Immobilization of Aspergillus niger Mycelium on Seeds for Glucoamylase Production

Mycelium of the glucoamylolytic mutant Aspergillus niger C-58-III was immobilized on wheat, rye, barley, pea, buckwheat and mustard seeds in repeated-batch flasks. After every 24th it was possible to obtain culture broth rich in glucoamylase. The highest yield of enzyme (66.4 U × ml⁻¹) was obtained on the mustard carrier. Immobilized cells were successfully reused with high level of enzyme formation being mantained for longer period (192h). Some of the variables influencing the enzymatic activity have been standardized. Enzyme productivity reached in immobilized cells of A. niger was 1.6-times higher in comparison with free cells.     

Synthesis of Glucoamylase by Somatic Diploids of Aspergillus niger in Submerged Culture Conditions

Out of twelve forced heterocaryons of A. niger, in nine cases somatic diploids were obtained comprising a total of 107 strains. All were initially examined with respect to general amylase activity in test-tube microcultures. Over 21% of all diploid recombinants reached a higher coefficient of amylase activity than the parent haploid highly active in this respect. Four most active diploids selected from this group were examined with regard to glucoamylase synthesis. Also in this case diploids were characterized by a certain predominance in their activity over the initial prototrophic haploid strain (maximally about 16%).     

Improvement of Aspergillus niger Glucoamylase Thermostability by Directed Evolution

Directed evolution was used to improve the thermostability of Aspergillus niger glucoamylase (GA) expressed in Saccharomyces cerevisiae. A starch‐plate assay developed to screen GA mutants for thermostability gave results consistent with those of irreversible thermoinactivation kinetic analysis. Several thermostable multiply‐mutated GAs were isolated and characterized by DNA sequencing and kinetic analysis. Three new GA mutations, T62A, T290A and H391Y, have been identified that encode GAs that are more thermostable than wild‐type GA, and that improve thermostability cumulatively. These individual mutations were combined with the previously constructed thermostable site‐directed mutations D20C/A27C (forming a disulfide bond), S30P, and G137A to create a multiply‐mutated GA designated THS8. THS8 GA is substantially more thermostable than wild‐type GA at 80°C, with a 5.1 kJ/mol increase in the free energy of thermoinactivation, making it the most thermostable Aspergillus niger GA mutant characterized to date. THS8 GA and the singly‐mutated GAs have specific activities and catalytic efficiencies (k_cat/K_m) similar to those of wild‐type GA.     

Glucoamylase Biosynthesis by Cells of Aspergillus niger C58-III Immobilized in Sintered Glass and Pumice Stones

A simple method of A. niger C_58-III cell immobilization is described. This strain produces extracellular glucoamylase. According to the proposed method A. niger spores were first immobilized by adsorption in sintered glass Rasching rings (RR) or pumice stones (PS). Growing out from spores, A. niger cells produced extracellular glucoamylase. This technique facilitates the culture growth in a filamentous spongy structure of the supports with a continuous accumulation of biomass. After every 24 h it was possible to obtain culture liquid rich in glucoamylase. This procedure can be repeated 30 times using the same sample of immobilized A. niger culture without any loss of glucoamylase activity in the liquid medium. In a 96 h period immobilized A. niger cells produced 300 units × ml⁻¹ whereas a shake culture of this fungus produced only 186 units × ml⁻¹.     

黑曲霉固态发酵产糖化酶的研究

以糖化酶活力为评价指标,对1株黑曲霉变异株固态发酵产糖化酶的培养基组成和发酵条件进行了研究,分别考察了碳源、氮源、原料粉碎度、培养基含水量、温度、起始pH值、发酵时间、接种量等对该菌株固态发酵产糖化酶的影响.结果表明,该菌株产糖化酶的最佳发酵培养基组成为:麦麸:豆饼粉=3:1,(NH4)2SO4含量为3%,K2HPO40.1%.原料粉碎度为过60目筛,起始pR5.0,培养基含水量为120%,培养温度为32℃,接种量为每瓶培养基接108/mL的孢子悬液2.5mL,培养时间为72h,最高产酶活力达17800U/g.     

