基于途径分析促进α-酮戊二酸过量积累的数学模型技术

在详尽分析α-酮戊二酸(α-KG)合成途径的基础上,结合数学模型,对光滑球拟酵母(Torulopsis glabrata)CCTCC M202019过量合成α-KG的合成途径及其调控因素进行全面分析与优化.全因子实验表明,硫胺素(B1)和CaCO3是影响α-KG过量积累的关键因素.在此基础上,采用最速上升实验得到α-酮戊二酸积累的最大响应区域为B10.016 mg/L、CaCO3 84 g/L附近.采用中心组合设计及响应面分析确定最优培养基组分为:硫胺素(B1)0.02 mg/L、生物素(Bio)0.05 mg/L、CaCO3 82 g/L和乙酸钠4 g/L.在最优培养基中,α-KG产量达到26.8 g/L,提高了34%.α-酮戊二酸发酵过程动力学分析表明,采用最优培养基使发酵延滞期缩短6 h,菌体比生长速率和α-KG比产物生成速率分别提高了45.4%和8.64%.图2表3参12     

一株高产灵菌红素粘质沙雷氏菌的分离鉴定及发酵条件优化

天然色素化合物灵菌红素具有抑制细菌、真菌、疟疾、藻类和肿瘤细胞等多种生物学活性;微生物发酵是生产灵菌红素的主要方法.从自然界植物根际土壤中分离得到一株产红色色素的菌株JWR.经形态和16S rDNA测序鉴定,菌株JWR为粘质沙雷氏菌(Serratia marcescens);用酸性甲醇溶液对发酵液萃取得到灵菌红素的粗提物,利用FT-IR和HPLC鉴定产物,证明该菌株所产红色色素为灵菌红素.通过单因素以及正交优化法对发酵培养基组分和发酵条件进行优化,确定发酵培养基配方为橄榄油1.5 mL/L、牛肉膏0.5 g/L、硫酸镁0.25 g/L,发酵条件为接种量2.5%(V/V)、温度28℃、转速220 r/min.优化后灵菌红素产量提高到6.011 g/L,相比优化前提高了2.845倍.本研究分离粘质沙雷氏菌并优化发酵条件,提高了灵菌红素产量,可为其工业化生产奠定基础.     

Enterobacter cloacae B5产转糖基β-半乳糖苷酶发酵条件优化

β-半乳糖苷酶是一类非常重要的糖苷水解酶,已被广泛应用于食品工业降低乳制品中的乳糖含量.某些种类的β-半乳糖苷酶还具有转糖基活性,近年来被应用于合成低聚半乳糖和半乳糖苷化合物.通过单因子试验和正交试验,对肠杆菌Enterobacter cloacae B5产生转糖基β-半乳糖苷酶的培养基组成及发酵条件进行了优化.结果表明,以无机盐溶液Ⅰ(CaCl2 0.011%、MnSO4 0.0001%、MgSO4·7H2O 0.03%、KH2PO4 0.005%、FeSO4·7H2O 0.003%)、乳糖1.5%、酵母粉2%、蛋白胨0.5%、起始pH 8.5的培养基在25℃培养E.cloacae B5菌株38 h,产酶量达到最高值4.663U mL-1,大约是未优化时的3倍.通过优化产酶条件提高了全细胞酶源的酶活量,不仅为功能性低聚半乳糖的生产降低了成本,同时也为商业化β-半乳糖苷酶的大量提纯降低了成本.图2表4参12     

响应面法优化锰氧化细菌发酵培养基

Bacillus sp.WH4是一株锰氧化能力强的锰氧化细菌,在锰污染的水源净化方面具有较好的应用前景.为提高发酵液中Bacillus sp.WH4的活菌数,应用响应面法优化其发酵培养基.Plackett-Burman设计获得对活菌数有显著影响的4个因素:豆粕粉、KH2PO4、MgSO4、FeSO4;最陡爬坡试验确定中心组合实验的最大响应区域;中心组合设计和响应面分析优化出4个显著因素的最佳浓度.优化后的最适培养基(w/%)组成为:蔗糖2、豆粕粉0.96、K2HPO4 0.8、KH2PO4 0.19、MgSO4.7H2O 0.11、CaCl2 0.02、NaCl 0.5、FeSO4.7H2O 0.02.发酵试验表明,最优培养基的活菌数可达4.2×109 CFU mL-1,比初始发酵培养基提高近3倍.     

