巴斯德毕赤酵母(P.pastoris)高密度发酵研究进展

高密度发酵已经成为提高毕赤酵母目的蛋白表达量的关键技术之一,而其中发酵工艺又是高密度发酵的一个重要因素。主要从巴斯德毕赤酵母遗传学特性、表达宿主菌、表达载体、外源蛋白的表达方面进行了阐述,并从毕赤酵母工程菌的选择、培养基的优化设计、发酵过程关键参数调控以及甲醇诱导等方面阐述毕赤酵母的高密度发酵。最后,提出了巴斯德毕赤酵母高密度发酵过程中存在的问题并进行展望。     

毕赤酵母表达巴曲酶发酵条件的优化研究

目的对表达重组巴曲酶的巴斯德毕赤酵母的发酵条件进行优化,确定最佳的发酵控制条件以获得重组巴曲酶的最高表达量。方法通过多因素正交实验确定巴曲酶发酵培养的最适培养条件。结果表达温度在25℃,pH值为7．0,加入甲醇的量为10g／L时为最优发酵条件,诱导表达时间为84-96h。结论重组巴曲酶可以开发为止血药,代替临床应用的从蛇毒中提取的巴曲酶。     

重组人抗凝血酶发酵条件的优化

目的:通过实验室发酵条件优化工作,实现在巴斯德毕赤酵母中高效表达重组人抗凝血酶。方法:在摇瓶培养条件下,应用正交实验方法考查人抗凝血酶重组毕赤酵母菌pPIC9K-AT-02的培养温度、培养基pH值、接种比例、甲醇补加间隔时间及甲醇补加浓度等5种因素对重组人抗凝血酶活性的影响,确定最优发酵条件。结果与结论:筛选出的最终发酵条件为培养温度30℃、培养基pH6.0、接种比例20%、甲醇补加间隔时间24 h、甲醇补加浓度2%,重组人抗凝血酶在巴斯德毕赤酵母中表达活性为4098 U/L,比原始活性提高了150%。     

外源基因在毕赤酵母中表达的优化

巴斯德毕赤酵母是近年来成功的外源基因表达系统,已表达出众多外源蛋白。它既能像原核生物一样快速生长高密度发酵,又能进行真核翻译后修饰,并且蛋白分泌量大,因此应用越来越广泛。如果对它的表达载体,转化诱导条件和目的基因内部结构,发酵条件等方面进行优化,能够进一步发挥它的优势,更好地表达需要的外源蛋白。本文就毕赤酵母表达系统表达优化进行总结综述。     

毕赤酵母高密度发酵研究进展

巴斯德毕赤酵母表达系统是近年发展起来的一种优秀的真核表达系统.本从培养基、温度、pH值、溶氧、甲醇的流加等角度对国内外毕赤酵母高密度发酵的研究进展做了较详细的综述,并提出了目前毕赤酵母高密度发酵过程中存在的问题.     

重组巴斯德毕赤酵母发酵生产内切几丁质酶的培养条件优化

探讨重组巴斯德毕赤酵母发酵生产内切几丁质酶的最适培养条件,以期获得最佳的内切几丁质酶活力。以内切几丁质酶活力为指标,通过部分因子试验设计以及响应面法优化确定重组巴斯德毕赤酵母高产内切几丁质酶的最适培养条件。部分因子试验设计筛选的影响重组巴斯德毕赤酵母高产内切几丁质酶的3个关键因子为甲醇、油酸和吐温-80。响应面法优化的上述3个关键因子的最佳浓度分别为0.71%、0.086%和0.31%。重组巴斯德毕赤酵母发酵生产内切几丁质酶的最适培养条件为：酵母膏1%、酵母氮碱（YNB）1.34%、蛋白胨2%、甲醇0.71%、油酸0.086%、吐温-80 0.31%、PTM₁ 0.8%、pH 6.0。在上述培养条件下,重组巴斯德毕赤酵母产内切几丁质酶的活力高达30.92U/mL。与未优化前相比,酶活力提高了1.44倍。研究结果为内切几丁质酶的产业化生产和应用奠定了良好基础。     

巴斯德毕赤酵母表达系统的研究进展和前景展望

巴斯德毕赤酵母经过近二十来年的发展,已经成为表达外源基因的优秀表达系统之一,成功地表达了许多重组异源蛋白。从表达菌株,表达载体等方面详细综述了毕赤酵母表达系统的优点,如：营养要求低、可高密度发酵、遗传稳定性高等;分析了可能影响巴斯德毕赤酵母表达系统的相关因素,这些因素包括外源基因的特性、基因拷贝数、产物稳定性及发酵策略等,结合这些因素和具体实践经验,就如何提高外源基因在巴斯德毕赤酵母中表达量进行了阐述;讨论了该表达系统存在的不足之处并且展望了其发展前景。     

