营养缺陷型酿酒酵母AY生长代谢的热动力学研究

用微量热法研究了尿嘧啶缺陷型酿酒酵母AY,和该菌株分别经穿梭质粒pYLZ2、重组质粒pYLZ2/f27、pYLZ2/622转化的3种菌株的生长代谢热动力学.尿嘧啶缺陷型酿酒酵母AY在基本培养基中不生长,没有代谢热效应产生,而在葡萄糖蛋白胨培养基和丰富营养培养基(YPD)中生长,并且在YPD中生长最好;向基本培养基中加入尿嘧啶后,AY能够生长,而且随着尿嘧啶浓度的增加,其生长速率常数增大;经质粒转化的3个菌株均能在基本培养基中生长,代谢产热曲线各不相同,与转入质粒的结构、功能密切相关.研究结果表明了各菌株遗传特征的差异.     

酵母高稳定载体的构建及人-αA干扰素在酵母中的高表达和分泌

本文利用酿酒酵母天然2μm质粒的SnaBI位点与pEMBL Yi27质粒的SmaI位点经平末端连接,构建成酿酒酵母高稳定质粒载体pHC11,经测定表明,其酵母转化子在非选择培养条件下连续生长50世代后,仍有82％的细胞保留该质粒,此结果证实了2μm质粒中非功能区的存在,将人-αA干扰素基因表达分泌单元插入pHC11,构建成重组质粒转化酵母后,在完全培养基中发酵培养,经分析测定,培养上清液中表达产物占总蛋白量的36.8％,干扰素效价达2.6×10~(10)u/L,表明利用高稳定载体pHC11使人-αA干扰素在酵母中得到了高表达和分泌。     

黑曲霉葡萄糖淀粉酶基因在酿酒酵母中的表达和分泌

将黑曲霉葡萄糖淀粉酶GAI的cDNA插入到质粒pMA91的酵母PGK基因的启动子和终止序列之间,构成含葡萄糖淀粉酶基因的表达载体pMAG69。用原生质体转化法引入酿酒酵母GRF18,实现黑曲霉葡萄糖淀粉酶基因在酿酒酵母中的表达,利用基因本身的信号序列使99％以上的酶蛋白分泌至胞外,构建的酵母菌株在含10％淀粉的培养基中2天内能水解87％的淀粉,对酵母转化子的稳定性检测显示,重组质粒pMAG69可在酿酒酵母中较稳定地存在。     

产甘油基因工程菌的构建

利用途径工程的基本原理,在大肠杆菌中构建一条产甘油的新代谢途径。从酿酒酵母Sacchdromyces cerevisiae INVSc1菌株总DNA克隆3-磷酸甘油脱氢酶基因(gpdl)和3-磷酸甘油酯酶基因(hot2),构建由两个杂合启动子trc启动基因的双表达盒的重组质粒pGEM-Cgpd1-Chor2,后者转入E．coli JM109菌株,构建的重组菌株就具有一条直接将葡萄糖转化为甘油的新代谢途径,将该重组菌株以葡萄糖为底物进行摇瓶发酵,甘油产率为1．18g／L。该研究结果为进一步构建生产1,3-丙二醇工程菌打下了基础。     

苏云金芽胞杆菌含不同质粒和不同基因工程菌的生长代谢

用微量热法研究了世界上产量最大的微生物杀中心剂苏云芽胞杆菌(Bacillusthuringiensis)野生菌株YBT－1463,无晶体突变株BMB160和无质粒、无晶体突变株BMB171以及含量不同杀虫蛋白基因工程菌BMB－304－171－A和BMB－304－171－B的生长代谢动力学变化。结果是,含有14个质粒的野生菌株YBT－1463的生长代谢热比无晶体含6个质粒的变株BMB160和无晶体、无质粒突变株BMB171低;将杀虫晶体蛋白基因vryⅠAc和cryⅠC分别转入受体菌BMB171中后,两个工程菌BMB－303－171－A和BMB－304－171－B生长代谢热与受体菌BMB171相比也吸显降低;表明质粒形成一个耗能过程,当受体菌BMB171转入杀虫晶体蛋白基因后,工菌比受茶杯菌的放热大幅度减少,表明基因编码杀虫晶体蛋白也是一个耗能过程,但是含基因cryⅠAc和cryⅠC工程菌BMB－304－171－A和BMB－304－171－B之间的生长代谢没有明显差异。首次报道这些热动力学变化,对杀虫剂发酵生产过程的代谢控有重要指导意义。     

重组质粒对大肠杆菌HB101生长影响的微量热研究

用微量热方法研究了嗜麦芽假单胞菌AT18, 受体菌大肠杆菌HB101, mel基因工程菌——大肠杆菌HB101/pWSY8和携带克隆载体pUC18质粒的大肠杆菌HB101等的生长代谢过程. 实验结果从热化学和热动力学上阐明了细菌的生长速率常数与其所含质粒的大小呈负相关. 探讨了低温处理对含不同质粒大肠杆菌生长的影响, 发现低温处理对工程菌生长影响最大.     

