Production and structural features of a water-soluble polysaccharide from a mutant strain of Agrobacterium sp.

We have developed a mutant strain derived from Agrobacterium sp. ATCC 31750, which produces a water-soluble polysaccharide having potential utility to the food, feed, pharmaceutical and cosmetic industries. A high concentration of product (15 g/L) is obtained by 48 h cultivation of the mutant strain under optimized fermentation conditions. The water-soluble polysaccharide obtained from cultures of the mutant strain beta82 has Glc:Man:Gal in approximate molar ratios of 5.8:6.7:1.0. The molecular weight of the polysaccharide was determined to be approximately 1000 kDa by HPSEC analysis. Linkage analysis contained 3-Glcp, 3-Manp, terminal Glcp and terminal Manp, as well as a small proportion of 3- and 3,4-Galp, and 4,6-Manp residues. Based on analyses using FT-IR and ¹³C NMR spectrometers, most glycosidic bonds joining these sugar residues are of the α-type, and acetyl groups are apparently attached to the polymer chain at random.     

磷细菌突变株生理特性的研究

本文对经过筛选培育出的一株解磷能力较强的磷细菌突变株Ｄ０１７Ｐ的生理特性进行了研究。其中,最佳氮源为麦麸汁,与出发菌株所用马铃薯汁相比,生物量增加９．８倍,解磷能力提高７１．４％,最佳碳源为蔗糖,与出发菌株所用碳源葡萄糖比较,解磷能力提高２８．８％,金属离子,培养温度,ｐＨ,气／液比等也对突变株的生长有一定影响,其结果可作为选定工业化条件的参考。     

睫状神经营养因子突变体的克隆、表达、纯化及生物学活性

目的设计具有更高生物学活性的睫状神经营养因子突变体,并对其进行表达、纯化及生物学活性检测。方法以计算机分子模拟系统设计突变体,重叠延伸PCR方法获得突变体的编码区DNA序列,克隆入表达载体pThioHisA,转化E.coliBL21,以IPTG诱导表达。复性纯化后,用鸡胚背根神经节无血清培养法和小鼠减重法进行生物学活性测定。结果目的蛋白以包涵体形式存在,表达量在35%以上,纯化后的纯度达95%以上,能有效地促进鸡胚背根神经节的生长,并能使正常小鼠的体重降低,脂肪指数下降。结论所设计的突变体经表达及纯化后,具有良好的生物学活性,为进一步研究突变体蛋白的促神经生长和减肥作用奠定了基础。     

刺桐胰蛋白酶抑制剂突变体在大肠杆菌中表达及在tPA纯化中的应用

目的构建刺桐胰蛋白酶抑制剂突变体(rserETI)原核表达载体,制备表达产物,用于tPA的纯化。方法设计合成rserETI编码序列DNA片段,以PCR法扩增出全长编码区序列,经酶切后克隆至pET9a表达载体,转化大肠杆菌JM109DE3,以IPTG诱导表达。表达产物经变性、复性、QFF层析纯化后,测定其活性,并与溴化氰活化的Sepharose偶联合成亲和层析柱,纯化rtPA。结果rserETI的表达量约占大肠杆菌菌体总蛋白的30%,复性率为80%,经QFF层析纯化后,蛋白浓度为1.58mg/ml,SDS-PAGE检测显示无杂蛋白污染,纯度大于95%,对rtPA突变体的抑制比活性为4×104IU/mg。偶联合成的rserETI-Sepharose亲和层析柱能特异性地纯化rtPA,rtPA突变体纯度达96%,比活性为5.07×105IU/mg。结论已成功构建了rserETI原核表达载体,并在大肠杆菌中高效表达,制备的表达产物可用于rtPA的纯化。     

Retention of inorganic arsenic by coryneform mutant strains

The natural resistance mechanisms of corynebacteria to respond to the environments containing high levels of arsenic were successfully adopted to develop inexpensive and selective extractants for submicrogram amounts of arsenic. Kinetic and equilibrium characteristics were evaluated, and a preliminary exploration of the capability of these strains to be used for arsenic speciation was also made in this work. Three kinetics models were used to fit the experimental data. It was found that the pseudo-first-order kinetics model was not quite adequate to describe the retention process, while the intraparticle diffusion and the pseudo-second-order kinetics models provide the best fits. The equilibrium isotherm showed that the retention of arsenic was consistent with the Langmuir equation and that the Freundlich and Dubinin-Radushkevich models provided poorer fits to the experimental data. The maximum effective retention capacity for arsenic was about 15.4 ng As/mg biomass. The amount of arsenic retained was directly measured in the biomass by forward planning a slurry electrothermal atomic absorption spectrometric procedure.     

