响应面法优化fHBP-A蛋白双水相萃取工艺的探究

目的 采用双水相萃取技术对重组B群脑膜炎奈瑟菌H因子结合蛋白A(fHBP-A)进行分离纯化,研究不同双水相萃取体系对fHBP-A粗提纯化的影响,应用响应面法优化fHBP-A在PEG/(NH₄)₂SO₄双水相体系中的萃取工艺。方法 首先建立PEG 2 000/(NH₄)₂SO₄、PEG 4 000/(NH₄)₂SO₄、PEG 6 000/(NH₄)₂SO₄和乙醇/(NH₄)₂SO₄ 共4种双水相萃取体系相图;然后考察PEG相对分子质量、PEG质量分数、(NH₄)₂SO₄质量分数、萃取体系pH这4个因素对fHBP-A萃取率、下相中fHBP-A纯度的影响;最后采用Box-Behnken试验设计以及响应面...     

不同盐碱类型胁迫对红地球/贝达葡萄植株离子分布的影响

以盆栽红地球/贝达葡萄为试材,定量浇灌NaCl、Na₂SO₄、NaHCO₃、NH₄Cl和(NH₄)₂SO₄,筛选导致葡萄叶片黄化的盐、碱离子,研究不同盐碱类型胁迫对葡萄植株离子分布的影响.结果表明： NaHCO₃对植株影响最大,叶片在处理14 d时出现黄化症状,而NaCl和NH₄Cl处理28 d时出现黄化症状.NaHCO₃和NaCl处理均显著增加了植株各器官中Na^+含量,NaHCO₃处理根中Na⁺含量是对照的 6.4倍;这两种盐处理均降低了除叶片外其他器官中的K⁺含量,NaHCO₃处理显著降低了各器官中K/Na,根中K/Na仅为0.1,NaCl处理降低了除茎外其他器官中K/Na;这两种盐处理还降低了Ca²⁺、Mg²⁺、Fe²⁺向地上部的运输.NH₄Cl、(NH₄)₂SO₄和Na₂SO₄处理降低了植株各器官中K/Na,以NH₄Cl处理显著.碱性盐NaHCO₃对葡萄叶片黄化影响最大,其次是中性盐NaCl,再次是NH₄Cl,而(NH₄)₂SO₄和Na₂SO₄影响较小.     

黑曲霉生产β-葡萄糖苷酶发酵条件的研究产

经多项式回归分析,研究了不同浓度N源、C源、无机盐等对酶产量的影响,确定出最佳培养基配方为:麸皮4.9%,(NH₄)_2>SO₄0.4%,KH₂PO₄0.29%,CaCl₂0.05%,MgSO₄·7H₂O0.04%,FeSO₄·7H₂O 5mg·L^-1,ZnCl₂1.4mg·L^-1,0.2%油酸钠。并对培养温度、时间、培养基初始pH、通气量、接种量、接种方式等培养条件进行优化,使黑曲霉生产β葡萄糖苷酶的产量由17U·ml^-1增至21.3U·ml^-1.     

铜铁试剂对菜青虫多酚氧化酶的抑制作用

以菜青虫Pieris rapae (L.)为试材,冰浴匀浆,4℃下6 403 ×g 离心,取上清液,经35%饱和度(NH₄)₂SO₄沉淀,Sephadex G100凝胶过滤柱层析等分离步骤,获得部分纯化的菜青虫多酚氧化酶制剂。研究不同浓度铜离子及铜铁试剂对该酶的影响,结果表明：Cu₂₊在0～0.100 mmol/L范围内对酶活力表现激活作用;浓度大于0.125 mmol/L时表现抑制作用,其IC₅₀为0.651±0.022 mmol/L;铜铁试剂对该酶抑制作用的IC₅₀为0.100±0.012 mmol/L。抑制动力学研究结果表明：铜铁试剂对该酶表现为可逆抑制效应,为竞争性抑制类型,其抑制常数K_i为0.076±0.013 mmol/L。     

