L—苹果酸产生菌筛选及高产突变株诱变选育

通过广泛收集和分离,获得根霉属、曲霉属及裂褶菌属等属菌侏８９７侏。产酸指示平板上的变色圈测定结果表明,它们中间６２８株为产酸菌。通过纸层析对产酸菌发酵液酸谱的分析,获得１２９株Ｌ－苹果酸产生菌,经进一步测定发酵液中Ｌ－苹果酸的含量,筛选出以葡萄糖为原料,摇瓶发酵１４０小时,Ｌ－苹果酸产率４８．３７ｇ／Ｌ,对糖转化率４８．３７×１０＾－２的菌株ＬＭ０２。经初步鉴定,这一菌株为曲霉。以ＬＭ０２作为出发     

L一精氨酸产生菌的选育及其发酵条件的研究

以黄色短杆菌（Brevibacterium flavum)AT174(AHV^r,TA^r)为诱变出发株,经亚硝基胍（NTG）诱变处理,获得一株能够积累大量L－O精氨酸的菌株AG77（AHV^r,TA^r,SG^r)。在20L发酵罐中,以葡萄为碳源,以硫酸铵等为氮源的培养基中培养4d,产酸率可达31.3g/L。     

苹果酸—乳酸发酵能量产生机理

苹果酸－乳酸发酵（MLF）是现代葡萄酒酿造工艺中重要的生物降酸手段。MLF是L－苹果酸在乳酸菌的苹果酸－乳酸酶催化下转变成L－乳酸的酶促反应过程,该过程没有底物水平的磷酸化,但在MLF过程中苹果酸确实能够刺激细菌的比生长速率,增加细菌生物量。进一步的研究表明,代谢能的产生并非由于苹果酸的脱羧反应,而主要源于跨膜的L－苹果酸摄入和（或）L－乳酸的流出,从而产生跨膜质子电动势（pmf)(一小部分能量可能由L－苹果酸的代谢中间产物丙酮酸产生）。按照化学渗透理论,pmf驱动F0F1-ATPase合成ATP用于细菌生长。本文还对苹果酸的运输机制进行了综述。     

黄曲霉发酵薯干粉水解液生产L－苹果酸

经选育和诱变得到一株产苹果酸的黄曲霉菌ＴＨ５００７,通过发酵条件的优化,以薯干粉的水解液为主要原料,发酵５ｄＬ－苹果酸可达到６％左右。在总酸中苹果酸含量平均达７２％以上,杂有机酸主要是柠檬酸,延胡索酸的含量在０．０２％以下。补糖发酵比分批发酵产酸性能更佳。     

黄曲霉发酵薯干粉水解液生产L—苹果酸

经选育和诱变得到一株产苹果酸的黄曲霉菌ＴＨ５００７,通过发酵条件的优化,以薯干粉的水解波为主要原料,发酵５ｄＬ－苹果酸可达到６％左右。在总酸中苹果酸含量平均达７２％以上,杂有机酸主要是柠檬酸,延胡索酸的含量在０．０２％以下。补糖发酵比分批发酸产酸性能更佳。     

以抗结构类似物筛选高产L—赖氨酸酵母突变株

以啤酒酵母（Saccharomycescerevisiae)I菌株为出发菌株,经紫外线处理后,在含2.0mg/L的5-（2-氨基乙基）－L一半胱氨酸（AEC）的平板上获得18株生长良好的抗性突变林,编号为I－1～18,其中Ⅰ-Ⅱ株抗AEC达10～15mg／L,是出发株抗性最高浓度的10倍左右,并且Ⅰ—11株的L-赖氨酸含量（重量与重量比,100％）较出发株提高3.89％。以Ⅰ－11株作亚硝酸诱变处理,在含30～120mg／LAEC的培养基上又获得18株抗性突变株,其中Ⅰ11－L和Ⅰ11－MAEC的耐受浓度可达1300mg/L,其赖氨酸的含量较Ⅰ菌株的提高了27．42％和40．28％。     

产氢紫色非硫光合细菌的分离与筛选

从不同来源的样品中分离到5株紫色非硫光合细菌,从中筛选出2株产氢量较高的菌株——菌株D和菌株H。对其产氢动力学研究表明,在含有20mmol/L谷氨酸钠和50mmol/L D,L-苹果酸培养基中,两株菌可连续稳定产氢5—8d,其产氢速率分别为10.18和20.61μ/h·mg细胞干重。     

