗稳定液中;经蜗牛酶0．2％、纤维素酶0．1％、溶菌酶0．2％的混合酶酶解15min。采用He-Ne激光诱变茁芽短梗霉原生质体筛选得到普鲁兰高产菌株J208,其蔗糖转化率达到53．3％,是原始菌株的10．6倍。结论：用激光诱变茁芽短梗霉原生质体是获得普鲁兰高产菌株的新途径。     

复合诱变原生质体选育耐热碱性蛋白酶高产菌

以地衣芽孢杆菌(Bacillus licheniformis)53号为原始菌株,在原生质体形成和再生的最佳条件下制备原生质体,对原生质体进行复合诱变,对大量再生突变株进行筛选,最终获得了高产、稳定、耐热的碱性蛋白酶产生菌53-G38-6,产酶活力由1104U/ml提高到22080U/ml。适宜的发酵条件:培养基(％)胰蛋白胨1,酵母膏0.5,玉米粉5,Na_2HP0_4·12H_20 0.4,KH_2P0_4 0.03,Na_2CO_3 0.1,自然pH。42℃旋转培养44～48h,得到的蛋白酶热稳定性强,60℃处理1h剩余酶活55％。酶反应最适条件:62℃,pH10.0,在pH9～10.5范围内稳定。     

聚半乳糖醛酸酶高产菌的产酶条件研究

通过单因素及正交试验对米曲霉（Aspergillus orza）C491产生聚半乳糖醛酸酶的发酵条件进行了优化。该菌株的摇床发酵滤液以桔子果胶为底物时酶活力可达344．8U／mL。产酶最适培养基组成为：麦芽汁（糖度6％）中添加6％桔皮粉,2％硫酸铵（w/v）。最适培养条件：起始pH4（灭菌前）,30℃,r/min,培养112h。Tween80可以促进C－491产酶。     

氧化铁鞘细菌铁氧化酶最适产酶条件及其酶学特性的研究

对氧化铁鞘细菌FC990 1菌株的铁氧化酶最适产酶条件及酶学特性进行了研究。菌株最适产酶培养基为 (g/L) :柠檬酸铁胺 10g ,NaNO3 1.2g,MgSO4·7H2 O 0 .5g ,K2 HPO4·7H2 O 0 .5g ,CaCl2 0 .0 15g ,ZnSO4·7H2 O 0 .0 0 0 5g。最适产酶条件为 :温度 30℃ ,起始pH7.0 ,接种量 2 % ,15 0mL三角瓶装 5 0mL ,15 0r/min振荡培养 72h。铁氧化酶最适pH为7.5 ,最适温度为 30℃。金属离子Ca2 、Mg2 、Zn2 对酶有激活和稳定作用 ;Cu2 、Hg2 、Al3 则抑制酶的活性 ;Fe2 、K 、Na 对酶活性影响不明显。     

嗜碱芽孢杆菌XE22—4—1碱性弹性蛋白酶发酵条件的研究

从西藏天然碱湖中筛选到一株产碱性弹性蛋白酶（alkaline elastase)菌株,最适生长pH为10．0,经鉴定为Bacillus sp.,编号XE22－4－1.该菌产酶最适碳源为2％葡萄糖,氮源为0．25％酵母粉,豆饼粉对发酵产酶有促进作用。2L发酵罐实验表明,溶解氧是影响该菌株产酶的重要因素。通过提高通气量和改变搅拌速度,该菌株可在发酵48h内达到产酶高峰,酶活力最高达266u/mL。     

高产超氧化物歧化酶菌株发酵条件的研究

从1348株芽孢杆菌(Bacillus cereus)中分离和选育出高产超氧化物歧化酶(superoxide dismutase)菌株C328,并进行了产酶条件的研究。结果表明,该菌株最佳产酶的最适温度为30～35℃,培养基最适起始pH为8．0～8．5。金属离子Cd^2 、Mn^2 、Zn^2 ,表面活性剂吐温80、司班80、琥珀酸钠和无机盐CaCl2、CaCO3对产酶有抑制作用。适宜浓度的K2HPO4、EDTA、金属离子Fe^3 和表面活性剂NaAC对产酶有明显的促进作用。发酵培养用250mL三角瓶以30mL装液量酶活力最高。适宜种龄为8～12h,适宜接种量为0．9％～3％,最佳接种量为1％～2％。     

高温蛋白酶产生菌的筛选及其产酶条件和酶学性质分析

从徂徕山温泉附近土样中分离到9株产高温蛋白酶菌株,选取一株碱性蛋白酶高产菌株L7为出发菌株,进行显微形态、16S rRNA基因序列分析,将其初步鉴定为短芽孢杆菌(Brevibacillus sp.)。研究该菌株发酵条件,确定产酶的最佳培养基组成为葡萄糖40 g/L,蛋白胨20 g/L,磷酸氢二钠1.4 g/L,氯化钙0.6 g/L,硫酸镁0.4 g/L,通过培养基优化,酶活达到103.08 U/mL。最佳培养条件为250 mL三角烧瓶中装液量50 mL、pH8.0、培养温度为55℃、培养时间为24 h。对该菌株酶学性质研究,L7菌株所产高温蛋白酶的最适温度为55℃,最适pH为10,并且具有良好的温度稳定性和pH稳定性,酶活性受PMSF强烈抑制。     

极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究

从深海的38份水样和泥样中筛选到一株产蛋白酶的菌株DY－A,其最适生长温度为10℃,能适应较大范围的pH值和盐度,此菌株只在酵母膏存在的情况下产酶,不利用单一氮源,最适产酶条件为,pH10.0,10℃,接种量0．5％,200r/min摇床培养40－72h,粗酶液的最适作用温度为40℃,最适pH值为10,在pH9-12内稳定,是一碱性蛋白酶。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Effect of soil salinity and water stresses on growth and content of nitrogen,chloride and phosphate of wheat and triticale

Summary Three wheats and one triticale were grown, up to flowering stage, in pots on calcareous soil adjusted to a range of salinities (S₁=3.5, S₂=6, S₃=8.5, and S₄=11 mmhos/cm, 20°C, soilpaste extract) by adding solution consisting of 3∶2∶1 of Na-, Ca- and Mg chlorides in chemical equivalent amounts. Moisture in the pots was kept at 100% (W₁), 40% (W₂) and 20% (W₃) of the available water. The vegetative growth, nitrogen and phosphate were affected by S and W treatments, chloride was affected only by S. The interaction S×W affected only dry weight. Varietal effect was observed between wheat as a group and triticale. Multiple quadratic regression equations of these properties on salinity and water revealed that the higher the available water the wider the range of tolerable salinity. Triticale was relatively more tolerant to water stress. Salinity increases Cl and decreases N, whereas water stress enhances N accumulation to a certain extent. However, in triticale at S₃ and S₄ the effect of water stress on N was overshadowed by the excessive salinity. This did not occur for the wheat (Florence). P trends were described. R² for P was low (0.7435–0.3603) which made interpretations rather difficult.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.