细菌变异株生长热谱研究

用微量热法研究微生物的生长与代谢,是生物热力学一个新的研究领域.我们已经测得细菌生长的完整热谱,这种热谱具有良好的重现性和明显的特征性,可以作为细菌的“指纹图”对其进行种的分类和鉴定[1～5].但在研究中发现,同种细菌不同变异株的生长图谱略有差异,若培养基选择?..     

苏云金芽胞杆菌含不同质粒和不同基因工程菌的生长代谢

用微量热法研究了世界上产量最大的微生物杀中心剂苏云芽胞杆菌(Bacillusthuringiensis)野生菌株YBT－1463,无晶体突变株BMB160和无质粒、无晶体突变株BMB171以及含量不同杀虫蛋白基因工程菌BMB－304－171－A和BMB－304－171－B的生长代谢动力学变化。结果是,含有14个质粒的野生菌株YBT－1463的生长代谢热比无晶体含6个质粒的变株BMB160和无晶体、无质粒突变株BMB171低;将杀虫晶体蛋白基因vryⅠAc和cryⅠC分别转入受体菌BMB171中后,两个工程菌BMB－303－171－A和BMB－304－171－B生长代谢热与受体菌BMB171相比也吸显降低;表明质粒形成一个耗能过程,当受体菌BMB171转入杀虫晶体蛋白基因后,工菌比受茶杯菌的放热大幅度减少,表明基因编码杀虫晶体蛋白也是一个耗能过程,但是含基因cryⅠAc和cryⅠC工程菌BMB－304－171－A和BMB－304－171－B之间的生长代谢没有明显差异。首次报道这些热动力学变化,对杀虫剂发酵生产过程的代谢控有重要指导意义。     

微量热法和荧光显微法研究CdTe量子点敏化的纳米TiO2对大肠杆菌的抗菌活性及细胞损伤机制

通过水热合成法和自组装方法分别合成了CdTe(QDs)量子点与CdTe(QDs)敏化的纳米TiO₂复合物,并将CdTe(QDs)/TiO₂与TiO₂均配成浓度为1×10^-2 mol/L的悬浮液. 取一小环大肠杆菌(E. coli)的营养肉汤培养基至5 mL LB培养基中,则E. coli的浓度配成大约1×10⁶ cell/mL的悬浮液以作备用. 在37.0 ℃条件下,利用LKB-2277生物活性检测仪并采用停流法进行检测,研究了CdTe(QDs)/TiO₂对E. coli的抗菌行为. 通过测定和分析产热功率-时间曲线,获得细胞生长速率常数(k)、最大产热功率(P_m)、传代时间(t_G)以及抑制率(I)等热动力学常数. 结果表明：在0～4.0×10^-5 mol·L^-1浓度范围内,生长速率常数(k)和抑制率(I)均与浓度成一定的线性关系. 生长速率常数(k)和最大产热功率(P_m)随着CdTe(QDs)/TiO₂的浓度增加而下降,而传代时间(t_G)和抑制率(I)随着CdTe(QDs)/TiO₂ 的浓度增加而增加;其中,CdTe(QDs)/TiO₂的生长速率常数斜率(0.0001)小于TiO₂的生长速率常数斜率(0.0005),可以初步判断CdTe(QDs)/TiO₂对E. coli的代谢影响较TiO₂要大. TiO₂和CdTe(QDs)/TiO₂对E. coli代谢活性均有一定的抑制作用. 在相同浓度时,CdTe(QDs)/TiO₂的抑制率斜率(0.84)大于TiO₂的抑制率斜率(0.26),进一步说明了CdTe(QDs)/TiO₂相比TiO₂具有对E. coli 更强的细菌抑制作用;结合Hadama荧光显微方法,进一步佐证了CdTe(QDs)/TiO₂相对于TiO₂表面结构的改变可能是导致其抗菌效果较好的主要原因之一. 从而为深入揭示纳米材料影响微生物生长代谢过程的热动力学规律和细胞损伤机制提供理论支撑.     

抗重金属真菌的分离及特性研究

从四川省彭州市龙门山镇铜尾矿土壤中分离得到了三株高抗重金属铜离子的真菌.本文对其中一株具有较好重金属抗性的菌株Y2进行了研究,根据形态学特征和18S rDNA序列分析将其鉴定为青霉菌属.在PDA培养基上,Y2菌株能够耐300mmol/LCu2+,限制Y2菌株生长的其它金属盐的最小浓度(mmol/L)依次为:Zn2+,600;Cd2+,<20;Cr3+,<3;Ni2+,<20;Pb2+,<50.在含3种金属盐混合物的PDA中的Y2菌株生长的正交实验结果表明:金属盐之间存在显著的毒性协同效应,不仅与盐的种类也与组成盐之间的浓度相关;Y2菌株具有一定的抗三种金属盐混合物的能力.原子吸收测定Y2菌株对重金属污染土壤的处理,结果表明菌株Y2对土壤中铜有一定吸附作用.     

