米曲霉pyrG 基因克隆及其同源转化系统的建立

建立以乳清酸核苷-5＇- 磷酸脱羧酶基因(pyrG)为选择标志,以米曲霉为宿主菌的同源转化系统。以米曲霉基因组为模板,通过PCR 扩增获得pyrG 基因,将该片段与表达载体pMD18-T 相连,转化大肠杆菌DH 5α,经蓝白斑筛选、PCR 快速筛选、酶切和测序验证,获得重组质粒pMD-pyrG,完成pyrG 基因的克隆。序列分析表明该基因与米曲霉KBN616 的pyrG 基因编码序列的同源性为99.9%,两者经推导的氨基酸的同源性为99.6%。以米曲霉 pyrG 营养缺陷株为受体菌,通过PEG/CaCl2 诱导的原生质体转化方法,将重组质粒导入该受体菌,使米曲霉pyrG 缺陷株发生基因转化,成为pyrG+,由此成功建立了以pyrG 为筛选标记基因、同型米曲霉pyrG 基因缺陷株为受体菌的基因转化系统。     

A novel amine oxidase-encoding gene from Aspergillus oryzae

We cloned a novel gene (aoxA) encoding amine oxidase (AOX) from Aspergillus oryzae. One cDNA clone showing extreme homology to the AOX-encoding genes was found in an expressed sequence tag (EST) library of A. oryzae. Molecular analysis revealed that the aoxA carried four exons interrupted by three introns and had an open reading frame encoding 672 amino acid residues. The deduced amino acid sequence showed about 83.5% identity to the Aspergillus niger AO-I. The strictly conserved residues for co-factor and copper binding in copper/quinine-containing AOXs were also preserved at Tyr 405, His 456, His 458 and His 617 in the cDNA sequence. When the aoxA was overexpressed in the homologous hyperexpression system of A. oryzae, AOX activity in the transformant was enhanced 75-fold. An apparent molecular weight of 159,000 by gel filtration and a subunit molecular weight of 75,000 by SDS-PAGE of the purified enzyme were estimated, suggesting that the enzyme molecule is a homo-dimer similar to other copper/quinine-containing AOXs. The A. oryzae AOXA preferentially oxidized aliphatic monoamines of C2-C6 rather than aromatic amines or diamines. From these results, the aoxA gene product obtained by homologous hyperexpression system of A. oryzae is undoubtedly a functional AOX.     

Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae

Isoamyl alcohol oxidase (IAAOD) is a novel enzyme that catalyzes the formation of isovaleraldehyde, which is the main component of mureka that gives sake an off-flavor (Yamashita et al. Biosci. Biotechnol. Biochem., 63, 1216-1222, 1999). We cloned the genomic DNA sequence encoding IAAOD from a koji mold, Aspergillus oryzae, using a PCR-amplified DNA fragment corresponding to the partial amino acid sequences of the purified protein as a probe. The cloned gene comprises 1903 bp of an open reading frame with three putative introns and encodes 567 amino acids with a presumed signal peptide consisting of 24 amino acids at the N-terminus. Moreover, nine potential N-glycosylation sites were present. Homology search on amino acid sequence showed that IAAOD has a region significantly similar to those conserved in FAD-dependent oxidoreductases. Southern hybridization analysis revealed that the cloned gene exists as a single copy in the A. oryzae RIB 40 chromosome. The cloned gene was overexpressed under the control of the amyB promoter in A. oryzae. The isovaleraldehyde-producing activity in the culture supernatant of one transformant was over 800 times as high as that of transformant with the control vector. This result demonstrates that the cloned gene encodes IAAOD. We named this novel alcohol oxidase gene "mreA".     

Cloning and functional analysis of the Aspergillus oryzae conidiation regulator gene brlA by its disruption and misscheduled expression

We cloned the brlA gene from Aspergillus oryzae genomic DNA using the A. nidulans brlA gene as a probe. A 3.1-kb EcoRI-BalI genomic DNA fragment was cloned and sequenced. The deduced amino acid sequence revealed 70% identity with A. nidulans BRLA and contained two C2H2 zinc finger motifs in its carboxyl terminus, and the promoter sequence contained a 43-bp highly conserved region, indicating that the cloned gene was an A. oryzae homologue of A. nidulans brlA. Disruption of the brlA gene by homologous recombination resulted in the loss of ability to form conidiophores. These results suggest that the brlA gene of A. oryzae plays a fundamental role in controlling conidiophore development. When the brlA gene was expressed under the control of the amyB promoter in A. oryzae transformants, highly differentiated and compact colonies were observed on a solid medium. The misscheduled expression of the brlA gene in submerged culture, in which conidiation does not normally occur, caused the development of complex conidiophore structures with vesicles, phialides and conidia.     

