激光诱变筛选高产植酸酶黑曲霉菌株的研究

用1.06um,15KHz高重复率声光调QNd：YAG激光以20W和15W的辐照功率分别照射产植酸酶的黑曲霉孢子液和原生质体液不同时间,检定再生菌落数并经透明圈初筛和摇瓶复筛,测定诱变菌株所产植酸酶酶活。结果表明：20W的功率辐照黑曲霉孢子1′以上,致死率近乎100…,15W功率辐照原生质体液20″-4′,存活率在8.571％-117.14％之间,正变率在27.45％-100％之间,正变辐度60.69％-124.96％,说明原生质体比孢子耐受激光诱变的能力强,并且诱变效果好。筛选到了一株酶活提高达3.75倍的单个变异菌株。     

Nd^＋3：YAG激光对黑曲霉的诱变效应

本文采用Ｎｄ＾＋３：ＹＡＧ激光辐照柠檬酸生产菌黑曲霉孢子,辐照后进行培养和发酵试验,分析测定不同辐照时间黑曲霉孢子的存活率、黑曲霉菌丝体生长繁殖及颓丧 酸的速度、主要代谢产物柠酸的产量及淀粉糖化酶活力等变化。     

N+注入和60Co-γ辐照对柠檬酸发酵菌黑曲霉的诱变效应

首次尝试了应用两种核技术手段(同时)对黑曲霉进行诱变,即将大剂量^60Co-γ辐照的黑曲霉孢于直接进行低能氮离子注入,使处于休眠状态下的黑曲霉抱子同时受至^60Co-γ辐照和N^ 注入的作用。通过溴甲酚绿指示性平板辅助筛选和摇瓶发酵。获得了1株产酸提高18．44％、转化率达1034．5％的M3代菌株CN05,为柠檬酸发酵菌黑曲霉的进一步育种工作提供了诱变参数和高产出发菌株。     

激光诱变黑曲霉原生质体选育高酶活生淀粉糖化菌的研究

本研究对生淀粉糖化菌黑曲霉S-1原生质体制备与再生因素进行了探讨。该菌菌丝体酶解2小时后,可制得2×108/ml原生质体,其再生率达13.3%。采用波长(λ₁=260hm,λ_2=266nm)能量8mj的激光直接照射该菌原生质体。结果表明:激光对原生质体的致死率比对孢子的致死率高;激光对原生质体的诱变效应也较好,其正突变率比孢子的正突变率高34%;与出发菌株相比生淀粉糖化酶活性平均提高37.4%,最高突变株酶活提高91%。     

黑曲霉菌株柠檬酸合成酶基因的敲除及功能分析

【背景】柠檬酸合成酶是碳代谢途径的中心酶,其在三羧酸循环(tricarboxylic acid cycle,TCA)、氨基酸合成和乙醛酸循环中发挥着重要作用,是柠檬酸合成的关键酶。本论文所选用的是一株高产柠檬酸的黑曲霉菌株CGMCC10142。【目的】克隆柠檬酸合成酶关键基因,构建柠檬酸合成酶的敲除菌株并鉴定其在黑曲霉菌株高产柠檬酸过程中的功能及影响。【方法】采用根癌农杆菌转化方法并利用同源重组原理,采用抗性筛选和致死型反向筛选的双重筛选方法获得正确敲除株。对转化子在不同碳源下的生长情况进行观察并对柠檬酸发酵过程中菌丝球变化和产酸量进行分析,最后通过荧光定量PCR分析柠檬酸合成酶基因对黑曲霉积累柠檬酸的影响,及其对主要代谢途径中重要酶相关基因和其他的表达量的影响。【结果】以柠檬酸高产菌株黑曲霉CGMCC10142为出发菌,构建一株遗传稳定的柠檬酸合成酶敲除的菌株T1-2。结果发现该菌株在以葡萄糖为碳源的培养基上生长缓慢并且产生孢子量减少。通过摇瓶发酵产酸实验,结果表明敲除菌在84 h产酸量为64.3 g/L,相对于出发菌的98.7g/L降低了34.85%。通过荧光定量PCR发现柠檬酸合成酶的表达量是下降的,同时重要酶的表达量都下降。【结论】该菌株的柠檬酸合成酶基因对柠檬酸积累具有重要作用,但存在其他同工酶基因,该基因敲除仅使产酸合成降低34.85%,同时发现该柠檬酸合成酶的顺畅表达有助于主代谢途径中各关键酶的高效表达,本研究可为研究黑曲霉高产柠檬酸机理奠定基础。     