温特曲霉转富马酸为L—苹果酸的初步研究

从大量霉菌在选育到一株具有较高富马酸酶活性的温特曲霉（Ａｓｐｅｒｇｉｌｌｕｓ ｗｅｎｔｉｉ）Ａ５－６１。在摇瓶培养条件下,３２℃ ９６小时,产Ｌ－苹果酸达１０．４９ｇ／１００ｍｌ,对富马酸的转化率达９０．８０％。利用菌体细胞,进行酶转化试验,结果表明：１．６ｇ湿菌体接入２５ｍｌ含富马酸１０．０％（用ＮａＯＨ中和至ｐＨ７．０）的转化液中,３５℃１６－２４小时,连续转化三次,分别产生Ｌ－苹果酸９．６１     

蛋氨酸高产酵母突变株的推理筛选

以啤酒酵母（Saccharomycescerevisiae）菌株Ⅰ为出发株,经紫外线处理后,在含可完全抑制出发株生长的乙硫氨酸（浓度为2g/L）的基本培养从上筛出14株生长良好的突变株,其中I－2,I－9株还能在无硫基本培养基上良好生长。经氨基酸自动分析仪检测,I－2株菌体蛋白中的蛋氨酸含量（质量由分比）由I株的1．93增年9．28。     

二十碳五烯酸的菌种选育及发酵条件

利用含亚麻子油的斜面培养基连续传代和逐渐降低培养温度,诱导筛选的方法,从大量丝状真菌中选育到一株产二十碳五烯酸（all－cis－5、8、11、14、17－eicosopnthenoicacid）较高的被孢霉菌（Motierellasp．）SM481。研究得到最适培养基及最适培养条件。在最适培养及产二十碳五烯酸条件下,细胞干重和二十碳五烯酸产量分别为28．8g／L和0.127g／L。     

Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor

Effects of various nutritional and environmental factors on the accumulation of organic acids (mainly L-malic acid) by the filamentous fungus Aspergillus flavus were studied in a 16-L stirred fermentor. Improvement of the molar yield (moles acid produced per moles glucose consumed) of L-malic acid was obtained mainly by increasing the agitation rate (to 350 rpm) and the Fe(z+) ion concentration (to 12 mg/L) and by lowering the nitrogen (to 271 mg/L) and phosphate concentrations (to 1.5 mM) in the medium. These changes resulted in molar yields for L-malic acid and total C(4) acids (L-malic, succinic, and fumaric acids) of 128 and 155%, respectively. The high molar yields obtained (above 100%) are additional evidence for the operation of part of the reductive branch of the tricarboxylic acid cycle in L-malic acid accumulation by A. flavus. The fermentation conditions developed using the above mentioned factors and 9% CaCO(3) in the medium resulted in a high concentration (113 g/L L-malic acid from 120 g/L glucose utilized) and a high overall productivity (0.59 g/L h) of L-malic acid. These changes in acid accumulation coincide with increases in the activities of NAD(+)-malate dehydrogenase, fumarase, and citrate synthase.     

产L-苹果酸重组大肠杆菌的构建

基于产琥珀酸重组大肠杆菌E.coli B0013-1050的琥珀酸合成途径,利用Red同源重组技术结合Xer/dif重组系统敲除富马酸酶基因fumB、fumC,苹果酸酶基因maeB,构建L-苹果酸合成途径,最终得到重组大肠杆菌E.coli2030,该菌株在15 L发酵罐中,产L-苹果酸12.5 g/L,葡萄糖-苹果酸转化率为52.1%,同时对发酵产物中主要杂酸丙酮酸和琥珀酸的生产原因进行了初步的探讨与分析。为进一步提高L-苹果酸的转化率,整合表达来源于黄曲霉的苹果酸脱氢酶基因,构建重组菌E.coli 2040,在15 L发酵罐中产L-苹果酸14 g/L,葡萄糖-苹果酸转化率提高到60.3%。     

A simple plate-assay for the screening of l-malic acid producing microorganisms

A simple plate-assay has been developed to screen microorganisms for L-malic acid production. Acid producing organisms were identified, after microbial colony growth on media containing glucose or fumaric acid as sole carbons sources, by formation of a dark halo of formazan. The halo was observed when the plate was covered with a soft agar overlay containing NAD(+)-malate dehydrogenase, NAD+, phenazine methosulfate (PMS) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay developed is simple, specific for L-malic acid and therefore can be used to identify L-malic acid producing filamentous fungi using glucose as carbon source (e.g. Aspergillus strains). The assay is also applicable for screening bacteria with high fumarase activity, able to convert fumaric acid to L-malic acid.     

