Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase genes in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A

The diversity of polyketide synthase (PKS) genes in Aspergillus ochraceus NRRL 3174 and Aspergillus carbonarius 2Mu134 has been investigated using different primer pairs previously developed for the ketosynthase (KS) domain of fungal PKSs. Nine different KS domain sequences in A. ochraceus NRRL 3174 as well as five different KS domain sequences in A. carbonarius 2Mu134 have been identified. The identified KS fragments were distributed in five different clusters on the phylogenetic tree, indicating that they most probably represent PKSs responsible for different functions.     

抗氯嘧磺隆黑曲霉乙酰乳酸合成酶（ALS）酶学特性研究

本文比较了不同黑曲霉（Aspergillus niger）菌株对大豆田除草剂氯嘧磺隆的耐药性,并且对抗性菌株中靶标酶乙酰乳酸合成酶（ALS）的酶学特性进行了研究。结果发现TR-H为抗性菌株,其ALS酶活性显著高于其它菌株。酶学特性分析显示,TR-H菌株的最佳培养时间为84h,最佳反应温度为35℃,最适反应pH为7.3,酶反应的初速度范围为0～60min。以丙酮酸钠为反应底物时,其米氏常数（Km）为39.41mmol/L,最大反应速度（Vmax）为232.2μg/mL·h。     

Quick guide to polyketide synthase and nonribosomal synthetase genes in Fusarium

Fusarium species produce a plethora of bioactive polyketides and nonribosomal peptides that give rise to health problems in animals and may have drug development potential. Using the genome sequences for Fusarium graminearum, F. oxysporum, F. solani and F. verticillioides we developed a framework for future polyketide synthases (PKSs) and nonribosomal peptides synthetases (NRPSs) nomenclature assignment and classification. Sequence similarities of the adenylation and ketosynthase domain sequences were used to group the identified NRPS and PKS genes. We present the current state of knowledge of PKS and NRPS genes in sequenced Fusarium species and their known products. With the rapid increase in the number of sequenced fungal genomes a systematic classification will greatly aid the scientific community in obtaining an overview of the number of different NRPS and PKS genes and their potential as producers of known bioactive compounds.     

Cloning and identification of rutin-degrading enzyme genes from Aspergillus niger in wheat Qu

Tartary Buckwheat Huangjiu, one of the oldest wines in China, has unique health-promoting properties. One of its specific ingredients is rutin, which has antioxidative and anti-ageing activities and plays important roles in the treatment of human diseases. During Tartary buckwheat Huangjiu brewing, however, rutin is degraded by fungi in wheat Qu, thereby leading to a decrease in the wine's nutritional value. The consequent aim of this study was to identify effective rutin-degrading fungi and degrading enzyme genes in the Tartary buckwheat Huangjiu brewing process. Among these fungi isolated from wheat Qu, only Aspergillus niger exhibited rutin-degrading ability. Following protein purification, SDS-PAGE, mass spectrometry, Blast analysis and clone verification, we ultimately identified glucoamylase and α-L-rhamnosidase encoding genes were related to rutin degradation. To our knowledge, this is the first report of rutin-degrading enzyme genes in Tartary buckwheat Huangjiu. Our results provide insights for regulating the nutritional value of Tartary buckwheat Huangjiu.     

Chemical Destruction of Aspergillus niger Conidiospores

SUMMARY: Destruction of A. niger conidiospores at 20°C (68°F) by 20 ppm NaClO and 20 ppm iodine as iodophor yielded D values of 0.61 min and 0.86, respectively at pH 3.0 and 1.31 and 2.04 min, respectively at pH 7.0. On the basis of mojar concentrations, iodine was slightly more effective than chlorine. A D value of 0.026 min was obtained with 4% NaOH at 60°C (140°F) indicating 4% NaOH at 60°C to be far more germicidal than 20 ppm of either halogen compound at 20°C. One per cent NaOH at 30°C resulted in an immediate and rapid release of amino acids presumably from the spore wall during the first 2 min of contact and a slower rate of release of RNA, with DNA released at the slowest rate.     

Antioxidants and pigments of Aspergillus niger

Extracts of six fungi, four yeasts, and two species of Streptomyces were tested for antioxidant activity when added to lard. Extracts of Aspergillus niger, the microorganism that showed the strongest antioxidant activity, were subjected to adsorption and gel permeation chromatography. Two fractions that protected lard against oxidation were obtained by chromatography of A. niger extract on Sephadex LH-20 and Bio-Beads S-X2. One of these fractions contained a gummy brown pigment BR, the other a bright yellow crystalline pigment Y. Pigment BR showed strong carbonyl group and methyl-methylene group absorption in the infrared. Apparently, on the basis of spectral data, pigment Y has a linear naphthopyrone structure. Pigment BR was obtained consistently from A. niger mycelium, while pigment Y was formed sporadically. Results indicated that more than one substance in A. niger mycelium have antioxidant properties and that brown and yellow pigments apparently are associated with antioxidant activity. Synergistic effects may be important in the strong antioxidant activity of A. niger extracts.     

Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR

Aspergillus carbonarius is an ochratoxin producing fungus that has been considered to be responsible of the ochratoxin A (OTA) contamination in grapes and wine. In order to monitor and quantify A. carbonarius, a specific primer pair Ac12RL_OTAF/Ac12RL_OTAR has been designed from the acyltransferase (AT) domain of the polyketide synthase sequence Ac12RL3 to amplify 141 bp PCR product. Among the mycotoxigenic fungi tested, only A. carbonarius gave a positive result. This specific primer pair was also successfully employed in real-time PCR conjugated with SYBR Green I dye for the direct quantification of this fungus in grape samples. A positive correlation (R(2)=0.81) was found between A. carbonarius DNA content and OTA concentration in 72 grape samples, allowing for the estimation of the potential risk from OTA contamination. Consequently, this work offers a quick alternative to conventional methods of OTA quantification and mycological detection and quantification of A. carbonarius in grapes.     

黑曲霉单宁酶发酵工艺

用黑曲霉Aspergilhus niger QG 0301进行单宁酶发酵，制得酶制剂。实验结果表明：Aspergillus niger QG 0301进行单宁酶发酵的适宜培养基包括：混合碳源（或玉米淀粉）、硫酸铵、磷酸二氢钾、碳酸钙、硫酸镁、单宁酸；在30℃、120r/min振荡培养5天，单宁酶产量平均为18．55u／mL。     

黑曲霉菌体制备壳聚糖的工艺研究

研究了黑曲霉不同发酵条件(发酵温度、发酵时间、发酵pH值、摇床转速)对壳聚糖产率的影响。并对培养基组成和培养条件进行了优化试验,结果表明,最佳培养液成分为70mL玉米浆,4g葡萄糖,1.2g硫酸镁,接入8.0×108个/mL孢子;培养条件发酵温度27℃~31℃,发酵时间72h,发酵pH值7.4~7.6,摇床转速130r/min。     

黑曲霉果胶酶产生条件的初步研究

本文报道了黑曲霉A3.1在麦麸固体培养基的产酶条件,在不同氮源试验中以(NH4)2SO4为最佳,该菌产酶的最佳条件为28℃,含水量45%,起始pH5.0,时间3d.     

黑曲霉产木聚糖酶的特性

研究了木聚糖酶产生菌黑曲霉(A.niger)238的产酶特性和麸曲的浸提条件,并对其浸提酶液进行浓缩.结果表明,菌株黑曲霉(A.niger)238产木聚糖酶受诱导物的影响,发酵时间在68～72h期间达到产酶高峰,酶活力为286U/mL.最佳氮源为(NH4)2SO4,装料量为每瓶10g,接种量为每瓶0.3mL.其发酵麸曲用固液比为1∶5的2g/dLCaCl2溶液,30℃浸提1.5h;浸提液采用25℃,40kPa,冷却水温度18℃条件超滤浓缩可获得90%以上回收率.     

黑曲霉产木聚糖酶的研究

筛选了 1株高产木聚糖酶的黑曲霉 (Aspergillusniger)An 2 3 8菌株 ,研究了其在固态培养基中的产酶条件。该菌株发酵曲中除含有木聚糖酶 5 117U /g(干曲 )外 ,还有纤维素酶 42 5U/g(干曲 ) ,果胶酶 12 3 6U/g(干曲 ) ,蛋白酶 2 2 5 3 1U/g(干曲 )。     

黑曲霉xj菌株发酵液的抗菌活性研究

目的:研究黑曲霉xj菌株发酵液的抗菌谱及抗菌活性的稳定性;方法:以常见的病原细菌作为测试菌株,滤纸片法测定xj菌株发酵液的抑菌谱,通过温度、光照以及pH值的变化测定发酵液中抗菌成分的稳定性;结果:xj菌株发酵液对5种病原细菌均有不同程度的抑菌活性,其中时金黄色葡萄球菌和根癌农杆菌的抑菌效果最好,抑菌圈直径分别达到42.14 mm和38.76mm,发酵液中的抗菌成分对光照稳定,但对温度及pH值不稳定;结论:xj菌株发酵液的抗菌范围较广,提取发酵液的抑菌成分时应尽量在低于80℃以下,pH中性环境中进行.     

