绿色糖单孢菌降解木质纤维胞外酶的分泌条件及其生物漂白研究

以一株耐热耐碱放线菌-绿色糖单孢菌（Saccharomonospora viridis）为研究对象,探讨其产胞外木素过氧化物酶、木聚糖酶、纤维素酶的优化发酵条件。结果表明,其最佳碳氮源分别为葡萄糖和蛋白胨,最佳接种量为1％,不同的诱导底物对三种木质纤维降解酶有不同的诱导效果,其中麦草浆的诱导效果最好。在培养基中添加0．01mol／L的Mn^2＋和0．1％的土温80能够显著促进木质纤维降解酶的产生。在pH8．0,45℃条件下,培养120h后木素过氧化物酶的酶活达到最大0．36U／mL,培养156h后木聚糖酶和纤维素酶的酶活达到最大,最高酶活分别为18．46U／mL,10．42u／mL。用含有这三种酶的粗酶液对麦草烧碱蒽醌浆进行生物漂白表明,绿色糖单孢菌所产的木质纤维降解酶具有较好的漂白效果。     

粗糙脉孢菌(Neurospora crassa)纤维素酶液体发酵培养基的优化

粗糙脉孢菌是天然纤维素降解真菌,具有产纤维素酶能力,国内外对其纤维素降解机理和发酵产酶有一定的研究,但对其产酶的条件优化研究得不多,其产酶潜力需要进一步挖掘。以粗糙脉孢菌基因组测序菌株FGSC 2489为对象,采用响应面分析法对Neurospora crassa摇瓶发酵产纤维素酶进行培养基优化。采用Plackett-Burman(PB)实验设计考察发酵培养基中关键参数对产酶条件的影响,进而采用最陡爬坡实验逼近最大响应区域,并结合中心组合实验(central composite design,CCD)和响应面分析法对两个显著因素进行分析。PB实验结果显示:Peptone、Yeast extract对产纤维素酶有显著影响。通过响应面分析得到一元二次方程,对方程求解得到Peptone 7.27g/L、Yeast extract 5.51g/L。采用该优化培养基,最大纤维素酶活可达1.27FPU/ml,较优化前提高了2.03倍;CMC酶活14.15IU/ml,比优化前提高1.88倍;木聚糖酶活24.13IU/ml,比优化前提高1.86倍;葡萄糖苷酶酶活1.22IU/ml比优化前提高2.08倍。     

绿色糖单孢菌-木素过氧化物酶的发酵工艺研究

本研究以一株中度耐热耐碱放线菌--绿色糖单孢菌(Saccharomonospora viridis)为研究对象,用16 L发酵罐对该菌进行了木素过氧化物酶(lignin peroxidases, LiP)的诱导发酵,确定了最适的产酶工艺条件:接种量为10%,C/N为1∶3,搅拌速度为250 r/min,通气量为5 L/min,通过控制通气量和调整搅拌转速,使溶氧维持在35%以上,此条件下绿色糖单孢菌较摇瓶实验提前将近24 h达到产酶高峰,酶活最高可达0.41 U/ml;同时在发酵罐中测定该菌株的生长曲线和代谢曲线以确定其发酵代谢规律.     

具有高木二糖形成活力的木聚糖酶生产菌株的筛选与产酶培养基的优化

从土样中筛选出一株产木聚糖酶的青霉,该青霉所产木降糖酶具有很高的木二糖形成活力,经鉴定为顶青霉,其木聚糖酶的合成与分泌受木聚糖等木糖苷类物质的诱导,麸皮对其木聚糖酶的合成也有促进作用,优化产酶液体培养主要成分的配比为：麸皮：玉米芯木聚糖：玉米芯粉;蛋白胨（或尿素）＝1：1：1：0．6（0．4）,摇瓶96h达到最大酶活,最高木聚糖酶活达到289．3U／ml,该菌所产木聚糖酶的最适作用条件为45－50度,PH4．4,在PH4．4－8．0范围内稳定。     

重组毕赤酵母产木聚糖酶的发酵条件优化研究

采用单因素实验确定重组毕赤酵母产木聚糖酶生长相的最适条件,然后利用Plackett—Bur—man实验设计对诱导相培养基成分和培养条件的10个因素进行筛选,方差分析结果表明,影响木聚糖酶表达的主要因子为酵母膏、诱导pH和摇床转速;在此基础上,用Box—Behnken的响应面方法对3个因素进行进一步优化,当酵母膏为11．13彰L,pH为6．38,摇床转速为228r／min时酶活有最大值,为262．77u／mL,较优化前提高了175．44％。优化后的摇瓶发酵条件应用于7L发酵罐并连续诱导培养120h,发现诱导72h后的木聚糖酶酶活最高,为2054．89u／mL。     

产适冷木聚糖酶的海洋产青霉的筛选和诱变

从黄海深层海底泥样中分离到1株产低温木聚糖酶的青霉,经EMS诱变得到11株酶活性提高的菌株,对其中1株产低温木聚糖酶活力最高的菌株产酶性质进行了初步研究。所产木聚糖酶在pH4．6,45℃时酶活可达25．8u／ml,比出发菌株提高126％。诱变后菌株所产木聚糖酶在0℃仍有显著酶活性,达8．2u／ml。     