微生物絮凝剂产生菌的筛选、鉴定及其培养条件的优化研究

采用常规细菌分离方法从土壤中筛选出一株高效絮凝剂产生菌,编号为B6。经过形态学特征、生理生化反应及16S rDNA序列(GenBank accession No.DQ115363)同源性比较鉴定该菌株为恶臭假单胞菌,命名为Pseudomonas putidaB6。对其培养基进行研究,确定其最佳的碳源和氮源,并通过正交试验确定其最佳的培养基成分配比(ρ/gL^-1)为葡萄糖15、果糖3、KH₂PO₄1.0、K₂HPO₄3.0、MgSO₄0.1、NH₄NO₃0.8、(NH₄)₂SO₄1.5、酵母汁0.2、NaCl0.1。同时对其培养条件进行研究,确定最佳的培养基初始pH值为8.0、培养温度为30℃、摇床速度为160r/min,以及产絮凝剂的周期变化过程。用高岭土悬浮液对其絮凝条件进行研究,确定培养液最佳的絮凝条件。     

The response of soil respiration to different N compounds addition in a saline–alkaline grassland of northern China

不同形态氮化合物添加对中国北方盐渍化草地土壤呼吸的影响 持续增加的氮沉降在提高陆地生态系统生产力的同时也会对土壤微生物产生显著影响;土壤呼吸由植物根系呼吸和土壤微生物呼吸组成,因此影响植物生产力和微生物的因子都会影响到土壤呼吸。以往氮富集对土壤呼吸的研究主要在土壤中性的草地生态系统开展,而对于盐渍化草地土壤呼吸是如何响应氮沉降的研究尚不多见,这限制了全球变化陆地生态系统土壤呼吸模型预测的准确性和完整性。本研究以中国北方农牧交错带盐渍化草地为研究对象,通过3年(2017–2019年)野外监测土壤呼吸及相关生物和非生物因子的变化,探讨了不同形态氮化合物添加(NH₄NO₃、(NH₄)₂SO₄和NH₄HCO₃)对盐渍化草地土壤呼吸的影响及其调控机制。结果表明：(i)土壤呼吸受大气温度、土壤温度及降水的调控,呈现双峰的季节动态变化趋势和显著的年际差异。(ii)与对照相比,经过3年的处理,土壤呼吸在NH₄NO₃、(NH₄)₂SO₄和NH₄HCO₃添加处理下分别提高了19.9%、13.0%和16.6%。(iii)NH₄NO₃添加对土壤呼吸较高的促进作用与较高的地上生物量、地下生物量以及土壤NO₃⁻含量有关。(iv)在NH₄HCO₃ 添加处理下,土壤碳排放(土壤呼吸)显著增加而碳输入(净生产力)无显著改变,表明NH₄HCO₃添加会降低土壤碳的固持。(v)净地下生产力(BNPP)是盐渍化草地土壤呼吸的最主要调控因子,并且土壤阳离子浓度和pH值通过影响土壤微生物间接影响土壤呼吸。上述研究结果表明,草地添加NH₄NO₃的研究高估了氮沉降对土壤呼吸的影响,并且在碳循环预测模型中应充分考虑盐渍化草地土壤碳动态。     

绿色木霉在啤酒糟基质上产纤维素酶条件的优化

对绿色木霉接种到啤酒糟固态发酵产纤维素酶的培养基和培养条件进行优化,考察发酵物料起始含水量、发酵时间、起始pH值等发酵条件,以及啤酒糟培养基中添加麸皮、氮源种类对产酶的影响。结果表明,以啤酒糟为发酵基质接种绿色木霉生产纤维素酶是可行的。经单因素和正交试验获得最适固态发酵的培养条件为:起始pH 5~6,培养温度28~30℃,发酵4 d;最佳发酵培养基组合为:麸皮比例30%,培养基起始含水量50%,(NH₄)₂SO₄添加量为2.0%~2.5%。     