黑曲霉素J4对中低品位磷矿粉的溶磷效果及重金属释放的影响

磷矿是不可再生的资源,如何科学利用低品位磷矿是磷肥行业急需解决的问题。通过发酵培养试验,研究了黑曲霉J4(AspergillusNigerJ4)在不同培养基发酵中对磷矿粉的溶磷效果以及磷矿粉伴生性重金属元素释放的影响。结果表明,(1)各磷矿粉在不同培养基发酵中的溶磷量为6.50—17.23 g·kg~(-1),溶磷率为26.11%—68.31%,黑曲霉J4在培养基发酵中对磷矿粉的溶磷量和溶磷率大小为C培养基A培养基B培养基。(2)黑曲霉J4在3种培养基发酵磷矿粉中伴生性Pb和As溶出量和溶出率都表现为C培养基A培养基B培养基,而Cd溶出量表现为C培养基B培养基A培养基,平均Cd溶出率表现为C培养基A培养基B培养基。(3)黑曲霉J4在对不同培养基发酵中磷矿粉的溶磷率与伴生重金属的关系因培养基和重金属种类而异。黑曲霉J4对磷矿粉的溶磷率与各磷矿粉的溶磷量呈极显著正相关,在A培养基中,黑曲霉J4对磷矿粉的溶磷率与磷矿粉中伴生性Pb和As溶出率分别呈极显著和显著正相关,在C培养基中,黑曲霉J4对磷矿粉的溶磷率与伴生性As溶出率呈显著性负相关。由此可见黑曲霉J4在C培养发酵对磷矿粉有较好的溶磷作用,其伴生性重金属的溶出率也最低,可以作为高效利用中低品位磷矿粉开发生物磷肥的新途径。     

抗真菌多肽捷安肽素发酵条件的研究

对筛选出的一株芽孢杆菌ZK发酵产物抗真菌多肽捷安肽素的发酵培养基组分(碳源、氮源及无机盐)和工艺条件(发酵温度、起始pH,摇床转速,装液量)进行了摸索,通过单因素实验和正交优化实验,确定了ZK菌株发酵培养基最佳组成:生物氮素3.5％、葡萄糖3％、酵母膏0.08％、MgSO4·7H2O 0.5％、KH2PO4·3H2O 0.1％;最适温度为32℃;最适初始pH为8.0.在250 mL三角瓶中装50 mL培养基,于150 r min-1的旋转摇床上32℃振荡培养72 h,ZK菌株产捷安肽素的量达到最大.图8表5参14     

产氢新种Ethanoligenens harbinese B49的氮素营养与代谢特征

从氮源角度研究了一株产氢新种Ethanoligenens harbineseB49发酵葡萄糖和糖蜜的产氢特性及其营养需求,以及酵母粉对产氢菌E.harbinese B49生长和产氢的特殊效应.试验结果表明,在以葡萄糖为底物条件下,以酵母粉替代蛋白胨作为唯一氮源可以大大提高E.harbineseB49的产氢能力,单位体积产氢量从1700mL L-1培养基提高到2400mL L-1培养基,产氢能力提高40.35%.该条件下去除维生素液,E.harbinese B49的产氢能力不受影响.从E.harbinese B49产氢动力学分析可以看出,对数生长期处于12~22h期间.从对数期开始迅速产氢,并且一直持续到稳定前期,产氢速率达到16.8mL/h,生物气中最大氢含量为41%.驯化后的E.harbinese B49利用糖蜜为底物产氢,糖蜜COD为13g L-1时,单位体积最大产氢能力为1576mL L-1.当培养基中额外加入0.5g L-1酵母粉时,可以大大促进E.harbinese B49发酵糖蜜产氢的能力,单位体积氢产量达到1960mL L-1,提高了24.4%,比产氢率达到150.8mL(H2)/g(COD).以牛肉膏、蛋白胨、尿素为氮源时,E.harbineseB49产氢量提高较小.图4表2参16     