柠檬酸发酵菌黑曲霉的菌种改良述评

柠檬酸是微生物发酵法生产的重要有机酸,用途极为广泛。柠檬酸发酵菌黑曲霉的育种在柠檬酸工业中占有重要地位。该文论述了柠檬酸发酵菌黑曲霉的发酵机制、代谢调控及柠檬酸积累的机理,柠檬酸发酵菌黑曲霉的物理和化学手段诱变育种取得的成果,细胞工程和基因工程在柠檬酸发酵菌黑曲霉育种中的应用,分析了柠檬酸发酵菌黑曲霉育种的发展方向。     

紫外-激光复合诱变选育天冬氨酸转氨酶高产菌株

对天冬氨酸转氨酶产生菌大肠杆菌XJ-1原生质体进行紫外-激光复合诱变筛选,结果表明,复合诱变对该菌的原生质体有明显的致死作用。以致死率和正突变率为指标,确定了紫外和He-Ne激光照射的最佳时间分别为45 s和40min。在此条件下对大肠杆菌原生质体进行紫外-激光复合诱变,得到3株高产菌株,分别命名为XJ-1-45、XJ-1-86和XJ-1-99,酶活较出发菌株XJ-1分别提高了12.82%、17.37%和26.27%。传代培养表明突变株生产性能稳定。     

苹果渣基质柠檬酸高产菌株选育

分别以固态苹果渣、苹果渣固态酶解物、苹果渣酶解液和10%葡萄糖溶液为发酵基质研究了17株野生黑曲霉的柠檬酸产生能力,并对获得的4株柠檬酸高产菌进行了诱变育种。结果表明:17株黑曲霉在4种发酵基质中均能良好生长并发酵产生柠檬酸,不同菌株在同一发酵基质中产酸能力间存在差异。FG17、FG23、FG26、FG30等4株菌苹果渣基质柠檬酸产生能力较高,且在4种不同发酵基质中产酸性能稳定。4株菌分别经紫外线和60Co-γ射线诱变后得到的正向突变株柠檬酸产率均显著提高,突变株中FG26-15-4(UV)发酵苹果渣后柠檬酸产率最高,达11.32%,FG23-13-3(γ)发酵苹果渣酶解液后柠檬酸产率最高,达2.73 mg/mL,均高于现有研究报道,可作为不同类型苹果渣基质柠檬酸发酵用菌种。     

激光诱变选育高产PUFAs真菌菌株的研究

采用PIV400型Nd：YAG激光器,对少见报道的产PUFAs丝状真菌菌株Monoblepharis polymorpha Comu进行激光诱变育种。在激光照射剂量：波长532nm,功率50mW,时间10min下,孢子致死率为75％-80％。突变株中w-3系列PUFAs正突变率大于80％,w-3系列突变株FPAm59传代10代以上性能稳定,在土豆基质上摇瓶培养6d后,生物量达到8g/L,产油率53．4％,GLA343．1mg／L、从1209．0mg／L、EPA125．1mg／L、DHA119．4mg／L;分别比对照菌株提高了2．5倍、9．7倍、18．0倍、6．6倍、10．3倍、3．8倍。结果表明,激光诱变育种是获得PUFAs高产菌株尤其是生产w-3系列PUFAs菌株的有效方法。     

