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%。     

拉曼被孢霉F5发酵生产γ-亚麻酸的研究

对拉曼被孢霉突变株F5发酵生产γ-亚麻酸的最适碳源、氮源、发酵时间及温度、无机盐离子添加、最适碳源浓度及补加碳源时间等发酵条件进行了研究探讨.最适发酵培养基组成为(g/L):葡萄糖100,酵母浸出粉4,蛋白胨1,K2HPO4 1,CaCl2 1×10-2,MgSO45×10-2,FeSO4 1×10-2,ZnSO4 7.5×10-3,CuSO4 0.5 × 10-5,MnSO4 2×10-3,pH 6.0.培养温度为25℃,140r/min振荡培养10天,培养8天后(即收获前2天)补加5%葡萄糖.发酵结果为:DC24.59g/L,TL 10.84g/L,TL/DC44.09%,GLA/TL10.67%,GLA产量为1156.63 mg/L. GLA产量较初始结果提高156.15%.该菌株已达到工业化生产菌株要求.     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

面包酵母海藻糖积累条件的研究

对面包酵母的海藻糖积累条件及补料分批发酵进行了研究。结果表明 pH为 5 .0 ,温度 37℃有利于海藻糖积累。少量多次的补加葡萄糖可持续增加海藻糖的含量 ,但随着发酵时间的延长 ,葡萄糖对海藻糖转化率有逐渐减少的趋势。     

拉曼被孢霉F_5发酵生产γ—亚麻酸的研究

对拉曼被孢霉突变株F5发酵生产γ—亚麻酸的最适碳源、氮源、发酵时间及温度、无机盐离子添加、最适碳源浓度及补加碳源时间等发酵条件进行了研究探讨。最适发酵培养基组成为 (g/L) :葡萄糖 1 0 0 ,酵母浸出粉 4 ,蛋白胨 1 ,K2 HPO4 1 ,CaCl2 1× 1 0 - 2 ,MgSO4 5× 1 0 - 2 ,FeSO4 1× 1 0 - 2 ,ZnSO4 7.5× 1 0 - 3,CuSO4 0 .5× 1 0 - 3,MnSO4 2× 1 0 - 3,pH 6.0。培养温度为 2 5℃ ,1 4 0r/min振荡培养 1 0天 ,培养 8天后 (即收获前 2天 )补加 5 %葡萄糖。发酵结果为 :DC 2 4 .5 9g/L ,TL 1 0 .84g/L ,TL/DC 4 4.0 9% ,GLA/TL 1 0 .67% ,GLA产量为 1 1 5 6.63mg/L。GLA产量较初始结果提高 1 5 6.1 5 %。该菌株已达到工业化生产菌株要求     

D-核糖发酵过程中底物抑制及补料的研究

研究了初始葡萄糖浓度对D -核糖发酵的影响 ,证实了较高的葡萄糖浓度对D -核糖发酵的抑制作用 ,并确定了较为适宜的初始葡萄糖浓度为 1 0 0g·L-1 或 1 5 0g·L-1 。前者条件下D -核糖的转化率和生产强度均达最大 ,分别为 32 8g·kg-1 和0 .6 8g·L-1 ·h-1 ;后者条件下D -核糖的产量达最大值 39.4 8g·L-1 。针对底物抑制现象 ,研究了补料工艺对D -核糖发酵的影响 ,确定发酵 2 4h后补加 5 0g·L-1 的葡萄糖为较优的补料工艺 ,在此工艺条件下最终D -核糖产量相对于对照组提高了 4 8.3%。     

戊糖和己糖共发酵生产燃料乙醇条件研究

利用嗜鞣管囊酵母P-01对木糖和葡萄糖共发酵生产燃料乙醇的条件进行了试验研究,结果表明,木糖和葡萄糖混合液生产燃料乙醇的最佳条件为发酵液的pH值5.5、30℃、摇床转速120 r/min、接种量10%、发酵液初始糖浓度6%、葡萄糖与木糖之比为2、发酵周期为84h。在最佳发酵条件下,发酵醪液中的燃料乙醇浓度为2.101%,糖醇转化率为35%。     

补料发酵法制备β-葡萄糖苷酶的研究

研究了黑曲霉制备β-葡萄糖苷酶液态发酵过程中添加麸皮和纤维二糖的补料工艺.补料量及补料次数影响着β-葡萄糖苷酶的分泌.经过四次补加1%菌麸皮干粉,发酵液中β-葡萄糖苷酶活力由未补料的1.72 U/mL增加到5.07 U/mL,提高了近3倍.而且,添加麸皮浸提液能达到同样效果.采用分批补料工艺产酶时,初始浓度为3%的麸皮用量比较适宜,其蛋白含量和β-葡萄糖苷酶的活力均为最高,分别达到0.69nag,/mL和7.00 U/mL.在补料总量和补料次数相同的情况下,以递减方式补料效果最好,产酶达7.34 U/mL.补加纤维二糖对β-葡萄糖苷酶的分泌具有一定的诱导作用,在接种培养48h时补加0.2%纤维二糖比较适宜.研究表明,分批补料发酵有利于黑曲霉分泌β-葡萄糖苷酶,是一种很好的提高酶产量的方法.     

重组大肠杆菌的高密度发酵和甘油生产条件的初步研究

在摇瓶中进行重组大肠杆菌菌株BL21高密度发酵条件的研究,考察了葡萄糖浓度、盐离子浓度、温度、接种量、发酵时间等对该菌株生产甘油的影响。初步确定底物浓度为2.5%,盐离子浓度0.2%,温度为37℃,接种量为2%,经24h的摇瓶发酵,甘油产量最高达6.8g/L。在30L发酵罐实验中、按初步确定的优化条件发酵26h,甘油产量可达46.67g/L,是LB/葡萄糖培养基中甘油产量的2.06倍。     

林肯链霉菌合成林可霉素代谢调节的研究

在摇瓶条件下研究了葡萄糖、铵盐、磷酸盐对林可霉素产生菌林肯链霉菌的生长及林可霉素生物合成的影响。发酵过程中林可霉素的合成主要发生在菌体生长期,逐渐下降。使用6%的葡萄糖未发现通常所说的“葡萄糖效应”。0.2%铵盐有利于细胞生长,但0.8%NH+4对林可霉素的生物合成具有抑制作用。发酵48h后补加0.6% NH^＋_４,对林可霉素的生成没有显著影响。0.05%～0.1%磷酸盐对林可霉素合成具有较强的抑制作用。并就磷酸盐对菌体由初级代谢转向次级代谢的作用作了初步考察。     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Principles of organization and evolution of systems of regulation of functions

Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms. The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization followed the way of development of delivery pathways of chemical signal and development of mechanisms of its regulation. The mechanism of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning of physiological systems and organs of the living organism