高产纤维素酶的拟康宁木霉菌株8985固态发酵条件优化

木霉是重要的产纤维素酶真菌,在其可利用性评价筛选过程中,获得了一株在实验室条件下高产纤维素酶的拟康宁木霉菌株8985。采用响应面法对8985产纤维素酶的固态发酵条件进行了研究,以滤纸酶活为响应值,通过Plackett-Burman设计对11个因素进行了筛选,包括温度、湿度、发酵时间、K₂HPO₄、(NH₄)₂SO₄、Tween-80、麸皮添加量、CaCl₂、MgSO₄、乳糖和pH;分析结果表明,影响产酶的3个关键因素分别为发酵时间、K₂HPO₄浓度和麸皮添加量。进而采用最陡爬坡试验逼近3个关键因素的最大响应区域,采用Box-Behnken设计建立数学模型,获得最佳产酶条件为：发酵5d、K₂HPO₄ 5.18g/L、麸皮添加量0.281g/g,滤纸酶活性可达7.00IU/g,与理论值7.03IU/g接近,是优化前的1.37倍,是菌株里氏木霉QM9414该酶活的2.92倍。研究结果为木霉菌种资源的利用提供了有价值的科学信息。     

低氮胁迫诱导木薯幼苗花青素积累及其基因表达

为明确氮素浓度和形态与木薯花青素产生和积累的关系,基于氮素胁迫能够在拟南芥等植物中促进花青素产生的研究结果,以木薯品种Arg7为研究对象,研究木薯无菌幼苗在添加了(1)40 mmol/L NO₃^-+ 20 mmol/L NH₄⁺,(2)40 mmol/L NO₃^-,(3)20 mmol/L NH₄⁺,(4)0.4 mmol/L NO₃^-+ 0.2 mmol/L NH₄⁺,(5)0.4 mmol/L NO₃^-,(6)0.2 mmol/L NH₄⁺,(7)1 mmol/L(N),(8)5 mmol/L(N),(9)9 mmol/L(N),(10)13 mmol/L(N)10种氮素浓度和形态的MS培养基中生长40 d的农艺性状,以及对花青素合成相...     

Box-Behnken响应面法优化司替霉素B产生菌发酵条件

【背景】粗糙链霉菌(Streptomyces scabrisporus) HBERC-53204是本中心自主分离的一株链霉菌,经鉴定,其产生一种活性化合物司替霉素B (steffimycin B,SMB),对多种动植物重要病原菌具有良好生物活性。【目的】提高SMB发酵水平,拓宽放线菌活性天然产物在农牧业领域的研究及应用。【方法】以本实验室筛选出的一株产SMB的粗糙链霉菌HBERC-53204为研究对象,运用单因素试验筛选培养基的主效碳源、氮源、无机盐及各营养成分最适浓度,并基于单因素试验结果,通过Plackett-Burman(PB)试验设计筛选出显著影响因素,再结合最陡爬坡试验、Box-Behnken (BB)响应面法拟合显著因子与产量的非线性方程求解,进一步优化菌株产SMB的最佳发酵培养基配方。【结果】优化后最佳培养基配方为：葡萄糖36.22 g/L,蛋白胨8.00 g/L,酵母粉8.51 g/L,酸水解酪蛋白1.50 g/L,MgSO₄ 0.68 g/L,KNO₃ 1.00 g/L。经摇瓶验证,优化后SMB效价达到477.26 mg/L...     

