一株蝉拟青霉产N~6-(2-羟乙基)腺苷静置发酵条件的研究

为了提高蝉拟青霉122菌株中N~6‐(2‐羟乙基)腺苷(HEA)的产量。采用静置液体培养方法,以HEA产量为指标,筛选得到122菌株在查氏培养基上的HEA产量优于沙氏和PD培养基;以查氏培养基为基础培养基,在26℃培养40d时,HEA产量达到最大;在黑暗条件培养优于光照条件培养;最优碳源是蔗糖和葡萄糖;最优氮源是磷酸氢二铵;最优无机盐是KH_2PO_4;最优前体物为次黄嘌呤;最适氨基酸为L‐谷氨酸。正交实验结果表明蔗糖是影响122菌株产HEA的最主要的因子,在筛选出的优化培养基上得到122菌株HEA的最大产量为(130.22±0.60)mg/L,较优化前HEA的产量提高9.73倍。     

pcD-awte候选疟疾DNA疫苗发酵条件的探索

探索pcD-awte候选疟疾DNA疫苗发酵条件,为该疫苗制备工艺的建立做准备.通过摇瓶和5 L发酵罐水平,考察不同培养基组成及来源、补料变化和温度转换对工程菌生长及质粒产量的影响.结果表明在TB培养基组分中添加Mg2+、微量元素复合物、核苷等成分,补料培养基由葡萄糖代替甘油,对数中期温度由37℃升至42℃等条件,能提高工程菌株pcD-awte质粒的产量.在优化的培养条件下pcD-awte质粒产量可达125～130mg/L培养液.     

peD—awte候选疟疾DNA疫苗发酵条件的探索

探索pcD—awte候选疟疾DNA疫苗发酵条件,为该疫苗制备工艺的建立做准备。通过摇瓶和5L发酵罐水平,考察不同培养基组成及来源、补料变化和温度转换对工程菌生长及质粒产量的影响。结果表明在TB培养基组分中添加Mg^2＋、微量元素复合物、核苷等成分,补料培养基由葡萄糖代替甘油,对数中期温度由37％升至42℃等条件,能提高工程菌株pcD—awte质粒的产量。在优化的培养条件下pcD—awte质粒产量可达125—130mg／L培养液。     

玉米水解糖液体培养灵芝发酵条件的优化

目的：优化液体培养灵芝的发酵条件,提高多糖产量。方法：采用玉米水解糖为主要成分的培养基,通过单因素和正交实验,对赤芝G22菌株液体培养过程中影响多糖产量的发酵温度、摇床转速等工艺条件进行了研究。结果：经极差分析和方差分析确定了多糖高产的最佳发酵条件为：发酵温度27℃、摇床转速170r/min、培养基初始pH值6.5、发酵时间144 h。结论：通过优化液体发酵条件,可显著提高灵芝多糖的产量。在最佳发酵条件下液体培养G22菌株,灵芝总多糖产量由1.851g/L提高到2.439g/L,提高了31.0%。     

蛹虫草菌株退化特征及鉴别方法

蛹虫草菌株在继代培养和低温长期保藏过程中极易退化导致子实体产量下降,对产业造成重大影响。本研究以正常菌株、PDA斜面长期4 ℃保藏导致退化的菌株和连续继代培养的菌株为材料,观察其子实体、菌落、菌丝形态和分生孢子数量,对菌丝细胞核、线粒体、活性氧积累和芽生孢子内脂滴进行染色观察;并对菌丝中虫草素、腺苷和麦角甾醇含量进行比较分析。结果表明PDA斜面长期4 ℃保藏和连续继代培养导致的蛹虫草菌株退化表型大多数一致,即退化菌株较正常菌株子实体产量降低、菌丝粘连打结、分生孢子数量显著降低、菌丝活性氧含量升高、细胞内线粒体数量减少、芽生孢子脂滴由弥散的小脂滴融合为大脂滴。然而,在菌落见光转色方面,长期低温保藏退化菌株基本不转色,而继代培养退化菌株转色不稳定;长期保藏退化菌株菌丝细胞核数目无明显变化,继代培养退化菌株细胞核数量明显减少;长期保藏退化菌株菌丝中虫草素、腺苷和麦角甾醇含量较正常菌株降低58%、41%和70%,继代培养退化菌株麦角甾醇含量无明显变化。因此,显微观察菌丝是否出现粘连打结及氮蓝四唑NBT检测菌丝活性氧含量,操作简单、用时较短,可用于大规模生产中蛹虫草菌株退化的检测手段;线粒体和脂滴染色也可作为退化菌株的鉴别方法;而生产中常用的通过菌落见光转色判断菌株优劣的方法需要慎重。继代培养菌株第4代开始出现明显的退化特征,因此在生产中使用的菌株最好控制在继代培养3代以内。     

