融合表达HLH结构域对猪溶菌酶抗菌活性的影响

[目的]通过遗传操作提高猪溶菌酶抗革兰氏阴性菌的活性。[方法]对猪溶菌酶进行分子模拟,得到了包含功能肽的螺旋-回环-螺旋(HLH)结构域,将HLH编码基因与猪溶菌酶基因N端或C端进行融合,于大肠杆菌中诱导表达,经复性、纯化后检测其抗菌活性,并利用原子力显微镜和荧光染色对抗菌活性较高的融合蛋白杀菌机制进行初探。[结果]与对照组相比,N端和C端融合蛋白基本保持了猪溶菌酶对革兰氏阳性菌的活性;同时,两种融合蛋白对革兰氏阴性菌的抗菌活性均显著增强,其中N端融合产物活性更高,它对大肠杆菌ATCC 10798、大肠杆菌ATCC 25922、克雷伯氏菌TR5、铜绿假单胞菌ATCC 15442、沙门氏菌CMCC(B)50335的抗菌系数分别为1.64、1.24、2.56、1.72和1.42,最低抑菌浓度分别为90μg/mL、100μg/mL、40μg/mL、80μg/mL和100μg/mL。经检测,该融合蛋白能显著增强革兰氏阴性菌细胞膜的通透性。[结论]通过融合表达自身来源的HLH结构域,猪溶菌酶抗革兰氏阴性菌活性得到了显著提升,可为其它溶菌酶抗菌活性的提高提供参考。     

动物源溶菌酶研究进展

动物源溶菌酶是一种动物体内广泛存在的酶类,它可以水解细菌细胞壁肽聚糖中的β-1,4糖苷键,具有消化分解细菌、抑制外源微生物生长、增强机体免疫力的作用.目前溶菌酶已被用作研究蛋白功能、性质以及分子进化的模型.首先介绍了溶菌酶及其分子的晶体结构,溶菌酶基因及其蛋白研究进展,其次介绍了动物源溶菌酶的功能,包括溶菌酶生物学功能和重组蛋白功能活性,重点介绍了溶菌酶基因在转基因工程中的应用研究,最后对动物源溶菌酶研究进行了展望.研究动物源溶菌酶对于基础科学,并应用其转变成现实生产力具有重要的指导意义.     

溶菌酶的研究进展

溶菌酶普遍存在于动物、植物和微生物中。溶菌酶是一种小分子碱性蛋白,长期以来一直被作为一种“模型”体系．用于研究蛋白质的空间构象、酶动力学及其与分子进化、分子免疫间的关系。介绍了溶菌酶的来源、结构、性质、作用机制。并对近年来其在食品工业、医学和酶工程中的应用进行了综述;分析了溶菌酶应用中存在的主要问题,并对其应用前景进行了展望。     

眼镜蛇属神经毒素研究概况

正世界范围内眼镜蛇属约28种[1]。我国有舟山眼镜蛇(naja atra)和孟加拉眼镜蛇(naja kaouthia)两种,前者过去又称为中华眼镜蛇[2]。眼镜蛇是腺管牙类毒蛇,其毒腺分泌的毒液是以神经毒为主的混合毒,即神经毒和血循毒。眼镜蛇毒神经毒素(cobra neurotoxin,CNT)是其毒液中毒性最大的成分,它能使动物产生呼吸抑制和骨骼肌麻痹,甚至死亡[3]。随着分子生物学和蛋白质组学的发展,眼镜蛇毒神经毒素的一些组分已得到分离、纯化、测定,并广泛应用于科研和临床。本文就眼镜蛇毒组分中神经毒素的研究与应用进     

溶菌酶的研究进展

溶菌酶在自然界中广泛存在,是一种与单核-巨噬细胞系统有关的非特异性防御机制。人溶菌酶在临床上具有潜在的使用价值。由于来源有限,利用基因工程技术从细菌或酶母中生产人溶菌酶实现产业化,是解决其供需矛盾的有效途径。有关溶菌酶抗菌功能以外的其它未知生物学作用,也是当前研究的热点。     

10种动物指数参考值的测定与分析

所谓指数 ,是指动物各器官或各系统占其体重的百分数 [8] ,人们为什么要研究它呢 ,意义何在 ?原来 ,在人工养殖效果评估方面 ,在确定屠宰或捕捞的大小与时间的适宜度方面 ,在生理、病理分析时 ,在治疗时间和用药量等诸方面 ,都要涉及此类问题 ,正因为有研究的必要 ,因此国内外学者 ,在人 [6]、蚱蜢 [1 2 ]、海胆 [1 3]、鲫鱼 [9]、刺猬 [1 0 ] 、黄鼬[2 ] 、东北虎[7、8] 、蓝鲸[5] 和家猪[3] ……。都有过各项指数方面的报告 ,为丰富这一领域的内容 ,我们也测定了另外 1 0种动物的指数。1　材料和方法1 .1　种类　 1 0种动物 ,6种是无脊椎…     