小兴安岭酸性泥炭土硝化作用的类型及其驱动因子

以小兴安岭酸性森林泥炭土为研究对象,通过加入10 mL·L^-1乙炔及不同浓度的外源硫酸铵(0、1.2、6.0 mmol N·kg^-1)进行硝化培养试验,探究酸性泥炭土中硝化作用类型及主要驱动因子.结果表明:无论有无外加氮源,酸性泥炭土均存在较强的矿化作用(0.9～1.4 mg N·kg^-1·d^-1),经过2周的培养均发生了硝化作用(0.4～0.6 mg N·kg^-1·d^-1),且不同浓度硫酸铵处理之间无显著差异;而乙炔处理虽有较强的矿化作用(0.8 mg N·kg^-1·d^-1),但未发生明显的硝化作用(0 mg N·kg^-1·d^-1),说明该酸性泥炭土以自养硝化为主,外源无机氮源浓度对硝化作用无显著影响,硝化底物NH₃的主要来源不是外源硫酸铵,更可能来源于土壤中有机氮的矿化.培养0～14 d,无论有无外加氮源,酸性泥炭土氨氧化细菌(AOB)和古菌(AOA)丰度均显著增加,但不同浓度硫酸铵处理间无显著差异,表明外源无机氮浓度对氨氧化微生物的生长无显著促进作用.与不加乙炔的对照相比,乙炔处理AOB和AOA丰度随时间均无显著变化,推测AOA与AOB在该酸性泥炭土的硝化过程中都可能起一定的作用.     

Optimal production of sulphuric acid by Thiobacillus thiooxidans using response surface methodology

Response surface methodology was applied to determine the optimal medium composition for sulphuric acid production by an indigenous Thiobacillus thiooxidans. With a view to reducing the number of experiments and obtaining the mutual interactions between the variables, a 2⁴ full-factorial central composite design was employed for experimental design and analysis of the results. Four independent variables, KH₂PO₄, (NH₄)₂SO₄, MgSO₄ and elemental sulphur, in the growth medium were tested. Elemental sulphur was found to exhibit positive correlation with the sulphuric acid yield, whereas the other three variables showed no significant effect. No mutual interactions were found between elemental sulphur and each of the other three variables. The optimal composition of the growth medium to achieve the optimal production of sulphuric acid was determined as follows (g/l): KH₂PO₄=3.5, (NH₄)₂SO₄=4.9, MgSO₄=0.7 and elemental sulphur=23.7. The corresponding sulphuric acid production and dry cell weight were 38 662 ppm and 0.7 g/l, which are about 2.6- and 1.75-fold increase, respectively, compared with those using the conventional T. thiooxidans optimum growth medium.     

Diplatinum(III) complexes with bridging 1-methylcytosinate ligands and variable axial ligands, including guanine nucleobases

A series of diplatinum(III) complexes derived from cis-(NH₃)₂Pt^II and the model nucleobase 1-methylcytosine (1-MeC) has been prepared and X-ray structurally characterized, all of which contain two anionic base ligands (1-MeC⁻) in a head–tail (ht) arrangement: ht-cis-[(ONO₂)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(ONO₂)](NO₃)₂·HNO₃·3H₂O (2b), ht-cis-[(NO₂) (NH₃)₂ Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(OH₂)](ClO₄)₃·3.5H₂O (3), ht-cis-[(OH₂)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(OH₂)](ClO₄)₄·H₂O (4b), and ht-cis-[(9-EtGH-N7)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt (NH₃)₂(9-EtGH-N7)](NO₃)₄·9H₂O (7b) (9-EtGH=9-ethylguanine). Several other compounds, differing in the nature of the axial ligands, have been isolated and or observed in solution by ¹H and ¹⁹⁵Pt NMR spectroscopy. The chemistry of these diplatinum(III) compounds is dominated by facile substitution reactions of the axial ligands. Of particular interest in this context is the ready reaction of 2b or 3 with guanine nucleobases. Since similar compounds are not obtained with any of the other common nucleobases, 2b and 3 can be considered guanine-specific chemical probes.     