Polyploid Formation Between <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Trichoderma viride</Emphasis> For Enhanced Citric Acid Production From Cellulose

The first-stage heterokaryons, obtaining from intergeneric protoplast fusion between Aspergillus niger (Y-b) and Trichoderma viride (M5S51), showed slow growth and mixed morphologies on minimal medium. The fusants were classified into heterokaryon and prototrophic haploid, showing the morphology as that of A. niger. The heterokaryon strains formed conidia with the same nutritional requirements as those of the original auxotrophic mutant strains. After several subcultivations on minimal medium containing d-camphor, some heterokaryon strains formed larger two to seven nuclei/conidium as compared to one nucleus/conidium of the auxotrophic mutant and prototrophic strains, indicating that the new hybrids were generated. Interestingly, three fusant strains AT 11-2-3, AT 11-2-10, and AT 11-2-14 produce 19.2, 6.1, and 10.5 g/l citric acid, respectively, in semisolid culture containing cellulose, whereas A. niger Yang no. 2 could not use carboxymethyl cellulose as the sole carbon source for citric acid production. In addition, the average maximum beta-glucosidase and carboxymethylcellulase productions from AT 11-2-3, AT 11-2-10, and AT 11-2-14 were about 16- and 4-folds higher than those of A. niger, respectively.     

Invertase production on solid-state fermentation by Aspergillus niger strains improved by parasexual recombination

Invertase production by Aspergillus niger grown by solid-state fermentation was found to be higher than by conventional submerged fermentation. The haploid mutant strains Aw96-3 and Aw96-4 showed better productivity of various enzymes, as compared to wild-type parental strain A. niger C28B25. Here we use parasexual crosses of those mutants to increase further the productivity of invertase in solid-state fermentation. We isolated both a diploid (DAR2) and an autodiploid (AD96-4) strain, which were able to grow in minimal medium after mutation complementation of previously isolated haploid auxotrophic strains. Invertase production was measured in solid-state fermentation cultures, using polyurethane foam as an inert support for fungal growth. Water activity value (Aw) was adjusted to 0.96, since low Aw values are characteristic in some solid-state fermentation processes. Such diploid strains showed invertase productivity levels 5–18 times higher than levels achieved by the corresponding haploid strains. For instance, values for C28B25, Aw96-3, Aw96-4, DAR2, and AD96-4 were 441, 254, 62, 1324, and 2677 IU/(L·h), respectively. These results showed that genetic recombination, achieved through parasexual crosses in A. niger, results in improved strains with potential applications for solid-state fermentation processes.     

Improving the catabolic functions of desiccation-tolerant soil bacteria

Bacterial strains were selected from a desiccated polluted soil for their drought tolerance and their ability to grow on diesel oil in view of incorporating them in a bioaugmentation product. These products are useful in case of recal citrant xenobiotic pollution, where there is no intrinsic biodegradation activity in the soil. These strains grow on the easily degradable components of diesel oil. In troduction of new catabolic genes into these desiccation-tolerant bacteria in order to improve their catabolic functions was considered. Plasmid-borne catabolic genes coding for enzymes in volved in the degradation of more recalcitrant compounds (Isopropylbenzene, trichloroethene, 3-chloroben zoate, 4-chlorobiphenyl, biphenyl) were successfully introduced in some of the desiccation-tolerant strains by means of natural conjugation. Strains exhibiting good tolerance to desiccation and able to grow on the new carbon sources were obtained. The frequencies of integration of the plasmids ranged from 2×10⁻⁸ to 9.2 10⁻² transconjugants/acceptor. Drought-tolerance is indeed important for bioaugmentation because of its in trinsic ecological significance and because a bioaugmentation starter has to be conditioned in a desic cated form to ensure good shelf-life. The conservation of the properties during storage was evaluated by accelerated storage tests.     

IMMOBILIZATION OF Saccharomyces Cerevisiae USING POLY（ACRYLAMIDE） GEL FOR ASYMMETRIC SYNTHESIS OF R（-）-MANDELIC ACID

In this paper, the poly(acrylamide) hydrogel used to immobilize saccharomyces cerevisiae for asymmetric synthesis of R(-)-mandelic acid was prepared with free radical ploymerization in deionized water at room temperature under nitrogen atmosphere. The influence of the composition of hydrogel, loading amount of cells and culture conditions on the asymmetric synthesis was investigated. Results show that PAAm hydrogel is a feasible carrier for immobilization of cells which is a potential alternative method to prepare enantiomerically pure R(-)-mandelic acid.     