氧化亚铁硫杆菌的铁和硫氧化系统及其分子遗传学

氧化亚铁硫杆菌(Thiobacillus ferrooxidans)是典型的化能自养菌,由于能生长在亚铁,元素硫和硫化物矿物上,因而成为生物湿法治金(Biohydrometallurgy)中最有应用价值的一个种。目前南非,美国,加拿大等国应用它提取铜,铀,金等已获得工业生产,另外它在煤和原油的脱硫方面也展现了广阔的应用前景[1]。多年来,我们对氧化亚铁硫杆菌在冶金和煤脱硫方面的应用研究也取得一定进展,如铜,铀,锰,金等金属提取,有的已进行了扩大试验或半工业试验[2,3]。     

U23细菌溶解酶性质的研究

我们对U23细菌溶解酶[1]溶解细菌细胞壁的产物进行了测定和分析,结果证明这是一种属于糖苷酶类型的细菌溶解酶。该酶不溶解几丁质。分子量27,000,等电点pH11.3。酶的结晶具有类似于蛋清溶菌酶结晶的多形性质,因结晶条件不同,获得三种不同形状的酶结晶。     

用改进的热休克法制备大肠杆菌碱性磷酸单酯酶

碱性磷酸单酯酶(AKpase)[E.C.3.1.3.1]是专切磷酸单酯键的最常用工具酶之一,它广泛地存在于动植物组织以及微生物中。目前,从大肠杆菌中提取AKpase有四种方法:(1)热休克法;(2)渗透休克法;(3)溶菌酶-     

人溶菌酶工程菌株培养条件的研究

人溶菌酶在食品工业和医药上具有广泛的用途,最近发现它在癌症的继承性免疫治疗上也有作用。为使人溶菌酶达到工业化生产,我们已成功地人工合成厂人溶菌酶基因,并构建了人溶菌酶基因的重组质粒和工程菌株。为提高该工程菌人溶菌酶的表达水平,我们对影响该工程菌表达人溶菌酶的培养条件进行探讨。     

Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.     

The role of dissolved oxygen and function of agitation in hyaluronic acid fermentation

The role of dissolved oxygen (DO) and function of agitation in hyaluronic acid fermentation by Streptococcus zooepidemicus ATCC 39920 were explored. With controlled DO levels, it was found that the present strain grew as well under both aerobic and anaerobic conditions. Dissolved oxygen plays a role as a stimulant in the HA synthesis; the intrinsic factor affecting the efficiency of HA synthesis is DO level; and there existed a critical DO level of 5% air saturation for the HA synthesis. On the other hand, agitation functions to mix the broth, to enhance oxygen absorption, but not to release HA capsule. In addition, vigorous agitation would lengthen the operation time. It therefore suggests that the relevant criteria for scaling up the fermentor are to maintain DO level above the critical value, and to provide a mild agitation for homogeneity in the fermentor.     

Increase of xylitol production rate by controlling redox potential in Candida parapsilosis

The effect of redox potential on xylitol production by Candida parapsilosis was investigated. The redox potential was found to be useful for monitoring the dissolved oxygen (DO) level in culture media, especially when the DO level was low. An increase in the agitation speed in a 5 L fermentor resulted in an increased culture redox potential as well as enhanced cell growth. Production of xylitol was maximized at a redox potential of 100 mV. As the initial cell concentration increased from 8 g/L to 30 g/L, the volumetric productivity of xylitol increased from 1.38 g/L. h to 4.62 g/L. h. A two-stage xylitol production strategy was devised, with stage 1 involving rapid production of cells under well-aerated conditions, and stage 2 involving cultivation with reduced aeration such that the culture redox potential was 100 mV. Using this technique, a final xylitol concentration of 180 g/L was obtained from a culture medium totally containing 254.5 g/L xylose in a 3,000 L pilot scale fermentor after 77 h fermentation. The volumetric productivity of xylitol during the fermentation was 2.34 g/L. h.     

Effect of aeration on antibiotic production byStreptomyces clavuligerus

Summary During the rapid growth phase ofStreptomyces clavuligerus in a 10 litre fermentor, the level of dissolved oxygen (DO) was found to drop to almost zero for a period of approximately 10 h, delaying the appearance of and lowering the production of the antibiotic cephamycin C. Controlling the DO at either 50% or 100% throughout the fermentation did not significantly alter the specific growth rate of the culture, but did elevate final antibiotic levels two- and three-fold respectively. The improved oxygen availability affected antibiotic production both by increasing the rate of specific cephamycin C bisosynthesis and by maintaining this higher rate throughout the production period. These results demonstrate that controlling dissolved oxygen levels close to saturation during periods of rapid growth markedly improves the efficiency and duration of cephamycin C biosynthesis inS. clavuligerus.     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