菌种耦合发酵米渣生酱油的品质和抗氧化活性

以高盐稀态法酿造米渣生酱油,探究菌种耦合对其品质和抗氧化活性的影响。4组不同菌种耦合发酵的米渣生酱油品质测定结果表明其各项质量指标均达到GB 18186—2000《酿造酱油》中一级酱油的标准,其中S4生酱油(米曲霉耦合黑曲霉和鲁氏酵母发酵)游离氨基酸总量达到41.81 g/L,4种米渣生酱油的总氨基酸、必需氨基酸、呈味氨基酸和抗氧化氨基酸的质量浓度均高于同时发酵的大豆生酱油S5生酱油(米曲霉发酵)。S3生酱油(米曲霉耦合鲁氏酵母发酵)和S4生酱油的抗氧化活性普遍高于S1生酱油(米曲霉发酵)和S2生酱油(米曲霉耦合黑曲霉发酵),各结果均表明S3的抗氧化活性最强,清除羟自由基的能力达到等质量浓度VC的最高175.42倍,对1,1-二苯基-2-三硝基苯肼自由基清除能力达到VC的2.16倍。抗氧化活性受发酵温度影响明显,分别在35℃和50℃发酵时出现峰值。多菌种耦合发酵对米渣生酱油品质和抗氧化活性均有明显改善,其中鲁氏酵母发挥了明显作用。     

Expression of Aspergillus oryzae phytase gene in Aspergillus oryzae RIB40 niaD(-)

Aspergillus oryzae RIB40 niaD(-) was transformed using a plasmid constructed with the A. oryzae phytase gene and pNAN8142 vector. The culture broth of the transformant, which was grown in a medium containing starch as a carbon source and polyvinylpyrrolidone showed phytase activity of a maximum of 2.0 units ml(-1) at 37 degrees C, pH 5.5.     

DCE-01菌株果胶酯酶基因克隆与表达

从麻类脱胶高效菌株DCE-01中克隆果胶酯酶基因并进行原核表达。根据全基因组测序注释结果设计引物,PCR扩增果胶酯酶基因连接到pEASY-E1载体上,导入大肠杆菌进行诱导表达,采用水解圈法进行选择和定量分析。结果表明:克隆出全长1107bp的果胶酯酶基因（GenBank登录号:KC422449）,编码368个氨基酸;该基因表达蛋白质序列的前26个氨基酸为信号肽,前导蛋白的分子量约为39.6ku,成熟蛋白为36.9ku,pI为9.1;基因工程菌株以高度酯化橘子果胶为底物的发酵液粗酶活为1.5IU/mL,是原始菌株DCE-01的22.4倍。本研究成功发掘出果胶酯酶基因,其表达产物果胶酯酶能降解高甲氧基果胶,在低甲氧基果胶制备方面具有重要应用前景。      

两种微生物水解脱淀粉小麦中蛋白效果的比较研究

为研究枯草芽孢杆菌（Bacillus subtilis）和米曲霉（Aspergillus oryzae）对脱淀粉小麦中蛋白质的水解效果,采用前发酵和后发酵获得发酵液,比较两种微生物培养过程中性蛋白酶活力和发酵液的pH值、可溶性无盐固形物、氨基酸态氮含量、氨基酸组成,并对其发酵液进行感官评价。结果表明,在最适前发酵工艺的条件下,枯草芽孢杆菌和米曲霉中性蛋白酶活力分别为1 060.82 U/g和908.02 U/g;两种微生物最终发酵液pH值为7.08和5.37,可溶性无盐固形物为15.60%和18.98%,氨基酸态氮含量为0.73 g/100 mL和0.75 g/100 mL,总游离氨基酸含量为4.35 mg/mL和9.04 mg/mL,蛋白质水解度为55.99%和59.71%。枯草芽孢杆菌发酵液中必需氨基酸的组成和比例更符合氨基酸模式谱,必需氨基酸与总游离氨基酸的比值（EAA/TAA）、必需氨基酸与非必需氨基酸（EAA/NEAA）的比值也高于米曲霉。感官评价结果显示,米曲霉发酵液好于枯草芽孢杆菌。     

Isolation and characterization of a novel gene encoding alpha-L-arabinofuranosidase from Aspergillus oryzae