Streptomyces sp. WJS-825产谷氨酰胺转胺酶发酵条件优化及中试

应用五因子二次正交旋转回归试验设计,建立了微生物谷氨酰胺转胺酶 (TG)发酵生产过程中以酶活力和菌体细胞生长量作为目标函数的数学模型,并以此模型对链霉菌 (Streptomycessp. )WJS-825菌株发酵生产TG的培养条件进行优化,确定了影响TG生产的主要因子及其最适取值为多价胨 2. 1%、可溶性淀粉 1. 5%、初始pH 7. 0及培养温度 30℃.以该优化工艺条件进行了 5L发酵罐小试和 200L发酵规模的中试生产.结果表明,在中试发酵生产中使用以豆饼粉部分替代多价胨的经济性发酵优化培养基,以及发酵过程中在线监控pH、溶氧系数等多项发酵调控参数,并分段控制pH、温度、通气量和搅拌转速以及进行适时的流加补充碳源,该菌株生长繁殖能力强、产酶效果好,TG活性达 3. 2u/mL,而且连续重复的中试发酵生产试验的TG产量均稳定在 3. 2u/mL以上. 图 2表 3参 15     

拟威克酵母产生表面活性剂的发酵条件

通过单因子试验和正交试验,对拟威克酵母WickerhamielladomercqiaeY2A产生槐糖脂的发酵培养基组成和发酵条件进行了优化.培养基组成为:8.0%葡萄糖、8.0%菜子油、0.3%酵母粉;发酵条件为:初始pH值6.0、接种量2.5%、发酵时间120h.在此条件下,槐糖脂产量可达到41.3gL-1.图4表5参16     

一株烟草内生拮抗放线菌发酵条件优化

为提高烟草内生放线菌Y12发酵时产生抑菌物质的产量,通过摇瓶正交优化试验,确定了Y12的三角瓶发酵条件.发酵培养基组成为:黄豆粉1.5%,蛋白胨1.5%,葡萄糖1.5%,可溶性淀粉1.5%,MgSO4.7H2O 0.05%,(NH4)SO40.25%,NaCl 0.4%,KH2PO4 0.1%,CaCO3 0.5%;培养基初始pH 7.5,培养温度30℃,发酵时间72 h.经过优化发酵液的抑菌圈直径增加了44.9%.     

Resting egg formation and hatching of the S-type rotifer Brachionus plicatilis at varying salinities

Several live specimens of the blue-ringed octopus Octopus maculosus were collected from the Philippines in November 1985, and from Japan in February 1986, and the distribution of toxicity, along with toxin composition, in the posterior salivary gland and other soft parts were examined. Tetrodotoxin (TTX: 1400 mouse units g^-1) was detected in the posterior salivary gland of a Japanese specimen, while not only the salivary gland but other soft parts were toxic in the Philippine specimens. The Philippine specimens contained TTX and anhydrotetrodotoxin, the Japanese specimen TTX, 4-epitetrodotoxin, and an unknown toxin. The posterior salivary gland, intestine and other parts were excised from the Philippine specimens and examined for bacterial flora. Twenty-two dominant strains were isolated and cultured in a 2xORI medium (Ocean Research Institute, Simidu and Tsukamoto 1985) at 20°C for 20 to 48 h. Cells were harvested by centrifugation, and disrupted by ultrasonication. The toxins were partially purified from the cell lyzate by ultrafiltration and Bio-Gel P-2 column-chromatography. Instrumental analyses disclosed that 16 of the 22 strains produced TTX and/or related substances. Six strains which clearly exhibited TTX productivity were identified as Alteromonas (2 strains), Bacillus (2), Pseudomonas (1) and Vibrio (1), based on biochemical and biological characteristics. Of these, one strain each of Bacillus and Pseudomonas produced TTX at a level detectable by the mouse assay.     

Tetrodotoxin in sinking particles from coastal waters

The occurrence of tetrodotoxin (TTX) in marine sinking particles was investigated. Sinking particles were collected in 1991 using sediment traps in the coastal area of Aburatsubo Inlet, Japan. TTX and related substances were analyzed by tissue culture bioassay, high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). TTX and related substances were detected from six samples. The concentrations were estimated to be ca. 200 to 1000 ng g^-1 by the HPLC analyses. To our knowledge, this is the first report on the occurrence of TTX in particles in an aquatic environment. The present results indicate that sinking particles are one of the sources of TTX in the marine environment and that these particles play a role in the toxification of marine organisms.     