古尼虫草胞内多糖高产培养基优化研究

以古尼虫草Cordyceps gunnii胞内多糖产量为目标,利用单因素法筛选古尼虫草产胞内多糖的最适碳源、氮源和无机盐,运用正交试验筛选最佳培养基组合,用最佳培养基研究古尼虫草产胞内多糖的发酵动力学。结果表明：古尼虫草产胞内多糖最佳培养基为葡萄糖35g/L、蛋白胨15g/L、硫酸锌1g/L、KH₂PO₄ 1g/L、K₂HPO₄ 0.5g/L,用最佳培养基获得胞内多糖（5.169±0.274）g/L,产量是优化前的1.81倍;动力学研究表明,144h是古尼虫草胞内多糖最佳培养时间,此时产量最高为（6.794±0.221）g/L,是目前报道古尼虫草胞内多糖的最高产量。     

Effects of increasing polymer hydrophobicity on distribution ratios of TcO4 in polyethylene/poly(propylene glycol)-based aqueous biphasic systems

The partitioning behavior of the pertechnetate anion was studied in aqueous biphasic systems (ABS) formed from (NH₄)₂SO₄ and four types of polymers – poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), Pluronic (a PEG/PPG block copolymer), and polyvinylpyrrolidone (PVP). Phase diagrams are reported for five (NH₄)₂SO₄-polymer ABS systems including the polymers PEG-2000, PEG-3400, PEG-12 000, Pluronic-L64 (average molecular mass ≈ 2900), and PVP-K15 (average M_r≈10 000). Distribution ratios for the TcO₄⁻ anion in each of these ABS were investigated as a function of increasing salt concentration. In addition, the water-insoluble polymer PPG-2000 was studied. Pertechnetate partitions nearly quantitatively to the polymer-rich phase in each ABS, however, distribution ratios of near one were found for the PPG system. The relative ordering of the distribution ratios is PPGPVP-2000PEG-3400>PEG-12 000, exhibiting the expected increase in phase incompatibility with increasing polymer M_r. Investigation of pertechnetate partitioning in two additional ABS based on K₃PO₄ and NaOH with Pluronic-L64 revealed trends similar to those reported for PEG-2000; the distribution ratio (D) values increase in the order NaOH<(NH₄)₂SO₄3PO₄. Despite the higher distribution ratios from Pluronic-L64 at lower concentrations than found for PEG, the limited useable range of salt concentrations available may limit the practical utilization of this polymer in ABS separations.     

The stereochemistry of Co(III)-amine complexes. The use of nOe and COSY H NMR spectroscopy to determine structure in solution

It is shown how 1D nOe and 2D COSY ¹H NMR spectroscopy can be used to assign the stereochemistry of Co(III) amine complexes. By using d₆-DMSO as solvent together with a small quantity of DCl all non-equivalent N---H hydrogens can be distinguished at 300 MHz. Through-space (nOe), and through-bond (COSY), associations with other N---H and C---H hydrogens can then be determined. This leads to a complete assignment of structure in solution. The technique is applied to the complexes syn(N), anti(N)-[Co(cyclen) (NH₃)₂] (ClO₄)₃, syn(N), anti(Cl)-[Co(cyclen) (NH₃)Cl] (ClO₄)₂, anti(N), syn(Cl)-[Co(cyclen) (NH₃)Cl](ClO₄)₂, syn(N), anti(O)-[Co(Mecyclen)-(GlyO)](ClO₄)₂ and Δ-cis-[Co(δ-en)₂(NO₂)₂](NO₂).     