IMMOBILIZATION OF Saccharomyces Cerevisiae USING POLY(ACRYLAMIDE)GEL FOR ASYMMETRIC SYNTHESIS OF R(-)-MANDELIC ACID

1. INTRODUCTIONPoly(acrylamide) hydrogel (PAAm gel), which is able to swell, but cannot be dissolved in the aqueous environment, is a three-dimension network with repeating hydrophilic units -CONH2. Since PAAm gel has a good biocompatibility as well as a high mechanical strength, and can be easily separated from reaction medium, PAAm gel has often been employed to immobilize enzymes and cells so as to catalyze various of chemical and biochemical reactions [1,2]. So far, biosynthesis …     

基元通量模式预测酵母生长现象

本文通过EFM预测了基因突变后的酵母细胞生长现象, 模拟预测结果和实验结果吻合很好; 与FBA方法得到的模拟结果相比较, EFM方法能更好地把基因突变和其表型(生长)联系起来.     

Co-Expression of Recombinant Nucleoside Phosphorylase from <Emphasis Type="Italic">Escherichia coli</Emphasis> and its Application

The genes encoding purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase), and thymidine phosphorylase (TPase) from Escherichia coli K12 were cloned respectively into expression vector pET-11a or pET-28a. The recombinant plasmids were transformed into the host strain E. coli BL21(DE3) to construct four co-expression recombinant strains. Two of them had double recombinant plasmids (DUD and DAD) and the other two had tandem recombinant plasmid (TDU and TDA) in them. Under the repression of antibiotic, recombinant plasmids stably existed in host strains. Enzymes were abundantly expressed after induction with IPTG and large amount of target proteins were expressed in soluble form analyzed with SDS-PAGE. Compared with the host strain, enzyme activity of the recombinant strains had been notably improved. In the transglycosylation reaction, yield of 2,6-diaminopurine-2’-deoxyriboside (DAPdR) from 2,6-diaminopurine (DAP) and thymidine reached 40.2% and 51.8% catalyzed by DAD and TDA respectively; yield of 2,6-diaminopurine riboside (DAPR) from DAP and uridine reached 88.2% and 58.0% catalyzed by TDU and DUD respectively.     

A SECOND PHOTOREACTIVATION-DEFICIENT MUTATION IN SACCHAROMYCES CEREVISIAE

Abstract— A mutant of Saccharomyces cerevisiae was isolated which has a low capability for photoreactivating UV-induced lethal damage. The DNA photolyase activity of the cell-free extract is much less than in a wild-type strain. In vivo complementation of photoreactivation was demonstrated in crosses with phr1 mutant strains. Tetrad analysis and backcrosses suggest that this new mutation defines a second chromosomal gene PHR2 which is loosely linked to the PHR1 gene.     

The “bringer” strategy

Random mutagenesis constitutes a keystone in many strategies of directed evolution of biocatalysts and is often done by error-prone polymerase chain reaction (epPCR). Traditionally, the epPCR-generated DNA fragments are then subcloned into an expression vector to obtain a mutant library, which in turn is transformed into a suited host and screened for mutants that display the desired property. However, the vast majority of epPCR-generated fragments generally are lost during the subcloning step, making it the bottleneck in the mutant library construction procedure. Here we report a rapid and convenient strategy based on the epPCR amplification of a ring-closed expression plasmid that contains the gene of interest; after a DpnI digest the product of the epPCR reaction constitutes the mutant library and can be used directly for screening procedures. Primers binding to the beta-lactamase gene were chosen to allow application of the strategy to as broad a range of target plasmids as possible. The functionality of this approach was demonstrated by mutating the alpha-peptide coding region of the lacZ gene.     

l-Asparaginase II of Saccharomyces cerevisiae

The activity profile of the periplasmic asparaginase of Saccharomyces cerevisiae was determined during cell growth in an ure2 mutant; in an ure2 transformed with a plasmid containing the gene URE2 and, for comparison, in the strain D273-10B. Cells were cultivated in media presenting variable quantitative and qualitative nitrogen availability and the enzyme activity was evaluated in fresh and in nitrogen-starved cells. Nitrogen affected the asparaginase II level in fresh and starved cells of all strains. In the best condition, enzyme was produced by the wild-type cells at the late log-phase in the glucose/ammonium medium with a carbon to nitrogen ratio 4.3:1. Upon starvation, the activity doubled. The overall profile of the transformed strain was similar to that of the wild-type strain. In the ure2 mutant, highenzyme levels were observed during growth, as expected. However the activity level, upon starvation, in proline grown cells, increased sixfold, suggesting that in addition to the Ure2p-Gln3p system, another system regulates asparaginase II biosynthesis.