大菱鲆鳗弧菌灭活疫苗原液发酵条件的优化

为实现大菱鲆鳗弧菌灭活疫苗中试生产,通过对大菱鲆鳗弧菌菌株VAM003二级种子培养时间、盐度、培养基、接种量、补料等发酵条件的优化筛选,确定大菱鲆鳗弧菌菌株VAM003灭活疫苗发酵原液的发酵工艺条件。鳗弧菌菌珠VAM003接种于含2. 5%Na Cl的TSB液体发酵培养基,28℃振荡培养12～14 h,制备二级种子液,按发酵罐培养基总量的10%接种二级种子液,28℃补料发酵10～12 h。在该条件下鳗弧菌菌株VAM003发酵活菌数达到1. 20×1010cfu/m L,比优化前提高120%以上。     

一株产赭曲霉毒素A黑曲霉及其产毒条件

以甘肃河西走廊葡萄酒产区分离的曲霉属黑色组H1菌株为试验材料,依据形态特征和基于rDNA ITS、β-tubulin及calmodulin基因序列的系统发育分析,将其鉴定为黑曲霉Aspergillus niger。利用HPLC-FLD和UPLC-MS/MS检测分析确认H1菌株具有产生赭曲霉毒素A(OTA)的能力。进一步研究了培养条件对H1菌株产毒的影响。试验结果表明,YES培养基比CYA培养基更利于H1菌株的营养生长和产OTA;18–25℃,该菌生长较慢,但OTA产量高;18℃H1菌株在YES培养基上培养9d时OTA的产量达到最大值,为26.01μg/g YES;30–37℃该菌的生长速率显著提高,但OTA产量很低,甚至不产OTA。试验结果表明,温度和营养条件是影响H1菌株产生OTA能力的重要因素。     

粉被虫草静置发酵产N6-(2-羟乙基)腺苷培养基组分及前体物的筛选

在静置培养条件下,对粉被虫草cp菌株产N6-(2-羟乙基)腺苷（简称HEA）的培养基及其组分进行优化。采用单因素实验方法,以HPLC法对cp菌丝体中HEA进行检测,以HEA的产量为指标,结果表明查氏培养基有利于cp菌株产HEA,其产量是沙氏和PD培养基的3.7倍和4.48倍。以查氏培养基为基础培养基,进行碳、氮源的筛选中,最有利于cp菌株产HEA的是蔗糖和硝酸钠;在无机盐的筛选中,K2HPO4是促进cp菌株累积HEA最主要的无机盐,其HEA产量较CK增长了5 822.37%（提高了58.2倍）;在查氏培养基上添加不同前体物和氨基酸,发现其结构类似物腺苷、次黄嘌呤和腺嘌呤都有促进cp菌株累积HEA的能力,其中腺苷和次黄嘌呤能提高其产量60%以上;而组氨酸是最有利于cp菌株累积HEA的氨基酸,产量达到（45.56±2.8）mg/L,较CK提高HEA产量251.68%,其次是L-谷氨酸增产184.19%。     