黑曲霉泡腾片制备工艺优化

该研究采用粉末直接压片法制备黑曲霉（Aspergillus niger）泡腾片,以崩解时限、pH值、发泡量为评价指标,通过单因素试验和正交试验对黑曲霉泡腾片的工艺参数进行优化。结果表明,黑曲霉泡腾片的最佳制备工艺为酒石酸和碳酸氢钠比例1.00∶1.25、崩解剂添加量70%、黑曲霉孢子粉添加量15%、质量分数为10%的聚乙二醇6000乙醇溶液为润滑剂,添加量为3.15 g/mL,质量分数为2.5%为聚乙烯吡咯烷酮乙醇溶液为粘合剂,添加量为7 g/mL。在此优化条件下所得的黑曲霉泡腾片表面光滑,黏冲不明显,pH值为5.7,且崩解时限（23 s）、发泡量（40.79 mL/g）、硬度（4.3 kg）均符合2015版《中国药典》的规定,在水中可迅速崩解。     

代谢改造黑曲霉合成乙酰氨基葡萄糖

该研究以黑曲霉为底盘细胞,首先敲除N-乙酰氨基葡萄糖（N-acetylglucosamine,GlcNAc）摄取相关转运蛋白ngtA编码基因,获得GlcNAc的吸收利用缺陷型菌株,有利于胞外GlcNAc的积累。在此基础上,通过表达大肠杆菌来源的卤酸脱卤酶样磷酸酶编码基因yqaB,构建黑曲霉GlcNAc的完整合成途径,实现GlcNAc的合成,产量为1.78 g/L。通过共过表达氨基葡萄糖-6-磷酸乙酰转移酶基因gnaA和氨基葡萄糖-6-磷酸合成酶基因gfaA强化GlcNAc合成途径,GlcNAc的合成水平提升至3.64 g/L。为增加GlcNAc合成前体GlcNAc6P的胞内供给,利用RNA干扰技术对乙酰氨基葡萄糖-6-磷酸（GlcNAc6P）分解代谢途径中氨基葡萄糖-6-磷酸脱氨基酶基因nagB和乙酰氨基葡萄糖-6-磷酸脱乙酰酶基因nagA基因表达进行弱化表达,GlcNAc产量进一步提高至4.03 g/L。为减弱黑曲霉糖酵解途径与GlcNAc合成途径竞争共同前体物质果糖-6-磷酸,对磷酸果糖激酶基因pfkA进行弱化表达,GlcNAc的最终产量为4.61 g/L。     

糖化黑曲霉复壮方法的研究

采用透明圈法对糖化黑曲霉AS3.324和AS3.4309的复壮方法进行研究,选出了三种适合的筛选培养基,确定了具体的操作步骤.复壮后菌株的麸曲糖化酶活力分别比退化的出发菌株的麸曲糖化酶活力提高45%和44%.     

黑曲霉TNA-09中α-葡萄糖苷酶基因敲除的研究

以柠檬酸生产菌黑曲霉TNA—09为原始菌株,通过农杆菌介导的方法,将其α-葡萄糖苷酶基因敲除,得到基因敲除菌株TGA101。对原始菌株黑曲霉TNA-09和基因敲除菌TGA101 α-葡萄糖苷酶活力测定,结果相对于TNA—09菌株,TGA101菌株α-葡萄糖苷酶活力下降61．15％。在柠檬酸发酵试验中表现出相对于黑曲霉TNA-09,TGA101菌株发酵液中异麦芽糖含量降低84．67％,柠檬酸提高2．34％,糖酸转化率提高2．34％。     

黑曲霉VVTP84生产果糖转移酶

从产果糖转移酶的 11株菌株中筛选出一株黑曲霉VVTP84菌 .该菌株在含蔗糖的培养基中 ,最佳摇瓶发酵时间为 30h ,pH值为 7.0 ;当蔗糖质量浓度在 2 5 0g/L以内时 ,产酶与蔗糖质量浓度呈正相关 ;MgSO4 ·7H2 O和KH2 PO4 的添加量分别以控制在 1.5 g/L和 1.0 g/L为宜     

分光光度法测定黑曲霉孢子浓度的研究

采用分光光度计测定黑曲霉（Aspergillus niger）xj在不同生长期、不同稀释倍数下孢子悬液的OD600 nm值,对比血球板计数法得到的孢子悬液浓度,研究在对数期和稳定期OD600 nm值与孢子浓度之间的关系;采用微电影拍摄法观察黑曲霉xj产孢结构形成过程,并绘制固体发酵条件下的生长曲线。结果表明,在10～20倍稀释区间内,稀释倍数与OD600 nm值之间呈现良好线性关系（对数期R2 =0.984 8、稳定期R2 =0.991 3）,且OD600 nm值与孢子浓度之间也保持良好的线性关系（对数期R2 =0.995 3、稳定期R2 =0.993 6）;通过微电影拍摄法观察到黑曲霉xj产孢结构形成是分阶段进行的过程;在固体发酵过程中,孢子浓度呈现“S”型增长趋势。该研究为测定丝状真菌孢子浓度提供另一种可借鉴的方法。