单宁酶基因在黑曲霉ST31中的克隆与表达

利用PCR扩增得到米曲霉(Aspergillusoryzae)单宁酶(tannase)基因的编码序列,经DNA测序证实单宁酶基因已成功克隆,然后将其连接到黑曲霉的表达载体ANED2-SP2上构建单宁酶基因表达载体。将构建好的单宁酶基因表达载体通过原生质转化法导入黑曲霉菌株ST31中进行表达研究。结果表明重组菌株的单宁酶活力最高为104.02U/ml发酵液,比原始出发菌株米曲霉提高2~3倍。研究构建了黑曲霉的高效转化体系,提高了黑曲霉表达系统的应用水平,为其它新酶的研究提供有价值的参考。     

黑曲霉(Aspergillus niger)产菊粉酶菌株的筛选及培养条件的研究

Ak3个地区7个土壤样品中筛选高产菊粉酶的黑曲霉菌株,对筛选出的黑曲霉菌株进行了形态鉴定,探讨了温度、pH值对菊粉酶活力的影响,比较了固体和液体两种扩大培养法对黑曲霉菌株产菊粉酶的影响。结果表明：经过初筛和复筛得到A4、A6、A8、A12、At4、A15和A16共7株黑曲霉菌株,菊粉酶活力大于15．0U／mL,经数码生物摄影显微镜镜检符合黑曲霉的形态特征。A8菌株的菊粉酶酶活最高,达到25．0U／mL,在60℃、pH值5．0的条件下酶活性最大。     

强启动子glaA介导cbhB基因在黑曲霉中的表达

黑曲霉纤维素酶三类不同酶系中,纤维二糖水解酶基因表达处于很低水平,导致纤维素酶总体活力水平不高。为构建黑曲霉纤维素酶高产菌株,采用基因工程方法,全基因合成拼接黑曲霉高表达葡萄糖淀粉酶基因glaA的强启动子片段与纤维二糖水解酶基因cbhB编码区片段,然后将杂合基因克隆到二元载体pCAMBIA1301上,重组质粒通过农杆菌介导转化黑曲霉分生孢子,携带杂合基因的T-DNA片段插入到黑曲霉转化子的染色体上,共筛选到48个具有潮霉素抗性的转化子。纤维素酶活力水平测定结果显示,转化子A3-9的CMC酶活力最高,为野生型黑曲霉菌株的1.31倍;转化子B1-7与A3-6的滤纸酶活力最高,为野生型黑曲霉菌株2.51倍。另外,初步分析了杂合基因在黑曲霉中的表达所需的诱导条件。     

去壁酶与酶解方式对曲霉原生质体释放的影响

研究了纤维素酶、蜗牛酶、溶菌酶以及菌丝体培养方式和酶解方式对黑曲霉和米曲霉菌丝释放原生质体的效应。发现黑曲霉菌丝原生质体制备最佳条件为固体透析培养菌丝体,2%纤维素酶,在平皿中,28℃和80r/min条件下酶解3h;米曲霉原生质体制备最佳条件为2%纤维素酶+1%蜗牛酶+5mmol/L二硫苏糖醇,酶解时间6h,其它条件与黑曲霉的相同。     

聚氨酯泡沫固定化黑曲霉P-6021可同时产果胶酶和澄清果汁

以多孔聚氨酯泡沫(PUF)固定化黑曲霉P-6021可以实现菌丝体的摇瓶重复间歇发酵产酶,重复5个批次,菌体数目增多,产酶量上升,发酵液酶活达到664u/ml。以PUF固定化黑曲霉P-6021进行了同时产果胶酶和澄清苹果汁试验,以浑浊苹果汁基质发酵12h后, 产酶量可达到643u/ml,苹果汁基本澄清,透光率提高,粘度降低。表明固定化黑曲霉可以同时产果胶酶和澄清苹果汁,实现产酶与澄清过程的耦合。     

黑曲霉DB056产柚苷酶发酵条件初步优化

以α-鼠李糖苷酶活力和柚苷酶活力为考察指标, 研究了发酵条件对黑曲霉DB056产柚苷酶的影响。结果表明: 发酵温度、菌丝形态、培养基初始pH、接种量和装液量对黑曲霉DB056产柚苷酶都具有重要影响。初步优化得到黑曲霉DB056产柚苷酶的条件为: 培养基初始pH 8.0, 玻璃珠添加个数5个, 装液量45 mL, 接种量7%, 发酵温度34°C, 摇床转速190 r/min。采用此条件进行发酵, 高效液相色谱法检测α-鼠李糖苷酶活力最大可达1076.32 U/mL, 柚苷酶活力最大可达420.68 U/mL, 分别比初始条件提高了72.35%和78.03%。黑曲霉DB056不仅在对数生长期能快速合成柚苷酶, 在稳定期及衰亡期也会不断地分泌柚苷酶。阐明了发酵条件对黑曲霉DB056产柚苷酶的影响并获得了经过初步优化的发酵条件, 为进一步优化发酵条件, 提高黑曲霉DB056产柚苷酶的产量奠定了良好的基础。     