固定化黑曲霉生产柠檬酸动力学研究

本文研究了柠檬酸发酵过程动力学，建立了固定化黑曲霉生产柠檬酸的动力学模型，用最优化梯度法估算了模型参数，仿真运算结果表明所建立的数字模型能较好地描述实际发酵过程。     

高糖化酶活菌株的选育及其在山西老陈醋酿造中的应用

采用N~+注入、氯化锂-紫外线复合诱变方法对糖化酶生产菌株黑曲霉As3.4309反复进行诱变处理,获得了1株高糖化酶活的突变株IV5-66,其糖化酶活力为出发菌株的3.5倍,该菌株具有无霉腐味、不产生色素等特点,具有较高的应用价值。建立了利用该突变株制备高糖化酶活麸曲的工艺,并将高糖化酶活麸曲与我们已经构建的产酒生香酵母菌共培养液共同添加到山西老陈醋的酒醪发酵中,使淀粉利用率提高了27%,乙醇产量提高了34%,乙酸乙酯产量提高了1倍,酒醪的产量与质量均得到了大幅度的提高。     

不同碳源对黑曲霉产糖化酶活力的影响

碳源对黑曲霉产糖化酶活力有很大影响。研究可溶性淀粉、葡萄糖、蔗糖、麦芽糖、乳糖和β-环糊精为碳源时,分别对黑曲霉产糖化酶活力的影响。结果表明,可溶性淀粉为碳源时酶活力最高,最高可达350．34±14．06U／ml;其次是葡萄糖;蔗糖和麦芽糖为碳源时酶活力没有显著性差异;乳糖为碳源时酶活力低于蔗糖和麦芽糖;β-环糊精的酶活力最低。不同碳源发酵培养基中生物量研究表明,黑曲霉能充分利用可溶性淀粉、葡萄糖、蔗糖和麦芽糖,对乳糖的利用较差,几乎不能利用β-环糊精。     

Continuous Production of Glucose from Dextrin by Glucoamylase Immobilized on Porous Silica

Glucoamylase was covalently attached to porous silica particles and investigated for its applicability as an industrial catalyst in the production of glucose from starch hydrolysates. Reactivity at various reaction conditions, as well as enzyme loading, reaction kinetics, diffusional effects, and thermal stability, were determined in laboratory scale experiments. Pilot scale tests on continuous production of glucose were performed in a 1 cubic foot packed column reactor that could produce approximately 1,000 lbs/d glucose at 40°C. The maximum glucose concentration (based on dissolved solids) varied from 87 to 93% depending on the dextrose equivalent (D.E.) and degree of retrogradation of the feed dextrin. In 80 days of continuous operation no appreciable enzyme deactivation was observed. Initial sterilization of the reactor and continuous heat sterilization of the feed stream (120°C, 3–4 min) virtually ensured operation of the immobilized enzyme reactor at low levels of contamination, typical bacteria counts of the product effluent being 30–50/ml. Occasional interruption of the system resulted in much higher levels of microorganisms but did not affect the overall enzyme reactivity.     

黑曲霉糖化酶基因在荧光假单胞菌中的表达

黑曲霉糖化酶基因cDNA按正确的读码框架克隆到广宿主表达载体pBBR1MCS-2,构建出含黑曲霉糖化酶基因的表达载体pBBR1MCS-2GA,用三亲和接合转化法将重组质粒导入2-酮基-D-葡萄糖酸高产菌株荧光假单胞菌AR4。得到重组菌ARW4,经SDS-PAGE凝胶电泳和Westernblot分析显示,黑曲霉糖化酶基因在ARW4中得到表达,表达产物主要以不溶性包涵体形式存在。     

黑曲霉高产糖化酶的分子水平研究方法概论

黑曲霉是一株产糖化酶的特殊菌株,而糖化酶在制造葡萄糖、氨基酸、抗生素等发酵工业上有着广泛的应用。以黑曲霉出发菌株为研究材料,基因改造作为研究背景,通过综述诱变育种、基因敲除、RNA干扰、基因重组表达等分子改造技术,将改造后的菌株与出发菌株生产糖化酶的产量及活性分别进行测定评估。以此探讨高产糖化酶的黑曲霉分子改造的优良手段,为以后设计新的效率更好的高产糖化酶的黑曲霉分子改造研究提供技术支持。     

Scanning Electron Microscopy of Wheat Starch. IV. Digestion of large Granules by Glucoamylase of Fungal (Aspergillus niger) Origin)

Starch granules attacked by glucoamylase of fungal origin show a pattern of erosion which is different from that resulting from wheat alpha-amylase attack. The glucoamylase pattern, which is characterised by a relatively uniform erosion of the surface is discussed in comparison with that of alpha-amylase and possible reasons for the differences are suggested.     