产小檗碱诱变菌株培养条件的优化

S-NU-3-2菌株是对源自药用植物黄檗的内生真菌S6进行诱变获得的小檗碱产量比出发菌株有明显提高的突变株,本研究对其培养条件进行了优化,以期进一步提高其产量,为开发利用真菌发酵生产植物活性成分的新途径奠定基础。以菌丝生长和小檗碱产量为指标,筛选出了适宜该菌株发酵的基本培养基、碳源、氮源、光照和培养温度,并经进一步的正交试验优化,得出该高产菌株的最佳培养条件为豆芽汁基本培养基含蔗糖3%,酵母膏0.2%,pH7.0,温度26℃,全光照培养。与初始培养条件相比,在优化后的条件下,该菌株的小檗碱得率提高了47.2%,菌体生物量提高了24%。     

不同培养条件对粉被虫草产N6-(2-羟乙基)腺苷的影响

N6-(2-羟乙基)腺苷[N6-(2-hydroxyethyl)-Adenosine,HEA],又名茧草菌素,是一种钙离子拮抗剂,具有抗紫外辐射、抗血小板凝结和镇痛等效用,是集医药保健、化妆品等多项开发潜力于一身的生物资源,也是衡量虫草制品质量的一个重要生物活性物质指标。该研究采用高效液相色谱法对菌丝体中HEA进行检测,以HEA的产量为指标,考察不同培养方法、培养基及其组分、前体物和氨基酸对粉被虫草(Cordyceps pruinosaPetch)cp菌株产HEA的影响。结果表明:在沙氏培养基上,静置培养120 d,cp菌株HEA的产量明显优于摇床培养20 d和发酵罐培养3 d的产量,说明随培养时间的延长,cp菌株中HEA的累积会逐步增加。在静置培养条件下,cp菌株在查氏培养基上HEA的产量明显优于沙氏培养基和PD培养基,HEA的产量是沙氏培养基的3.7倍,是PD培养基的4.48倍。以查氏培养基为基础培养基,静置培养120 d进行碳、氮源的筛选中,最有利于cp菌株产HEA的是蔗糖和硝酸钠;在无机盐的筛选中,K2HPO4是促进cp菌株累积HEA最主要的无机盐,其HEA产量较对照(不添加无机盐的查氏培养基)提高了58.2倍;在查氏培养基上添加不同前体物和氨基酸,发现其结构类似物腺苷、次黄嘌呤和腺嘌呤都有促进cp菌株累积HEA的能力,其中腺苷和次黄嘌呤的效果最好,能提高HEA产量60%以上;添加的14种氨基酸中有组氨酸、L-谷氨酸、甘氨酸、天门冬酰胺、丝氨酸、亮氨酸、L-丙氨酸、赖氨酸和精氨酸等9种氨基酸能促进cp菌株产HEA的能力,其中组氨酸是最有利于cp菌株累积HEA的氨基酸,其HEA产量较ck(查氏培养基)提高251.68%,HEA产量达到(45.56±2.8)mg/L,菌丝体中HEA含量达到(4 633.10±65.65)μg/g。     

1,3-丙二醇产生菌的诱变育种及培养基优化

以实验室自然筛选的克雷伯氏杆菌(Klebsiella sp.)为出发株,采用紫外诱变及亚硝基胍和超声波协同处理获得一株1,3-丙二醇高产突变株。在摇瓶发酵中,其产1,3-丙二醇产量由17.39 g/L提高到24.11 g/L,提高38.64%。变异株经10次传代培养,发酵能力稳定。对发酵培养基成分进行了优化,优化后1,3-丙二醇产量为30.05g/L,为优化前的1.25倍。     

Production of Flavine-Adenine Dinucleotide from Riboflavine by a Mutant of Sarcina lutea

A study was made to develop a new method for the production of flavine-adenine dinucleotide (FAD) from riboflavine and adenine by a mutant of Sarcina lutea deficient in the enzyme adenosine deaminase. It was found that this strain could convert exogenously supplemented riboflavine to extracellular FAD. The yields of FAD were increased by addition of D-cycloserine in the culture medium. The culture conditions for FAD production were investigated under the addition of D-cycloserine, and increased production of FAD was observed with the addition of an appropriate amount of thiamine, acetate, and sodium ion. The yield of 0.7 g/liter was obtained in the optimal culture in 5 days. Accumulated FAD was readily isolated by adsorption chromatography and ion-exchange chromatography in a 70% yield.     