聚氨酯泡沫固定化黑曲霉P-6021可同时产果胶酶和澄清果汁*

以多孔聚氨酯泡沫(PUF)固定化黑曲霉P-6021可以实现菌丝体的摇瓶重复间歇发酵产酶,重复5个批次,菌体数目增多,产酶量上升,发酵液酶活达到664u/ml。以PUF固定化黑曲霉P-6021进行了同时产果胶酶和澄清苹果汁试验,以浑浊苹果汁基质发酵12h后, 产酶量可达到643u/ml,苹果汁基本澄清,透光率提高,粘度降低。表明固定化黑曲霉可以同时产果胶酶和澄清苹果汁,实现产酶与澄清过程的耦合。     

黑曲霉在生物质废弃物资源化中的应用

生物质废弃物因其量大、结构和组分复杂,需要多种酶进行协同作用达到将其资源化利用的效果。黑曲霉能产生水解生物质的纤维素酶、半纤维素酶、木质素酶和果胶酶等多种酶类,可以在生物质资源化中发挥重要作用。本文中,笔者综述了黑曲霉自身产酶及其作为细胞工厂的作用,介绍了黑曲霉利用生物质废弃物产酶和高附加值产品的研究进展,以期为生物质资源化提供参考。     

纤维素酶产生菌和糖化酶产生菌液体混合发酵的研究

应用正交试验设计研究了糖化酶高产菌株黑曲霉UV-11(Aspergillus niger UV-11)和纤维素酶高产菌株黑曲霉F_(27)(A.niger F_27)液体混合发酵条件。结果表明,发酵液糖化酶活力为6500U/ml,CMC酶活力为99mg葡萄糖/ml·h。与UV-11单菌发酵相比,糖化酶活力提高16％,CMC酶活力提高266％。     

黑曲霉htmA基因的分离鉴定及其功能分析

本研究通过分析比较黑曲霉基因组与人、哺乳动物和酿酒酵母基因组序列同源性,首次分离鉴定了黑曲霉htmA基因,该基因长3459bp,编码1083个氨基酸。已知真核生物的htmA基因编码一种类α-甘露糖苷酶I的非必须蛋白HTMA,在内质网中参与降解非正确折叠的糖蛋白,htmA基因的破坏会延迟非正确折叠糖蛋白的降解。为分析htmA基因在黑曲霉中的功能,运用同源重组技术敲除黑曲霉基因组中的htmA基因,获得htmA基因缺失突变菌株,并进行了缺失株外源漆酶分泌能力的检测。结果表明黑曲霉htmA基因的破坏延缓了外源漆酶的降解,由此推测黑曲霉htmA基因编码蛋白HTMA具有与酵母、哺乳动物HTM1P/EDEM类似的功能作用。     

N+注入选育黑曲霉益生菌及其突变菌株产酶条件的研究

以益生菌株黑曲霉AN01为材料,经N 多次诱变得突变益生菌株AN03。结果表明,出发益生菌株AN01酸性蛋白酶、纤维素酶和果胶酶的酶活分别由原来的71.6Ug、141.7Ug和264.8Ug相继提高到996.5Ug、940.4Ug和906.5Ug。突变益生菌株AN03经传5代培养,产酶特性稳定。试验还研究了变突变益生菌株AN03最佳产酶条件,培养基为每升含麸皮105g,玉米芯105g,豆粕105g,氯化铵16g,pH5.0。30℃培养4d。     

Purification and properties of NADP(+)-dependent glycerol dehydrogenases from Aspergillus nidulans and A. niger

Glycerol dehydrogenase, NADP(+)-specific (EC 1.1.1.72), was purified from mycelium of Aspergillus nidulans and Aspergillus niger using different purification procedures. Both enzymes had an Mr of approximately 38,000 and were immunologically cross-reactive, but had different amino acid compositions and isoelectric points. For both enzymes, the substrate specificity was limited to glycerol and erythritol for the oxidative reaction and to dihydroxyacetone (DHA), diacetyl, methylglyoxal, erythrose and D-glyceraldehyde for the reductive reaction. The A. nidulans enzyme had a turnover number twice that of the A. niger enzyme at pH 6.0, whereas inhibition by NADP+ was less (Ki = 45 microM vs 13 microM). It is proposed that both enzymes catalyse in vivo the reduction of DHA to glycerol and that they are regulated by the anabolic reduction charge.     