利用细胞工程筛选烟草抗赤星病突变体的研究——一种双层培养基筛选系统的建立

为有效筛选烟草抗赤星病细胞突变体,本研究建立了一种双层培养基筛选系统。即在下层培养基中接种烟草赤星病(Alternaria alternata),在上层培养基中接种烤烟品种 NC89和净叶黄的花药(NC89高感赤星病,净叶黄高抗赤星病)。在26±1℃培养40天后发现,NC89的花药出苗率和花药平均出苗数与对照(不接种赤星病的培养基)相比,分别由30%和53颗降到2%和12颗,而净叶黄的花药出苗率和平均出苗数基本没有变化。对筛选出来的 NC89花培突变体进行了田间赤星病抗性鉴定,其中80%对赤星病抗性较 NC89有所提高,甚至部分接近高抗。这说明该双层培养基系统可以有效地对抗烟草赤星病的细胞突变体进行筛选。本系统尤其适应于(1)需长期培养;(2)毒素不易制备或易失活的病原菌的抗性筛选。     

Hydrogen production by combining two types of photosynthetic bacteria with different characteristics

The double-layer photobioreactor using two types of photosynthetic bacteria, Rhodobacter sphaeroides RV and its reduced-pigment mutant, MTP4, was developed for efficient hydrogen production. The two types of bacteria had different characteristics on light energy, hydrogen production rate and conversion efficiency. MTP4 produced hydrogen more efficiently under high light conditions and RV did so under low light conditions. Illuminated light toward the surface of a photobioreactor quasi-exponentially declines as it penetrates into the reactor. When two types of bacteria were placed using the developed reactor according to this light distribution, the hydrogen production rate reached 3.64 l/m²/h at a light intensity of 500 W/m² in 24 h and the conversion efficiency of light energy to hydrogen was 2.18%. These values were 33% higher than those of only using RV. The low light in the deep part of the reactor was utilized efficiently, resulting in a higher hydrogen production rate.     

Efficient hydrogen production using a multi-layered photobioreactor and a photosynthetic bacterium mutant with reduced pigment

A multi-layered photobioreactor (MLPR), where the light paths were formed by the localization of bacterial cells, was constructed for efficient hydrogen production. The performance was investigated under several conditions in order to clarify the effect of this reactor on hydrogen production. An analysis of the hydrogen production profile showed that the MPLR utilizes both the light that directly illuminates its surface and the light induced and diffused from its light paths for hydrogen production. It was also found that the hydrogen productivity in the MLPR was more than twice that in a plate-type reactor. When a photosynthetic bacterium mutant with reduced pigment, MTP4, was used, the maximum hydrogen production rate reached 2.0 l/m² h, which was 38% higher than that of a conventional plate-type reactor. The synergistic effect of the improvement in the reactor and the modification of the bacteria was brought about by the combination of the MLPR and MTP4, and resulted in an improvement in the hydrogen production.     

蜂毒肽/变构hIL-2融合蛋白的原核表达及其对宿主菌生长的影响

目的构建蜂毒肽(Melittin)与变构hIL-2融合基因原核表达质粒,并检测表达的融合蛋白对宿主菌E.coli生长的影响。方法以质粒pGEX-4T-2/Melittin-IL-2(88Arg)为模板,通过PCR定点诱变为Melittin-IL-2(88Arg,125Ala),将PCR产物和pET-15b载体分别经双酶切后连接,构建重组表达质粒pET-15b/M-IL-2(88Arg,125Ala),转化E.coliBL21(DE3),IPTG诱导表达。SDS-PAGE及ELISA分析表达产物;检测诱导不同时间重组菌的A600值,绘制生长曲线,并进行活菌计数。结果PCR扩增的目的片段长542bp,重组表达质粒测序分析证明目的基因如预期突变;ELISA可检测到目的蛋白表达,但表达量较低;SDS-PAGE分析未见目的条带;诱导表达4h,重组菌A600值由0.8下降至0.6;诱导2h,活菌计数由108个/ml降至104个/ml。结论已成功构建了原核表达质粒pET-15b/M-IL-2(88Arg,125Ala),表达的融合蛋白可能对宿主菌具有毒性,从而杀伤宿主菌。     

集成干扰素突变体的构建、表达及纯化

目的构建高效且可供单甲氧基聚乙二醇马来酰亚胺(mPEG-MAL)定点修饰的集成干扰素突变体(IIFNm108),并进行表达、纯化及鉴定。方法采用同源序列对比、空间结构模拟并结合α干扰素与受体结合的特点设计突变位点。用重叠延伸PCR法,扩增IIFNm108基因,与pET-23b载体连接,构建重组表达质粒pET-23b-IIFNm108,转化E.coli BL21(DE3),IPTG诱导表达,所得包涵体经变性、复性、DEAE阴离子交换及凝胶过滤两步层析纯化后,进行各项鉴定。结果重组表达质粒经双酶切及测序鉴定证明构建正确。目的蛋白的表达量占菌体总蛋白的30%以上,主要以包涵体形式存在。纯化的IIFNm108蛋白纯度大于95%,比活性约为(2.08±0.17)×108IU/mg,N-端、C-端氨基酸序列与理论一致,相对分子质量为19500,可与mPEG-MAL成功交联。结论已获得一种高效且可供mPEG-MAL定点修饰的集成干扰素突变体IIFNm108。