Development of a new commercial-scale airlift fermentor for rapid growth of yeast

To grow yeast rapidly, it is necessary to supply sufficient oxygen to the yeast and to effectively remove the heat of the fermentation. We succeeded in developing a commercial-scale fermentor for growing a food yeast (Candida utilis) to produce RNA rapidly. This fermentor is an internal-loop airlift type with vertical heat transfer tubes between inner and the outer columns. The volume of the fermentor is 145 m³ (working volume 75 m³). The oxygen transfer rate (OTR) was 9.9 kg-O₂/m³/h using a superficial gas velocity of 30 cm/s based on the outer column. Much of the heat of fermentation and the energy resulting from aeration could be removed effectively by the heat transfer tubes. This unique airlift fermentor was driven at a dilution rate of 0.43 h⁻¹ for about 70 d, with the yeast concentration being maintained at 22.8 kg-dry cell/m³. The yeast production rate was 9.79 kg-dry cell/m³/h. Compared with a traditional stirred-type fermentor, two Vogelbush-type fermentors and another airlift fermentor, our fermentor was far superior with respect to OTR and yeast productivity.     

供氧对产丙三醇假丝酵母科丙三醇发酵研究

研究了产丙三醇假丝酵母（Ｃａｎｄｉｄａ ｇｌｙｃｅｒｏｌｇｅｎｅｓｉｓ）产丙三醇及副产物与氧供给的关系。摇瓶试验发现其它营养条件一定,玉米浆添加量决定酵母量。在０．４％的玉米浆和装液比０．０８时产丙醇最高,副产物乙醇、乙酸和乙酸乙酯最小,玉米浆和装液比影响丙三醇和副产物的形成。在５Ｌ的反应器中以搅拌转速控制供氧水平,菌体生长阶段比耗氧速率为２８ｍｇ／（ｇ．ｈ）,在发酵阶段比耗氧速率１６ｍｇ／（ｇ．     

Effect of oxygen transfer on lipase production by Acinetobacter radioresistens

The influence of oxygen on alkaline lipase production by Acinetobacter radioresistens was studied under two operating modes: controlled dissolved oxygen (DO) concentration and controlled aeration rate. Compared with cell growth, the lipase production depended more extensively on oxygen. The intrinsic factor determining cell growth and lipase production was oxygen transfer rate (OTR) rather than DO concentration. Improvements in OTR, either by aeration or agitation, resulted in an increase in lipase yield and/or a reduction in fermentation time. The formation of A. radioresistens lipase could be described by a mixed-growth-associated model, and the enzyme was mainly a growth-associated product. The overall productivity for the lipase, which depended more strongly on agitation than aeration, could be related with kLa. DO concentration could not be employed in this correlation, though it has been useful as a criterion for ensuring no oxygen limitation in an aerobic fermentation.     

重组毕赤酵母发酵牛肉风味肽的中试研究

研究分泌表达16拷贝牛肉风味肽（beefy meaty peptide, BMP）毕赤酵母工程菌株(Pichia pastoris GS115-16B2)的500L发酵罐中试发酵工艺。在500L发酵罐中采用三步发酵法进行了3批次中试发酵实验,设定菌体培养阶段温度为30℃,pH5.0,诱导表达阶段温度为28℃,pH3.0。在发酵程中通过调节搅拌速度、通气量和罐压等措施来使DO维持在20%~35%左右,总共发酵126h（诱导时间为96h）,当诱导88h后（发酵118h）,600nm波长下的光密度值(OD600)达到184.5,菌体湿重达到318.7g/L,目的产物BMP的表达量达到最高为172 mg/L,实现了BMP的高效表达。通过中试发酵初步建立了工程菌P. pastoris GS115-16B2的中试发酵工艺,为BMP的进一步研究开发奠定了良好基础。     

Improved poly-ε-lysine biosynthesis using Streptomyces noursei NRRL 5126 by controlling dissolved oxygen during fermentation

The growth kinetics of Streptomyces noursei NRRL 5126 was investigated under different aeration and agitation combinations in a 5.0 l stirred tank fermenter. Poly-epsilon-lysine biosynthesis, cell mass formation, and glycerol utilization rates were affected markedly by both aeration and agitation. An agitation speed of 300 rpm and aeration rate at 2.0 vvm supported better yields of 1,622.81 mg/l with highest specific productivity of 15 mg/l.h. Fermentation kinetics performed under different aeration and agitation conditions showed poly- epsilon-lysine fermentation to be a growth-associated production. A constant DO at 40% in the growth phase and 20% in the production phase increased the poly-epsilon-lysine yield as well as cell mass to their maximum values of 1,992.35 mg/l and 20.73 g/l, respectively. The oxygen transfer rate (OTR), oxygen utilization rate (OUR), and specific oxygen uptake rates (qO2) in the fermentation broth increased in the growth phase and remained unchanged in the stationary phase.