内切葡聚糖酶高产菌株的诱变选育

【目的】建立里氏木霉(Trichoderma reesei)高产突变菌株的快速筛选方法,选育出高产内切葡聚糖酶的突变株。【方法】对里氏木霉T306菌株的初筛培养基进行优化,建立快速筛选方法;通过紫外诱变手段选育内切葡聚糖酶高产突变菌株,并对突变菌株的产酶培养基进行优化。【结果】在初筛培养基中添加浓度为0.1%(W/V)的乳糖、蛋白胨及脱氧胆酸钠有利于菌株的筛选。诱变后筛选出菌落形态发生明显变化的内切葡聚糖酶高产突变株0516,其羧甲基纤维素酶活力(CMC酶)较出发菌株提高了38.9%。其产酶培养基经优化后,得到最适碳、氮源分别为:乳糖1.50%、硫酸铵0.14%、尿素0.05%、蛋白胨0.10%,优化后CMC酶活力达64.2 U/mL,较优化前提高了2.3倍。【结论】建立了里氏木霉高产突变菌株的快速筛选方法,通过紫外诱变育种获得了产内切葡聚糖酶能力高且遗传稳定的突变株0516。     

腺苷产生菌Xn151菌株的选育及发酵条件研究

以肌苷产生菌枯草杆菌GMI-741(Ade~(--))为出发菌株,通过多因子诱变选育出具有黄嘌呤、鸟嘌呤双重营养缺陷型、丧失腺嘌呤脱氨酶的腺苷产生菌Xn151。以Xn151为发酵菌株,采用正交实验设计法对其发酵基础培养基进行优化,确定最佳发酵配方。利用此培养及配方摇瓶发酵72h,腺苷产量可达12.3g/L。     

天麻萌发菌株的生物学特性研究

对4个天麻种子萌发菌菌株在不同培养条件下菌丝的生长进行了研究。结果显示,4个天麻萌发菌的最佳生长温度为25℃,适宜生长的pH值范围为5.0~6.0,最佳培养基为木屑-麸皮培养基。综合试验结果筛选出西乡为一株优良天麻萌发菌。     

利用原生质体紫外诱变技术选育可常温发酵的中国被毛孢新菌株

中国被毛孢(Hirsutella sinensis)是天然冬虫夏草[Cordyceps sinensis(Berk)Sacc.]的真正无性型,其生长温度低(低于20℃),生长缓慢(发酵周期长达20～30d),对人工发酵的技术条件要求很高,因而大大增加了工业化发酵生产的成本和技术难度。为了解决上述问题,实验中从天然冬虫夏草子座中分离到中国被毛孢菌株BS-1,并对其进行了原生质体紫外诱变处理。经过粗筛和精筛,从中选出一株可常温下(25～28℃)发酵、生长迅速(发酵周期4～5 d)、有效成分(甘露醇和腺苷)含量显著高于原始菌株的诱变株BS-5962。通过连续10代PDA斜面继代培养和摇瓶试验,以及连续3批的1t罐的中试试验,检查结果表明所得诱变株性状优异而稳定。可为降低中国被毛孢的液体深层发酵成本和技术难度提供一种有效的解决方案。     

The production of polyhydroxyalkanoate by Bacillus licheniformis using sequential mutagenesis and optimization