中性条件下超声波/零价铁协同降解活性艳红X-3B

考察了中性条件下超声波降解、零价铁还原及超声波-零价铁联用对活性艳红X-3B模拟废水的降解效果,通过对比降解过程中UV-Vis光谱的变化,探讨了超声波-零价铁协同处理活性艳红X-3B的可行性.结果表明,在中性条件下超声波对活性艳红X-3B降解缓慢,经过25min辐射后,活性艳红X-3B的分解率不足7.5%;零价铁直接还原速率较慢,反应25min后,活性艳红X-3B的分解率仅为48.82%;"超声波/零价铁"对降解活性艳红X-3B有明显的协同效应,25min后分解率达99.42%(600W),反应符合准一级动力学过程.与零价铁直接还原相比,在200W,400W和600W超声波的协同作用下,X-3B降解的表观反应速率常数分别提高了2.12,2.76和4.00倍,半衰期相应缩短.另外,添加H2O2会抑制协同效应.     

Occurrence of tetrodotoxin in the gastropodsRapana rapiformis andR. venosa venosa

Paralytic toxicity was detected in four of 17 specimens and six of 28 specimens of the gastropodsRapana rapiformis andR. venosa venosa, respectively, collected from Chiching (Kaohsiung City) and Nanfangao (Ilan County), Taiwan, in 1988 and 1989. The highest toxicities, calculated as tetrodotoxin (TTX) content, were 140 and 13 mouse units (MU) g^–1 digestive gland inR. rapiformis andR. venosa venosa, respectively. The toxins obtained from each species were purified by ultrafiltration and Bio-Gel P-2 column chromatography. The toxin's specific toxicities (as TTX) were 56 MU mg^–1 (R. rapiformis) and 52 MU mg^–1 (R. venosa venosa). Results of analyses by thin-layer chromatography, cellulose acetate membrane electrophoresis and high-performance liquid chromatography showed that TTX and anhydrotetrodotoxin were responsible for the toxicity. The alkali hydrolysate of each toxin showed maximum absorption at ca. 274 nm, which is the unique absorption for the C₉-base of TTX and its related substances (such as anhydrotetrodotoxin, 4-epitetrodotoxin, etc.). Here, we report for the first time the occurrence of TTX in the gastropodsR. rapiformis andR. venosa venosa.     

Predators usurp prey defenses? Toxicokinetics of tetrodotoxin in common garter snakes after consumption of rough-skinned newts

Snakes are common predators of organisms, such as amphibians, with toxic defenses that can be lethal to other predators. Because snakes do not have the option of dissecting prey into edible versus inedible components, they face a full dose of any chemical defenses encountered during attempted predation. This limitation has likely resulted in intense selection favoring the evolution of alternative mechanisms for dealing with prey toxins. These mechanisms can be physiological (e.g., resistance to prey toxins) or behavioral (e.g., toxin sampling and rejection). When physiological resistance arises, the possibility of bioaccumulation of a toxin results. We examined the coevolutionary interaction between the common garter snake (Thamnophis sirtalis) and the rough-skinned newt (Taricha granulosa), which contains a powerful neurotoxin called tetrodotoxin (TTX). In some populations syntopic with newts, individuals of T. sirtalis have evolved resistance to TTX. We examined the persistence of TTX in T. sirtalis after administration of an oral dose of TTX to investigate the possibility that snakes are sequestering TTX. The half-life of TTX in snake liver was estimated at 8.1?days. Accordingly, clearance of 99% of a single dose of TTX averages 61?days. Negative fitness consequences of intoxication during and after newt consumption may be balanced by co-opting the newts?? chemical defense for protection from the snakes?? own predators. Accounting of the coevolutionary dynamic between snakes and newts must incorporate post-consumption affects of lingering TTX.     