寄生曲霉CICC40365利用木糖产L-苹果酸的发酵条件优化

【目的】为提高L-苹果酸产量及木糖利用率,以寄生曲霉(Aspergillus parasiticus CICC40365)为菌种,木糖为碳源,对其发酵工艺及木糖代谢途径进行初步研究。【方法】采用单因素试验和响应曲面法(Box-Behnken设计)对培养基和发酵条件进行优化。【结果】获得最佳培养基配方为:木糖100.0 g/L、硫酸铵2.0 g/L、酵母浸粉3.0 g/L、硫酸镁0.20 g/L、硫酸锰0.15 g/L、硫酸亚铁0.08 g/L、碳酸钙80.00 g/L,L-苹果酸的产量为53.58 g/L,较优化前提高40.5%。发酵条件较好组合为:接种量为8%(体积比)、摇瓶装液量60 mL/250 mL、发酵温度32°C、摇床转速170 r/min、发酵周期8 d,L-苹果酸的产量为55.47 g/L。Mg2+、Mn2+对木糖代谢中相关酶的影响研究结果表明,木酮糖激酶在该菌株代谢木糖过程中起着重要作用。【结论】寄生曲霉CICC40365能够较好地利用木糖发酵产L-苹果酸,其产量及木糖的利用效率均得到提高。     

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.     

黑曲霉对黄曲霉生长、产毒及黄曲霉毒素B1的影响

目的研究黑曲霉对黄曲霉生长、产毒的抑制作用及对AFB1的降解作用。方法将黑曲霉分别与黄曲霉、AFB1共同培养,定期测定培养液pH、菌丝体干重、黄曲霉孢子数、AFB1含量。结果黑曲霉与黄曲霉混合培养时,黄曲霉孢子数、AFB1含量均比单独培养的低,2组之间差异有统计学意义(P<0.05),抑制率达到68.06%~91.52%;加入黑曲霉后,AFB1含量降低,实验组与对照组之间差异有统计学意义(P<0.05),降解率为46.19%。结论黑曲霉既能抑制黄曲霉生长、产毒,又能降解AFB1。     

Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp.

Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in some A. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intact veA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.     

曲酸高产菌株的诱变选育

以紫外线和氮离子注入作为诱变手段对曲酸产生菌黄曲霉CICC2242进行诱变处理,筛选出一株高产菌株N83。曲酸产量由初始的12．4g／L提高到19．4g／L,提高了56．5％;遗传稳定性实验结果表明,N83遗传性状稳定良好。该结果表明氮离子注入诱变是获得高产曲酸的有效途径。     

Osmotic and matric potential effects on growth, sugar alcohol and sugar accumulation by Aspergillus section Flavi strains from Argentina

AIMS: The effect of osmotic and matric potential stress on growth and sugar alcohols (polyols: glycerol, erythritol, arabitol and mannitol) and sugars (trehalose and glucose) accumulation in toxigenic and nontoxigenic colonies of Aspergillus flavus and A. parasiticus was evaluated. METHODS AND RESULTS: Growth of Aspergillus section Flavi with significant reductions at 20 and 30 degrees C was more sensitive to changes in matric potential, between 60 and 100% in the range of -7 to -14 MPa. No significant differences were found between toxigenic and nontoxigenic strains for both species. Total polyol accumulation in unamended maize meal agar medium (-0.75 MPa water potential) was higher at 30 than 20 degrees C. The major change in concentrations of endogenous sugars and total polyols was in matrically amended medium (with PEG 8000) at -7 and -10 MPa. Accumulation of glucose, arabitol, mannitol and erythritol content of A. flavus and A. parasiticus mycelial colonies was greater in normal unstressed maize meal agar medium (-0.75 Mpa) at 20 degrees C. This was modified by solute and matric stress. CONCLUSIONS: The data showed relative sensitivity to osmotic and matric potential, and temperature, and the impact on growth rates, polyol and sugar accumulation in mycelia of A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The matric potential effects on growth may be of particular importance for growth and survival in environments with low-matric potential stress. The tolerance of spoilage fungi such as Aspergillus section Flavi to such modifications could increase the potential for spoilage and mycotoxin production in such substrates. This knowledge is important for understanding the relative ecological fitness of these aflatoxigenic species and in the development of prevention strategies for their control.