重组textlinin-1抑制活性的性质研究

目的对毕赤酵母表达的textlinin-1(Q8008)进行分离纯化,并对其抑制plasmin活性的性质进行研究。方法构建表达Q8008pachia pastoris工程菌,经发酵、纯化得到纯度达95%以上的Q8008。考察不同的温度、pH缓冲液和金属离子等条件对Q8008抑制plasmin活性的影响。结果 Q8008在中性条件下比较稳定,其最适pH值为7.0,最适温度为40℃,在30~50℃相对酶活力在90.0%以上;有2个金属离子对Q8008活性测定方法的影响产生了显著性差异,Zn2+起激活作用,Co2+起抑制作用,其余金属离子均无显著性差异。结论 Q8008在中性pH下抑制活性稳定;在50℃以下,受温度变化影响较小;金属离子对其抑制活性测定有一定影响。     

重组巴斯德毕赤酵母产米根霉α-淀粉酶发酵条件的研究

为了提高重组菌的淀粉酶表达量,以可分泌表达米根霉α-淀粉酶的甲醇快速利用型巴斯德毕赤酵母重组菌为基础,采用摇瓶发酵方式对影响重组菌表达淀粉酶的多个因素进行了研究和优化。摇瓶发酵条件确定为:温度为30℃,pH值为6.0,接种量为2.0(OD_(600)),甲醇补加方式采用前72 h发酵时间内每隔12h添加至终浓度为1.0%,72 h以后每隔24 h添加至终浓度为1.0%,在此条件下获得的淀粉酶最高表达量为47.5 U/mL,且在无机盐培养基中和有机氮源培养基中获得的淀粉酶发酵单位相当。以摇瓶发酵数据为基础确定15 L发酵罐放大实验条件为:无机盐培养基,温度为30℃,pH值为6.0,接种量为10%,甲醇流加方式采用DO—Start法控制,在此发酵条件下获得的淀粉酶表达量为440 U/mL,约为摇瓶发酵方式获得的淀粉酶表达量的9倍。     

重组巴斯德毕赤酵母高密度发酵表达植酸酶

对巴斯德毕赤酵母的高密度发酵条件进行了试验,并根据摇瓶发酵的优化结果进行了补料方式的研究。在摇瓶发酵时,最佳种龄为16h,接种量为3％,甲醇的诱导浓度为15g／L,生长阶段最适pH为5．0,诱导阶段最适pH为5．5。在间歇补料、恒速补料、变速补料三种补料方式中以变速流加最优。     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium     

类球红细菌添加前体物产辅酶Q_(10)的工艺优化

目的:考察摇瓶与发酵罐水平下类球红细菌CU20-9产辅酶Q10添加前体物质的最优配比。方法:通过正交试验优化发酵前体配比,考察摇瓶发酵多种前体物质的添加对辅酶Q10产量的影响,并利用发酵罐进行中试规模实验。结果:优化后发酵前体配比为维生素B1 10 mg/L、乙酸钠100 mg/L、对羟基苯甲酸20 mg/L、花生油0.01%。结论:前体添加及优化配比后,摇瓶及中试发酵水平下辅酶Q10的产量均有显著提高。     

Methanogenic population dynamics during semi‐continuous biogas fermentation and acidification by overloading

Aims: This study was to investigate the methanogenic community in a biogas reactor from start‐up to acidification conditions. Furthermore, reliability and accuracy of the applied quantitative real‐time PCR method (Q‐PCR) was briefly evaluated. Methods and Results: A mesophilic (37°C), maize silage fed, continuously stirred tank reactor was surveyed. It was operated semi‐continuously with increasing daily organic loading rates (OLRs) to reach acidification. Gas production and organic acid composition were measured. Methanogenic community structure was determined by 16S rDNA‐based Q‐PCR to estimate the abundance of key methanogenic micro‐organisms. 16S rDNA of hydrogenotrophic Methanobacteriales was most abundant at OLRs of ≥3·7 g dry organic matter (DOM) l^?1 day^?1. By contrast, that of aceticlastic Methanosaetaceae predominated at lower OLRs but disappeared at OLRs of ≥4·1 g DOM l^?1 day^?1. At the same OLR, the propionate concentration increased dramatically indicating the acidification of the digester. Application of internal standards to examine Q‐PCR’s accuracy revealed that the detected amount of 16S rDNA may vary within one log cycle. Conclusions: These results suggest that the absence of Methanosaetaceae might be taken as biological indicator for process’ instability. Inhibitory effects on Q‐PCR analyses could not be determined based on the spiking experiments. Significance and Impact of the Study: In this study, reactors’ microbiology was observed over time using Q‐PCR. Insights into the abundance of different methanogens might be used to improve the performance of biogas reactors.     