Tetrodotoxin affects survival probability of rough-skinned newts (Taricha granulosa) faced with TTX-resistant garter snake predators (Thamnophis sirtalis)

Lethal chemical defenses in prey species can have profound effects on interactions with predators. The presence of lethal defenses in prey can correct the selective imbalance suggested by the life-dinner principle in which the fitness consequences of an encounter between predator and prey should be much greater for the prey species than the predator. Despite the apparent adaptive advantages of lethality the evolution of deadly prey presents a fundamental dilemma. How might lethal defenses confer an individual fitness advantage if both predators and prey die during interactions? We examined the interaction between the rough-skinned newt (Taricha granulosa), which contains a powerful neurotoxin called tetrodotoxin (TTX), and the common garter snake (Thamnophis sirtalis). In some sympatric populations, Th. sirtalis have evolved physiological resistance to TTX. Whether the newts’ toxin confers protection from snake predators or has been disarmed by the snakes’ physiological resistance has not yet been directly tested. In predator–prey trials, newts that were rejected by snakes had greater concentrations of TTX in their skin (4.52 ± 3.49 mg TTX/g skin) than those that were eaten (1.72 ± 1.53 mg TTX/g skin). Despite the plethora of taxa that appear to use TTX defensively, this is the first direct and quantitative demonstration of the antipredator efficacy of TTX. Because the survival probability of a newt (and thus fitness) is affected by individual TTX concentration, selection can drive the escalation of toxin levels in newts. The variable fitness consequences associated with both TTX levels of newts and resistance to TTX in snakes that may promote a strong and symmetrical coevolutionary relationship have now been demonstrated.     

The praying mantis (Mantodea) as predator of the poisonous red-spotted newt <Emphasis Type="Italic">Notophthalmus viridescens</Emphasis> (Amphibia: Urodela: Salamandridae)

A Chinese praying mantis, Tenodera sinensis, was observed feeding on a living red-spotted newt, Notophthalmus viridescens. Specimens of that newt’s population are known to contain high concentrations of tetrodotoxin (TTX), a specific blocker of voltage-gated sodium channels. After experimental oral administration of a TTX-solution (1 mg/ml) to adult specimens of four mantis species, all survived high TTX concentrations (up to 30.8 μg/g body mass) as revealed by analysis of their body extracts, but they are rapidly killed by intra-abdominal injection of 1 μg TTX. The toxin was found to be gradually excreted with faeces. As demonstrated by monoclonal antibody-based immunohistochemical technique, TTX does not penetrate the mid-gut membrane, since it was localized only in the gut lumen, but not in the epithelial cells. This prevents the toxin to reach its target, the sodium channels of the insect’s nervous system, and enables the mantids to feed on toxic prey without risking poisoning.     

织纹螺体内的河豚毒素及其衍生物

对2002年福建省引起食物中毒的织纹螺毒素进行研究。高效液相色谱-质谱联用(liquid chromatography/mass,LC/MS)的分析结果显示,织纹螺毒素的主要成分是河豚毒素类毒素(tetrodotoxin,TTX),另外还含有4-epiTTX、anhydroTTX、deoxyTTX等成分。生物法对织纹螺毒素的毒力测定结果表明:所收集的26个织纹螺样品中15个检测出毒性,毒素的毒力在5.7～47.3MU·g-1之间。毒力最高的织纹螺样品2003年5月1日来自福安,主要由半褶织纹螺和另一未定种组成。     

Chemical defense in pelagic octopus paralarvae: Tetrodotoxin alone does not protect individual paralarvae of the greater blue-ringed octopus (Hapalochlaena lunulata) from common reef predators

Some pelagic marine larvae possess anti-predator chemical defenses. Occasionally, toxic adults imbue their young with their own defensive cocktails. We examined paralarvae of the greater blue-ringed octopus (Hapalochlaena lunulata) for the deadly neurotoxin tetrodotoxin (TTX), and if present, whether TTX conferred protection to individual paralarvae. Paralarvae of H. lunulata possessed 150 ± 17 ng TTX each. These paralarvae appeared distasteful to a variety of fish and stomatopod predators, yet food items spiked with 200 ng TTX were readily consumed by predators. We conclude that TTX alone does not confer individual protection to paralarvae of H. lunulata, and that they possess an alternative defense. In larger doses, tetrodotoxin is a deterrent to the predatory stomatopod Haptosquilla trispinosa (mean dose = 3.97 μg/g). This corresponds to 12–13 paralarvae per predator based on the TTX levels of the clutch we examined. Thus, the basic assumption that individual paralarvae of H. lunulata are defended by TTX alone was disproved. Instead, functionality of TTX levels in paralarvae may arise through alternative selective pathways, such as deterrence to parasites, through kin selection, or against predator species not tested here.