The purpose of this study was to enhance the production of polyhydroxyalkanoate (PHA) by sequential mutation of Bacillus licheniformis PHAs-007, using UV and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). In addition, the effect of nutrient additions and environmental conditions were optimized to increase the production of PHA. Bacillus licheniformis PHAs-007 produced high amounts of PHA (64.09 ～ 68.80% of DCW) under both synthetic and renewable substrates. After mutagenesis treatment, mutant M2-12 was selected from 380 strains, based on its high biomass and PHA concentration. The mutant M2-12 gave the highest value of specific growth rate (0.09/h), biomass (22.24 g/L) and PHA content (19.55 g/L) under optimal conditions, consisting of 3% palm oil mill effluent, with no additional trace elements, at 45oC and pH 7. The mutant strain showed higher resistance to substrate concentrations, as well as pH and temperature, than the wild type. The accumulation of PHA was increased by 3.18-fold compared to the wild type, and the production of PHA by the mutant M2-12 was constantly retained over 12 times of cultivation. The mutation and optimization strategy appear to be suitable for producing high density PHA, reducing the medium cost and consequently lowering the production cost. Interestingly, the mutant strain could synthesize the novel PHA copolymers such as 3-hydroxyvalerate and 3-hydroxyhexanoate, which were not produced by the wild type.     

黄原胶降解菌BIT-BJ001培养条件的优化

黄原胶在采油工程中有重要的应用,但其难降解性质给采油工程带来很多问题。从塔里木油田胡杨木根部样品中分离得到1株黄原胶降解菌BIT-BJ001,对其发酵条件的研究表明,此黄原胶降解菌最适培养条件为:黄原胶0.3%,酵母粉0.5%,Na+浓度0.8%,Mg2+浓度0.8%,初始pH值为10,温度60℃。菌种BIT-BJ001降解黄原胶的能力与发酵时间、发酵液中还原糖浓度有关,发酵96 h,黄原胶降粘率达到最高,发酵液中还原糖浓度过高,将抑制菌株对黄原胶的降解。     

紫外线诱变选育聚丁二酸丁二醇酯（PBS）高降解活性菌株

以PBS降解菌HJ03（Alternariasp．）为出发菌株,通过紫外诱变,透明圈初筛及PBS薄膜复筛,获得一株降解能力增强且对温度和pH耐受力均得到提高的突变株HJ10。与出发菌株相比,HJ10在培养初期气生菌丝少,而培养7d时菌落致密且生长速度较快。经过连续继代培养7代后发现,突变株的降解活力保持了良好的遗传稳定性,其降解率较出发菌株提高百分比达14．4％以上;在最适降解温度范围内（25℃～30℃）和不适宜降解的温度条件下突变菌株Ⅻ10的降解率均高于出发菌株;各pH条件下,突变株对PBS的降解能力明显优于出发菌株,尤其在pH5．0时降解率提高了22．80％。     

Metabolite transport in mutants of Escherichia coli K12 defective in electron transport and coupled phosphorylation.

1. The uptakes of Pi and serine by whole cells of mutant strains of Escherichia coli K12, grown under both aerobic and anaerobic conditions, were studied. 2. Uptake by aerobic cells was low in a ubiquinone-less mutant but normal in two mutant strains unable to couple phosphorylation to electron transport. 3. One of these uncoupled strains, carrying the unc-405 allele, does not form a membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate, and it is concluded that the Mg2+-stimulated adenosine triphosphatase does not serve a structural role in the aerobic active transport of Pi or serine. 4. The other uncoupled strain, in which aerobic uptake is unaffected, carries a mutation in the uncB gene, thus distinguishing this gene from the etc gene, previously shown to be concerned with the coupling of electron transport to active transport. 5. The uptakes of Pi and serine by anaerobic cells were normal in the ubiquinone-less mutant, but defective in both the uncoupled strains. 6. The uptake of Pi and serine by anaerobic cells of the uncB mutant could be increased by the addition of fumarate to the uptake medium. The unc-405 mutant, however, required the addition of fumarate for growth and for uptake. 7. The uncB mutant, unlike the unc-405 mutant, is able to grow anaerobically in a minimal medium with glucose as sole source of carbon. Similarly a strain carrying a mutation in the frd gene, which is the structural gene for the enzyme fumarate reductase, is able to grow anaerobically in a glucose-minimal medium. However, a mutant strain carrying mutations in both the uncB and frd genes resembles the unc-405 mutant in not being able to grow under these conditions.