We cloned and characterized a novel gene (abfA) encoding alpha-L-arabinofuranosidase (alpha-L-AFase) from Aspergillus oryzae. One clone homologous to the alpha-L-AFase gene of Thermotoga maritima was found in an expressed sequence tag (EST) library of A. oryzae and a corresponding gene was isolated. Molecular analysis showed that the abfA gene carried six exons interrupted by five introns and had an open reading frame encoding 481 amino acid residues. The amino acid sequence similarity at active sites to the alpha-L-AFases from other organisms indicated that the alpha-L-AFase encoded by abfA was classified as a family 51 glycoside hydrolase. When the abfA was overexpressed in the homologous hyperexpression system of A. oryzae, a large amount of alpha-L-AFase was produced as intracellular protein. The apparent molecular mass of the purified enzyme was estimated to be 228,000 by gel filtration and that of its subunit as 55,000 by SDS-PAGE, suggesting that the enzyme is a tetramer. The enzyme hydrolyzed p-nitrophenyl-alpha-L-arabinofuranoside but not other p-nitrophenyl glycosides. These results demonstrated that the abfA gene encodes a functional alpha-L-AFase.     

一种米曲霉蛋白酶的分离纯化及酶解特性研究

对米曲霉固态发酵所产蛋白酶分离纯化,采用硫酸铵盐析、DEAE-FF层析、Butyl-HP层析和Superdux 7510/300GL凝胶层析得到一种电泳纯的蛋白酶,SDS-PAGE显示分子量大小为27 ku左右。以酪蛋白为底物时,该蛋白酶Km=1.23 g·L-1,Vm=27.03μg·m L-1·min-1,最适反应条件为50℃,p H9.0。该蛋白酶对酪蛋白水解活性最高,而对牛血清蛋白的水解活性很低;对牛胰岛素B链上-Phe-Val-,-Cys-Gly-,-Glu-Ala-和-Arg-Gly-组成的肽键有较强的切割能力,酶切位点较多,对疏水性氨基酸具有较高的选择性,为米曲霉所产蛋白酶在食品上的应用提供有力的参考。      

Identification of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene.

The partial sequences of the mitochondrial (mt) cytochrome b gene (402 bp) were determined for species of Aspergillus section Flavi. On the basis of identities of DNA sequences, 77 strains were divided into seven DNA types, from D-1 to D-7. The type strains of A. sojae, A. parasiticus, A. flavus and A. oryzae together, A. tamarii, and A. nomius were placed in DNA types D-1. D-2, D-4, D-5 and D-7, respectively. These species could be differentiated from each other. Furthermore, two other DNA types, D-3 and D-6 were found. DNA type D-3 was closely related to A. parasiticus (D-2) and included one strain that deposited as A. flatus var. flavus and produced aflatoxins B and G. DNA types D-6 included one strain named A. flavus and closely related to A. tamarii. The observations of conidial wall texture by SEM (Scanning Electron Microscopy) supported the relationships derived from the cytochrome b gene. The production of aflatoxins was also examined. Using the DNA sequence of cytochrome b gene, several strains were reidentified. The derived amino acids sequences were all the same in the studied strains. The mt cytochrome b gene is useful and reliable in distinguishing and identifying the species in Aspergillus section Flavi.     

两株米曲霉菌种的发酵性能研究

将2株米曲霉扩大培养后用于生产试验,并将其发酵酱油的性能进行对比。结果表明,米曲霉1号酱油的氨基酸态氮、pH值、A530 nm、总氮低于米曲霉2号菌,总酸高于米曲霉2号;米曲霉2号酱油的游离氨基酸总含量为6.05 g/100 mL,是米曲霉1号（4.18 g/100 mL）的1.45倍;米曲霉1号酱油中醇类物质、酸类物质和醚类物质相对含量分别达61.53%、14.16%和1.26%,高于米曲霉2号的57.3%、13.07%和0.46%,而酯类物质（3.73%）、酮类物质（1.83%）和醛类物质（16.33%）低于米曲霉2号的3.96%、1.95%和21.31%;米曲霉1号酱油香气浓郁偏醇香味,米曲霉2号酱油香气浓郁偏酯香;米曲霉2号酱油的加热沉淀达到204 mm2,远高于米曲霉1号酱油的25 mm2。加热后过滤可去除米曲霉2号酱油中的沉淀。     

耐盐米曲霉CICIM F0899在酱油酿造过程中的应用

耐高盐环境的蛋白酶对酱油工业有着积极作用。该实验对1株产耐盐蛋白酶的米曲霉CICIM F0899在高盐稀态酱油发酵过程中的作用进行了研究,并同沪酿3042进行了比较。结果表明,在酱醪发酵阶段,微生物代谢产酸使酱醪pH持续下降,影响了蛋白酶的活性,导致酱油中氨基态氮和风味物质含量的变化,在180天时氨基态氮含量达到0.62 g/100 mL,比沪酿3042高11.11%。利用气相色谱-质谱联用(GC-MS)对挥发性成分进行了分析鉴定,检测出酱醪中7大类共49种挥发性化合物,其中酯类、杂环类化合物的含量明显高于沪酿3042,而醛类物质的含量相对较少。因此,米曲霉CICIM F0899在氨基态氮含量和风味组成方面都具有更加明显的优势。     