黑曲霉作为细胞工厂:知识准备与技术基础

黑曲霉是一种重要工业微生物,在酶制剂、异源蛋白、有机酸等领域应用广泛。2007年黑曲霉基因组的公布将黑曲霉的研究引入后基因组时代,各种组学数据如雨后春笋般涌现,人们对黑曲霉高效生产机制的理解上升到系统、分子层次;与此同时,黑曲霉遗传操作系统也不断成熟,为系统地研究和改造黑曲霉、将黑曲霉打造成通用细胞工厂奠定了基础。     

Exploiting unusual affinity of usual polysaccharides for separation of enzymes on fluidized beds

Two polysaccharides, alginate and chitosan, showed unusual affinity and bound alpha-amylase (from various sources) and Aspergillus niger cellulase, respectively. The beads prepared from these polymers were successfully used for the purification of the respective enzymes by fluidized bed affinity chromatography. alpha-amylase from wheat germ could be purified by 58-fold with about 90% recovery of activity. Aspergillus niger cellulase, on the other hand, was purified by 30-fold with 80% recovery of enzyme activity. Both purified preparations show single band on SDS-PAGE.     

Influence of dissolved oxygen concentration on intracellular pH for regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor

In this paper we report the regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor. For this, the influence of dissolved oxygen concentration in a medium on intracellular pH values and consequently on overall microbial metabolism was emphasized. Intracellular pH of mycelium grown under different concentrations of dissolved oxygen in the medium was determined. Sensitivity of proteins toward proton concentration is well recognized, therefore pH influences on the activities of key regulatory enzymes of Aspergillus niger were determined at pH values similar to those detected in the cells grown under lower dissolved oxygen concentrations. The results have shown significantly reduced specific activities of hexokinase, 6-phosphofructokinase and glucose-6-phosphate dehydrogenase in more acidic environment, while pyruvate kinase was found to be relatively insensitive towards higher proton concentration. As expected, due to the reduced specific activities of regulatory enzymes under more acidic conditions, overall metabolism should be hindered in the medium with lower dissolved oxygen concentration which was confirmed by detecting the reduced specific growth rates. From the studies, we conclude that dissolved oxygen concentration affects the intracellular pH and thus growth rate of Aspergillus niger during the fermentation process.     

Enzymes with new biochemical properties in the pectinolytic complex produced by Aspergillus niger MIUG 16

A comprehensive study on the purification and characterization of pectinolytic enzymes produced by Aspergillus niger MIUG 16 on raw materials solid-state fermentation is reported. Five pectinolytic enzymes were purified using a combination of chromatographic techniques. The properties of these homogenous enzymes were analyzed. The purified enzymes were classified with respect to their biochemical properties and substrate specificity. Among these proteins, one revealed polygalacturonase activity, another appeared to be a pectin methylesterase and three were identified as pectate lyases. The capacity of the fungus A. niger to produce pectate lyases with optimum pH in acidic domain was reported for the first time.     

Taxonomy and significance of black aspergilli

Members of Aspergillus section Nigri (formerly A. niger group) are distributed worldwide and are regarded as common food spoilage fungi. Some of them are widely used and studied for industrial purposes. They are common sources of extracellular enzymes and organic acids to be used in food processing and are also used in the production of traditional foods, especially in the Orient. Products produced by strains of Aspergillus niger hold the GRAS (Generally Recognised As Safe) status from the FDA. However some species in Aspergillus section Nigri can produce ochratoxin A, a nephrotoxic mycotoxin. In spite of their industrial importance, the taxonomy of black aspergilli ( Aspergillus section Nigri ) is not clear and many attempts have been made in order to find suitable taxonomic criteria. The aim of this paper is to provide an overview of the significance of black aspergilli focusing on all the approaches made in the taxonomy of this group of fungi. Some species, such as A. carbonarius and uniseriate species can be easily recognised. In the A. niger aggregate, although speciation at molecular level has been proposed, no morphological differences can be observed and species identification will therefore remain problematic. Phylogenetic analyses of ITS and 5.8S rDNA gene region of representative black Aspergillus species and a simple key to the most common species that can be easily distinguished by morphological criteria are also included.     

Characterization of a polyketide synthase in Aspergillus niger whose product is a precursor for both dihydroxynaphthalene (DHN) melanin and naphtho-γ-pyrone

The genome sequencing of the fungus Aspergillus niger uncovered a large cache of genes encoding enzymes thought to be involved in the production of secondary metabolites yet to be identified. Identification and structural characterization of many of these predicted secondary metabolites are hampered by their low concentration relative to the known A. niger metabolites such as the naphtho-γ-pyrone family of polyketides. We deleted a non-reducing PKS gene in A. niger strain ATCC 11414, a daughter strain of A. niger ATCC strain 1015 whose genome was sequenced by the DOE Joint Genome Institute. This PKS encoding gene we name albA is a predicted ortholog of alb1 from Aspergillus fumigatus which is responsible for production of the naphtho-γ-pyrone precursor for the 1,8-dihydroxynaphthalene (DHN) melanin/spore pigment. Our results show that the A. nigeralbA PKS is responsible for both the production of the spore pigment precursor and a family of naphtho-γ-pyrones commonly found in significant quantity in A. niger culture extracts. The generation of an A. niger strain devoid of naphtho-γ-pyrones will greatly facilitate the elucidation of cryptic biosynthetic pathways in this organism.