Purified silk fibroin‐L‐asparaginase bioconjugates show increased thermostability and resistance to trypsin digestion

Bioconjugation protocols have been recently used to improve the therapeutic properties of the anti‐leukemic enzyme L ‐asparaginase. In this study, we study the variation of reaction factors, such as mass ratio, pH value, reaction temperature and time, and the concentration of cross‐linker, in the modification reaction of L ‐asparaginase with silk fibroins. The optimum reaction parameters were obtained as follows: pH 7.0, mass ratio of silk fibroin to L ‐asparaginase 5:1, reaction time 8 h, and temperature 4°C. The rate of ε‐amino group modification was 57.88% and the recovery of modified L ‐asparaginase was 66.58% under these reaction conditions. We isolated and purified the silk‐L ‐asparaginase conjugates with two consecutive chromatography steps: anion‐exchange (XK 16×20, Q Sepharose FF) and gel filtration (Tricorn 10×600, Sephcryl S‐300 HR) chromatography. Finally, the stabilities of the enzymes were investigated. The results showed that modified L ‐asparaginase had a higher thermostability and higher resistance to trypsin digestion.     

Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of α‐synuclein

The discoveries of mutations in SNCA were seminal findings that resulted in the knowledge that α‐synuclein (αS) is the major component of Parkinson's disease‐associated Lewy bodies. Since the pathologic roles of these protein inclusions and SNCA mutations are not completely established, we characterized the aggregation properties of the recently identified SNCA mutations, H50Q and G51D, to provide novel insights. The properties of recombinant H50Q, G51D, and wild‐type αS to polymerize and aggregate into amyloid were studied using (trans,trans)‐1‐bromo‐2,5‐bis‐(4‐hydroxy)styrylbenzene fluorometry, sedimentation analyses, electron microscopy, and atomic force microscopy. These studies showed that the H50Q mutation increases the rate of αS aggregation, whereas the G51D mutation has the opposite effect. However, H50Q and G51D αS could still be similarly induced to form intracellular aggregates from the exposure to exogenous amyloidogenic seeds under conditions that promote their cellular entry. Both mutant αS proteins, but especially G51D, promoted cellular toxicity under cellular stress conditions. These findings reveal that the novel pathogenic SNCA mutations, H50Q and G51D, have divergent effects on aggregation properties relative to the wild‐type protein, with G51D αS demonstrating reduced aggregation despite presenting with earlier disease onset, suggesting that these mutants promote different mechanisms of αS pathogenesis.

     

First Syntheses of Melophlins P,Q, and R,and Effects of Melophlins on the Growth of Microorganisms and Tumor Cells

The marine tetramic acid (=1,5‐dihydro‐4‐hydroxy‐2H‐pyrrol‐2‐ones) derivatives melophlin P, Q, and R ( 1p – 1r , resp.) were synthesized for the first time in only four steps. Together with the congenerous melophlins A–C and G, they were also tested for antimicrobial and cytotoxic effects. Melophlins B, C, P, Q, and R, which share a 5‐Me residue, showed some antibacterial activity, mainly in Gram‐positive bacteria. Melophlins B, C, and R, which have Me‐branched 3‐acyl side chains in common, inhibited the growth of cells of human KB‐3‐1 cervix carcinoma, A‐498 kidney carcinoma, and U‐937 leukemia with IC₅₀ values <10 μM . They were similar in activity to cisplatin. Melophlin Q, also Me‐branched, was astoundingly specific in inhibiting A‐498 kidney cancer cells, while melophlin P inhibited U‐937 leukemia cells particularly well. The position of the Me branch is decisive for the magnitude of the antiproliferative effect of the melophlin couples B/C and R/Q.     

快速提取类球红细菌中辅酶Q10的方法研究

目的：建立一种从类球红细菌中快速分离纯化辅酶Q10的方法。方法：对影响超声提取辅酶Q10的各因素,包括提取试剂、超声频率、循环次数及工作时间的最佳条件进行正交试验,比较超声破碎法与碱醇皂化法提取辅酶Q10的差异。结果：在超声提取中,提取试剂和循环次数对辅酶Q10提取效果具有显著性影响;在超声频率0.5s、丙酮提取3min、循环3次的条件下提取的辅酶Q10的含量比碱醇皂化法提高了近6倍。结论：超声破碎法是一种简单、迅速、高效的提取辅酶Q10方法。