柑橘皮渣废物利用发酵生产单细胞蛋白饲料

传统柑橘果实加工后剩下的大量柑橘皮往往被随意丢弃,对环境造成污染的同时也造成浪费。利用其中的纤维素及果胶类物质可以提供碳源生产单细胞蛋白,并提高其中关键氨基酸（苏氨酸、甲硫氨酸、赖氨酸）的产量,以及促进单细胞蛋白在作为饲料时的吸收效率。经单因素实验可使关键氨基酸的总产量最大:苏氨酸、甲硫氨酸、赖氨酸分别为2.03×10-3、3.58、1.49×10-2g/L,共3.60g/L,占总氨基酸14.77%,优化后的培养基为60g/L柑橘皮渣、21g/L尿素,接种量为20%（V/V）的混菌（黑曲霉∶啤酒酵母=1∶1）,最优发酵时间为5d。      

2 种米曲霉发酵酱油风味物质比较

为进一步比较传统菌株米曲霉沪酿3.042和前期实验诱变菌株米曲霉100-8酿造酱油的差异。通过高效液相色谱、气相色谱-质谱联用仪比较酱油中有机酸、氨基酸和风味物质成分的差异。通过发酵终期酱油有机酸的结果比较发现：米曲霉100-8发酵的酱油中苹果酸含量增加,柠檬酸和琥珀酸含量下降。通过酱油发酵在大曲阶段和后期阶段的氨基酸比较发现,使用米曲霉100-8菌株发酵的酱油大曲及其后期发酵为酱油阶段,天冬氨酸、谷氨酸和精氨酸这3 种氨基酸含量都明显升高。这些有机酸和氨基酸含量之间比例的差异性是造成酱油风味不同的原因之一。对风味物质成分的分析发现：与对照相比,米曲霉100-8发酵的酱油中醇、醛、酸和吡嗪类物质都有所增加,其中风味物质增多最明显的是吡嗪类。通过米曲霉100-8酿造的酱油与传统米曲霉沪酿3.042比较,其有机酸、氨基酸和风味物质都有明显的不同。     

高产高纯度果胶甲酯酶黑曲霉工程菌的构建

为获得一株高产高纯度的果胶甲酯酶的黑曲霉工程菌,提高果胶甲酯酶的产量,从果胶酶生产菌种中克隆了果胶甲酯酶基因pmeA,通过同源重组的原理,冻融法转化农杆菌、农杆菌介导法转化黑曲霉方法,成功构建了分泌表达果胶甲酯酶的纯合重组菌株TH-2（glaA::pmeA）。基本发酵培养基中发酵第9 d上清中最高酶活达到467.77 U/mL。进一步敲除重组菌株TH-2（glaA::pmeA）中背景蛋白酸稳定的α-淀粉酶的编码基因asaA,获得纯合重组菌株TH-2（glaA::pmeA?pyrG?asaA）。该菌株在添加1%的硫酸铵的发酵培养基中培养7 d后,发酵液上清中主要的背景蛋白均消失。但是与纯合重组菌株TH-2（glaA::pmeA）相比,果胶甲酯酶表达量有所下降,最高酶活为255.40 U/mL。重组果胶甲酯酶的最适作用温度为50 ℃,适合的温度范围是40~80 ℃,在80 ℃下仍能维持其酶活性的70%以上,适合的pH范围是3.0~5.0,最适pH为4.0。最终获得了一株温度和pH作用范围较宽的高产高纯度果胶甲酯酶的黑曲霉工程菌。     

Cloning and functional analysis of the orotidine-5'-phosphate decarboxylase gene (PbrURA3) of the pathogenic fungus Paracoccidioides brasiliensis

A genomic clone encoding the Paracoccidioides brasiliensis orotidine monophosphate decarboxylase gene (PbrURA3) was isolated by screening a subgenomic plasmid DNA library of this fungus, using a PCR amplification product of the gene as a probe. Sequence analysis revealed that the gene contains an open reading frame of 855 bp with a single intron (162 bp), and encodes a putative 285 amino acids polypeptide of estimated molecular weight 31.1 kDa and isoelectric point 6.5. The deduced amino acid sequence predicted a 73.4% identity with orotidine monophosphate decarboxylase of Aspergillus nidulans. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae ura3 null mutant.