固定化黑曲霉增殖细胞生产低聚果糖的研究

黑曲霉孢子经海藻酸钙包埋３３ｈ后得到的固定化增殖细胞其酶活性达到最高，机械强度仍保持较高水平（为起始时８０％），最适ｐＨ为５．０，与游离酶相同，最适温度为５５℃，比游离酶高５℃；有害离子对其影响较小，其Ｋｍ（以蔗糖为底物）为８２ｍｍｏｌ．将固定化增殖细胞装入填充式反应柱，产品中低聚果糖含量在５０％～５５％，操作一个月活性保持不变。     

Production of Citric Acid from Beet Molasses by Immobilized Cells of Aspergillus niger

Citric acid was produced from beet molasses by immobilized cells in shake flasks and glass bioreactor. Maximum concentration of citric acid (35 g/L) was observed from immobilized A. niger cells in shake flasks after 28 days fermentation. In repeated batch fermentations, the A. niger cells entrapped in Ca-alginate gel beads retained ability to produce citric acid for up to 84 days.     

碳源对固定化黑曲霉生产柠檬酸影响的研究

以海藻酸钙为载体包埋固定化黑曲霉AspergillusnigerW1-2细胞，用于生产柠檬酸，研究了碳源（蔗糖、葡萄糖和乳糖）及其浓度对固定化细胞生产柠檬酸的影响。结果表明，最适宜的碳源为蔗糖，其次为葡萄糖，固定化细胞利用乳糖时只生成少量柠檬酸。利用固定化细胞生产柠檬酸时，最适蔗糖浓度为120g／l，较利用游离细胞低（140g／L），并且，蔗糖浓度对固定化细胞生产柠檬酸的影响较利用游离细胞更为明显。     

Glucoamylase Produced by Submerged Culture of Aspergillus oryzae

Aspergillus oryzae grown in wheat bran submerged culture produced three forms of glucoamylase, designated as glucoamylase I, II and III. They were obtained in the ratio of 51: 3: 1. Glucoamylase I readily hydrolyzed soluble starch to above 90% and had a strong debranching activity. Glucoamylase II and III hydrolyzed soluble starch to about 75% and had weak debranching activity. Glucoamylase I was further purified by rechromatography, gel filtration, preparative isoelectric focusing on carrier ampholyte and again gel filtration. Glucoamylase I was fairly stable at pH-range between 5.5 to 7.0 and temperature up to 40°C. The limit of hydrolysis of soluble starch was 80% and the liberated product was only glucose.     

黑曲霉H9-30全细胞催化合成低聚异麦芽糖

以黑曲霉H9-30全细胞为催化剂转化麦芽糖生产低聚异麦芽糖,通过单因素实验确定其摇瓶最佳转化条件为:温度48℃,初始p H值4. 2,麦芽糖质量浓度为600 g/L,黑曲霉细胞添加量为15 g/L。此条件下,有效三糖(异麦芽糖、潘糖和异麦芽三糖)占总糖质量分数的50. 5%,总低聚异麦芽糖含量为63. 3%,达到低聚异麦芽糖工业生产标准。研究结果表明,利用黑曲霉全细胞催化生产低聚异麦芽糖具有较好的操作稳定性,生产IMO-50的半衰期达到20批次(10 d),有望应用于工业化生产低聚异麦芽糖。     

Intensification of Amylase Synthesis with Aspergillus niger by Way of Multistage Mutagenization

Conidia of amylolytically active Aspergillus niger C strain were subjected to four-stage mutagenization, using different combinations of mutagens in each stage (stage I – UV irradiation + ethyleneimine, stage II – UV irradiation + nitrosomethylurea, stage III – UV irradiation + N-methyl-N′-nitro-N-nitrosoguanidine, stage IV – acryflavine). In all, 378 strains were isolated after mutagenization, which were initially evaluated for the total amylolytic activity by the method of test-tube microculture. Nine most active mutants were then tested in submerged culture determining the activity of glucoamylase and α-amylase in post-culture liquid. The activity of α-amylase increased from over 7 to nearly 31%, and that of glucoamylase from 4 to over 61%.