嗜热绿色糖单孢菌木聚糖酶XyN10A基因在毕赤酵母中的分泌表达及其酶学性质研究

木聚糖酶(Xylanase)可降解自然界中大量存在的木聚糖类半纤维素,由于其在饲料、食品、造纸等领域的广泛应用,越来越受到人们的关注。利用分子生物学技术首次成功构建了一株高效表达嗜热绿色糖单孢菌(Saccharomonospora viridis)木聚糖酶SviXyN10A的重组工程菌,建立了SviXyN10A的毕赤酵母表达体系,实现了SviXyN10A在毕赤酵母(Pichia pastoris)中的分泌表达,并对表达产物进行了诱导表达及纯化,以及酶学性质的研究。结果表明,重组木聚糖酶SviXyn10A用甲醇诱导2 d后酶活即达到7.53IU/mL,最适反应温度为70℃,pH7.0。pH作用范围较广(pH5.0-9.0),具有较强的热稳定性和耐碱性,几乎没有纤维素酶活性,故可以应用在很多工业领域,尤其是在纸浆造纸行业具有很大的潜在应用价值。     

诱导蜂房哈夫尼菌产L-赖氨酸脱羧酶条件的研究

用响应面法对蜂房哈夫尼菌（Hafnia alvei）L-赖氨酸脱羧酶产酶诱导条件进行优化。首先通过单因素实验对产酶体系的pH、震荡培养时间、静置培养时间、诱导物添加量和Ⅷ添加量进行优化。在此基础上,用部分因子重复试验筛选出对酶活影响显著的3个因素（静置培养时间,诱导物添加量,VB6添加量）,再通过Box-behnken实验对这三个因素进行优化,得出最优值。最终得到产酶最佳诱导条件为：震荡培养阶段培养基pH6．5,静置培养阶段pH5．5;摇床震荡培养11h后静置培养7．5h,诱导物L一赖氨酸加入量为5．18dL,维生素B6加入量为1．38g／L时酶活最高,达到71．2U／mL,为优化前（1．74u／mL）的41．8倍,在单因素的基础上提高了19％。     

壳聚糖酶产生菌的筛选及固定化细胞产酶

旨在筛选得到一株壳聚糖酶产生菌,并研究固定化细胞产酶的条件。在培养基中以壳聚糖为唯一碳源,对土壤样品进行筛选,获得一株无花果沙雷氏菌(Serratia ficaria CH-0203),该菌可被壳聚糖诱导产生壳聚糖酶。固定化细胞产酶的研究结果表明,多孔玻璃可以有效吸附CH—0203菌细胞。在最适发酵条件下(pH6．5,培养基与载体的总体积48ml,载体与培养基的比例为1．5g/4．0ml,吸附时间是20h-26h),发酵液酶活达到4．5U／ml,比游离细胞发酵提高了16％。采用半连续发酵的方式,固定化的细胞可以稳定发酵产酶120h左右。固定化细胞产酶的效率大大高于游离细胞。     

麦芽糖诱导软化芽孢杆菌α-环糊精葡萄糖基转移酶在枯草杆菌中的表达

通过PCR扩增软化芽孢杆菌α-环糊精葡萄糖基转移酶基因,将基因片段克隆到大肠杆菌-枯草杆菌穿梭载体pGJ103中,转化枯草杆菌WB600得基因工程菌进行外源表达。在1.5%的麦芽糖初始发酵培养基上摇瓶培养,48 h后重组枯草杆菌产酶活性为6.1U/ml。通过单因素分析和响应面分析对重组枯草杆菌产CGT酶摇瓶发酵条件进行优化。分析得到培养基关键组分麦芽糖,玉米淀粉和酵母粉三者最佳浓度分别为:15.5g/L,13g/L和20g/L。在此条件下,摇瓶培养36h后α-CGT酶活性为17.6U/ml,5L罐分批发酵30h后酶活达到20U/ml (水解活性为1.4×10⁴ IU/ml)。     

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、...     

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,对于揭未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。     

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.     

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。     

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     

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.     

Morphology and putative function of the colon and cloaca of marine and freshwater snakes

Among tetrapods, evidence for postrenal modification of the urine by the distal digestive tract (including the colon and cloaca) is highly variable. Birds and bladderless reptiles are of interest because the colon and cloaca represent the only sites from which water and ions can be reclaimed from the urine secreted by the kidney. For animals occupying desiccating environments (e.g., deserts and marine environments), postrenal modification of the urine may directly contribute to the maintenance of hypo‐osmotic body fluids. We compared the morphology and distribution of key proteins in the colon, cloaca, and urogenital ducts of watersnakes from marine (Nerodia clarkii clarkii) and freshwater (Nerodia fasciata) habitats. Specifically, we examined the epithelia of each tissue for evidence of mucus production by examining the distribution of mucopolysaccharides, and for evidence of water/ion regulation by examining the distribution of Na⁺/K⁺‐ATPase (NKA), Na⁺/K⁺/Cl^? cotransporter (NKCC), and aquaporin 3 (AQP3). NKCC localized to the basolateral epithelium of the colon, urodeal sphincter, and proctodeum, consistent with a role in secretion of Na⁺, Cl^?, and K⁺ from the tissue, but NKA was not detected in the colon or any compartment of the cloaca. Interestingly, NKA was detected in the basolateral epithelium of the ureters, suggesting the urothelium may play a role in active ion transport. AQP3 was detected in the ureters and coprodeal complex, consistent with a role in urinary and fecal dehydration or, potentially, in the production of the watery component of the mucus secreted by the coprodeal complex. Since no differences in general cloacal morphology, production of mucus, or the distribution of ion transporters/water channels were detected between the two species, cloacal osmoregulation may either be regulated by proteins not examined in this study or may not be responsible for the differential success of N. c. clarkii and N. fasciata in marine habitats. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.