凋落叶多样性对杉木幼苗生长及吸收15N标记硫铵的影响

利用¹⁵N硫铵研究了凋落叶多样性对杉木幼苗生长及养分吸收的影响.结果表明,凋落叶多样性的增加有利于盆栽杉木幼苗的生长.杉木、火力楠、红栲和刺楸4种凋落叶混合处理后,杉木幼苗的生长量最大;杉木、火力楠、刺楸3种凋落叶混合处理后的杉木幼苗生长量次之,其它依次为杉木、火力楠、红栲3种凋落叶混合处理>杉木和刺楸凋落叶处理>杉木和红栲凋落叶处理>对照>杉木和火力楠2种凋落叶混合处理>杉木凋落叶处理.就杉木幼苗对硫铵氮的吸收率而言,不作任何处理的杉木幼苗吸收率最高,其次为杉木、火力楠、红栲和刺楸4种凋落叶混合处理,其它依次为杉木、火力楠、刺楸3种凋落叶混合处理和杉木、火力楠、红栲3种凋落叶混合处理>杉木和刺楸凋落叶处理>杉木和红栲凋落叶处理>杉木和火力楠2种凋落叶混合处理>杉木凋落叶处理.另外,用凋落叶处理后,土壤中硫铵氮的残留量比不作凋落叶处理的土壤多,硫铵氮的总回收量也比不作凋落叶处理的土壤大幅增加,而且凋落叶多样性的增加也会增加硫铵氮的残留量.     

超嗜热古菌Thermococcus eurythermalis A501编码B家族DNA聚合酶的生化特性及PCR应用

DNA聚合酶广泛应用于PCR技术,在生命科学研究及相关领域发挥重要作用。但目前商业化DNA聚合酶仍不能完全满足科研需要,有必要寻求高性能DNA聚合酶。文中克隆表达了超嗜热古菌（Thermococcus eurythermalis）A501来源的B家族DNA聚合酶基因（NCBI数据库基因登录号为TEU＿RS04875）、表征该重组蛋白的生化特性、评价了其PCR应用。将删除intein蛋白序列的DNA聚合酶（Teu-PolB）进行体外重组表达,经亲和层析和离子交换层析纯化获得Teu-PolB蛋白;利用5′端带荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳鉴定Teu-PolB的生化特性;以噬菌体λDNA基因组为模板,探究Teu-PolB的PCR应用。结果显示,Teu-PolB具有DNA聚合酶活性和3′→5′核酸外切酶活性,该酶在98℃下的半衰期约为2 h,热稳定性高。使用Teu-PolB进行PCR扩增,最适PCR缓冲液为50 mmol/L Tris-HCl pH 8.0,2.5 mmol/L MgCl₂,60 mmol/L KCl,10 mmol/L （NH<...     

Structure and physicochemical properties of barley non-starch polysaccharides—II. Alkaliextractable β-glucans and arabinoxylans

Three fractions containing hemicellulosic material were obtained by sequential extraction of barley residue (left after removal of water-extractable polysaccharides) with saturated barium hydroxide [Ba(OH)₂ fraction], distilled water [Ba(OH)₂/H₂O fraction], and 1 m sodium hydroxide [NaOH fraction]. The yields of the fractions were 1.6, 1.7, and 2.6% (w/w), respectively, of the dry barley grist. The Ba(OH)₂ fraction contained mainly arabinose and xylose, 35.8% and 60.9%, respectively. The Ba(OH)₂/H₂O fraction in addition to 26.7% Ara and 36.6% Xyl contained also 34.8% Glc. The NaOH fraction was composed of 14.2% Ara, 44.0% Xyl, and 40.9% Glc. The Ba(OH)₂/H₂O and NaOH extracts were further fractionated by stepwise (NH₄)₂SO₄ precipitation into several subfractions with varying amounts of β-glucans and arabinoxylans. β-Glucans in Ba(OH)₂/H₂O and NaOH fractions were characterized by high ratios of β-(1→4)/β-(1→3) linkages, large amounts of contiguously linked β-(1→4) segments, and high ratios of cellotriosyl/cellotetraosyl units. The alkali-extractable arabinoxylans, especially those NaOH-extractable, were characterized by a very low degree of substitution, high xylose/arabinose ratio, and a small content of doubly substituted xylose residues. Some populations of arabinoxylans displayed structural features that would enable them to self-associate or to interact with β-glucans.