梅岭霉素产生菌抗药性突变标志诱变筛选模型的初步研究

本文通过梅岭霉素 (Meilingmycin)产生菌南昌链霉菌NS 4 1 80菌株孢子对 6种抗生素敏感性测定 ,采用诱变剂EMS四种不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含致死浓度链霉素的高氏平板上。然后从抗药性突变标志菌株中进一步筛选梅岭霉素高产菌株。在 150 0多个抗药性突变株中通过初筛获得了比诱变出发菌株的产素能力提高 50 %以上菌株。通过诱变剂量分别与抗药性突变率和突变株产素产量的变势统计分析表明 ,菌株抗药性突变与产素突变密切相关 ,产素突变的EMS诱变剂量高于抗药性突变诱变剂量 ,在 0 .0 3mol/LEMS剂量作用下 ,菌株致死率为 99.4 3% ,抗药性突变率为 0 .0 4 4 0 % ,建立了梅岭霉素产生菌抗药性突变标志诱变推理性筛选模型。为南昌链霉菌高产菌种选育研究作了有益的尝试 ,并有助于其它链霉菌属的抗生素产生菌育种研究。     

Citric acid production by a novel Aspergillus niger isolate: I. Mutagenesis and cost reduction studies

Ultraviolet-irradiation (UV), ethyl methane sulfonate (EMS) and acridine orange (AO) were used to induce citric acid overproduction mutations in Aspergillus niger UMIP 2564. Among 15, eight of the mutant derivatives, were improved with respect to citric acid production from sucrose in batch cultures. Maximum product yield (60.25%) was recorded by W5, a stable UV mutant, with approximately 3.2-fold increase when compared to the parental wild type strain. In terms of the kinetic parameters for batch fermentation processes, the mutation doubled the specific substrate uptake rate and achieved 4.5- and 7.5-fold improvements in citric acid productivity and specific productivity, respectively. For reduction of the fermentation medium cost, corn steep liquor and calcium phosphate pre-treated beet molasses were successfully used as substituents of nitrogen and carbon sources in the growth medium, respectively. These medium substitutions resulted in a W5 citric acid fermentation culture with a product yield of 74.56%.     

N+注入选育产蛋白酶益生菌及其生物学效应研究

试验研究了N+注入选育产蛋白酶益生菌及其生物学效应.经N+反复注入诱变筛选,突变高产菌株的酶活由出发菌株的75.8IU.g-1提高到631.6IU.g-1;利用中心组合设计原理摸索出了突变菌株最佳产酶条件即麸皮24.83%,豆粕23.68%,水50%,硫酸铵1.49%,发酵温度30℃,发酵时间66h,培养基pH 5.5.生物学效应研究发现真空处理对活细胞有明显伤害;孢子存活率先是随着注入剂量加大而迅速降低,当注入剂量加大到50×2.6×1013 ions/cm2时存活率出现平缓回升,注入剂量超过100×2.6×1013ions/cm2时存活率又再次开始下降;注入剂量越大,孢子突变率越高,中等注入剂量30×2.6×1013N+/cm3～80×2.6×1013N+/cm2可引起较高的正突变率;ESR谱研究结果表明,N+注入前和注入后都有自由基ESR波谱单一峰,随着注入剂量的增大,孢子内的自由基ESR波谱的强度也逐渐增大;SEM观察发现,未经N+注入的孢子较为完整、饱满且表面光滑,而经注入后的孢子表面粗糙,可见明显的刻蚀损伤和破壁现象,并且注入剂量越大,伤痕也越深宽.试验结果提示N+注入是一种有效的动物益生菌选育方法.     

Citric acid production by Aspergillus niger ATCC 9142 from a treated ethanol fermentation co-product using solid-state fermentation

Aims:  To investigate the ability of the citric acid-producing strain Aspergillus niger ATCC 9142 to utilize the ethanol fermentation co-product corn distillers dried grains with solubles for citric acid production following various treatments.
Methods and Results:  The ability of A. niger ATCC 9142 to produce citric acid and biomass on the grains was examined using an enzyme assay and a gravimetric method, respectively. Fungal citric acid production after 240 h was higher on untreated grains than on autoclaved grains or acid-hydrolysed grains. Fungal biomass production was enhanced after autoclaving and acid-hydrolysis of the grains. Phosphate supplementation to the grains slightly stimulated citric acid production while methanol addition decreased its synthesis. Using the phosphate-supplemented grains, the optimal incubation temperature, initial moisture content of the grains and the length of fermentation time for ATCC 9142 citric acid production were determined to be 25°C, 82% and 240 h, respectively.
Conclusions:  A. niger ATCC 9142 synthesized citric acid on corn distillers dried grains with solubles. The phosphate-treated grains increased citric acid production by the strain.
Significance and Impact of the Study:  The ethanol fermentation co-product corn distillers dried grains with solubles could be useful commercially as a substrate for A. niger citric acid production.     

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 +/- 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-guanidine (MNNG). Out of 3,2-deoxy-D-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 +/- 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 x 10(-5) M MgSO4 x 5H2O into the fermentation medium reduced the Fe2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 +/- 5 g/l).     

Improved production of citric acid by a diploid strain of Aspergillus niger

Aspergillus niger strain CGU 87 was treated with UV radiation and some auxotrophic mutants were obtained. These mutants were less productive than CGU 87, which produced an average of 7.4% citric acid. All possible crosses in pair wise combinations were carried out between these auxotrophs, and three heterokaryons were synthesised. Finally, one heterozygous diploid was isolated from each of them. These heterokaryons and diploids showed improved productivity when compared with their component parents, but except in one diploid D5, all others produced less citric acid than CGU 87. The yield of D5 exceeded that of CGU 87 by 1.2 times and it produced 9% citric acid. This is a significant improvement and the increased productivity seems to be the result of successful adaptation of D5 to its fermentation environment.     

Effect of volume of culture medium on enhanced citric acid productivity by a mutant culture of Aspergillus niger in stirred fermentor

AIMS: The present study deals with the effect of volume of culture medium on enhanced citric acid productivity by a mutant culture of Aspergillus niger. METHODS AND RESULTS: A laboratory scale stirred fermentor of 15-l capacity was employed for all microbial cultivations. Blackstrap molasses, a by-product of sugar industries is easily and abundantly available for its exploitation as a carbon source in fermentation processes. The parental culture of A. niger was improved by mutation using ultraviolet radiations and N-methyl N-nitro N-nitroso guanidine, i.e. mutagen MNNG. Six MUV and eight MNNG-treated mutant strains were isolated after extensive screening and optimization. Mutant strain of A. niger MNNG-2 showed enhanced citric productivity (87.60 g l-1) over the parental strain BTL-45 (19.53 g l-1) and other mutant derivatives (49.85 g l-1 citric acid in case of mutant MUV-5 and 76.82 g l-1 in case of mutant MNNG-7). The optimal sugar level was found to be 150 g l-1 (optimum volume of the medium, 60%) after 6 days of inoculation, which is economically significant. Specific productivity of the mutant culture MNNG-2 (qp = 0.057 g/g cells h-1) was several folds higher than other strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study are of commercial level. All kinetic parameters including yield coefficients and volumetric rates revealed the hyper-producibility of citric acid by mutant MNNG-2 using blackstrap molasses as the basal medium in stirred fermentor.     

Attempts at improving citric acid fermentation by Aspergillus niger in beet-molasses medium

Natural oils with high unsaturated fatty acids content when added at concentrations of 2% and 4% (v/v) to beet molasses (BM) medium caused a considerable increase in citric acid yield from Aspergillus niger. The fermentation capacities were also examined for production of citric acid using BM-oil media under different fermentation conditions. Maximum citric acid yield was achieved in surface culture in the presence of 4% olive oil after 12 days incubation.     

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACM 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30°C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe²⁺. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.