白曲酸性蛋白酶在无孢黑曲SH-2中的克隆表达及酶学性质分析

白曲霉酸性蛋白酶在白酒酿造发酵过程中具有溶解发酵原料的颗粒,促进微生物繁殖,分解蛋白质生成香味物质,降解酵母菌体蛋白等多种功能,可以提高白酒风味,因此被广泛应用于白酒生产中。本研究利用经基因工程改造的低内源蛋白背景的黑曲霉宿主SH-2,表达白曲霉酸性蛋白酶基因pepB。通过PCR技术获得pepB以及表达元件糖化酶启动子PglaA、糖化酶终止子TglaA、乳清酸核苷-5-磷酸脱羧酶标记基因pyrG,在pMD18-T载体基础上,构建了pepB表达载体,通过PEG介导转化法转化无孢黑曲酶SH-2。重组菌株经发酵罐发酵240 h,发酵粗酶液酶活达9722 U/mL,是报道的白曲原始菌株酶活的8.5倍,SDS-PAGE结果显示表达产物分子量约为47 ku。酶学性质分析结果表明,该酸性蛋白酶最适反应温度为35℃,最适pH为4.0,Mn~(2+)、Cu~(2+)对酶活有显著地激活作用。最后,探究了在不同发酵初始pH下重组菌株酶活的变化,结果显示,在pH 4.5~6.5范围内,适当提高发酵初始pH,酸性蛋白酶酶活会提高。以上结果表明,本研究成功构建了一株能胞内高效表达白曲酸性蛋白酶的菌株。     

一株产复合酶真菌菌株的筛选、鉴定及发酵产酶研究

从土壤样品中分离获得一株产复合酶的真菌菌株,将其命名为HKS11,进行了菌株形态特征观察,并在此基础上进行了分子生物学鉴定,经PCR扩增后测定菌株的18S rDNA基因测序,由18S rDNA基因序列比较分析,得知菌株HKS11与黑曲霉菌(Aspergillusniger)和泡盛曲霉(Aspergillus awamori)亲缘关系最为接近,18S rDNA基因相似性达到99%以上,可初步判断为曲霉属(Aspergillus)。结合形态特征观察综合比较分析后,将其鉴定为曲霉属(Aspergillus)的黑曲霉菌(Aspergillus niger)。其18S rDNA GenBank中登录序列号为GenBankJX112703。并对其发酵产酶进行了研究。     

Digestion of DNA regions to discriminate ochratoxigenic and non-ochratoxigenic strains in the Aspergillus niger aggregate

Aspergillus strains belonging to the Aspergillus niger aggregate, either isolated from Italian grapes or received from public collections, were analysed in order to discriminate between the ochratoxin A (OTA) producing and the non-producing strains by means of the analysis of Internal Transcribed Spacers (ITS), Intergenic Spacers (IGS) and of a beta-tubulin gene portion. A. niger and Aspergillus awamori were identified observing the macro- and microscopic features of the colonies and the strains ochratoxigenicity was evaluated through Thin Layer Chromatography and/or High Performance Liquid Chromatography. PCR amplification of ITS, IGS and beta-tubulin gene portion produced 600, 440 and 550 bp amplicons, respectively, in all the analysed strains. The digestion of the IGS amplicon with Hinf I and of the other two amplicons with Rsa I, revealed one main profile type ("T-C-C") associated with the A. niger aggregate strains which lack the ability to produce OTA.     

Detection of ochratoxin A in animal feeds and capacity to produce this mycotoxin by Aspergillus section Nigri in Argentina.

Ochratoxin A (OA) is a mycotoxin detected in a variety of food and feeds mostly from countries with a temperate climate because of the fungi that produce it, mainly Aspergillus ochraceus and Penicillium verrucosum. In Argentina, there is no available information about the natural occurrence of OA and ochratoxigenic fungi from feedstuffs. The aim was to evaluate the natural occurrence of OA in poultry, pig and rabbit feeds over 8 months. Likewise, the capacity to produce OA by Aspergillus section Nigri was investigated. Mycotoxin analysis showed that in some months of sampling, OA was detected in three feeds. OA was found in 38% of the poultry feed samples tested with levels ranging from 25 to 30 ng g(-1). From rabbit feed samples, 25% contained OA and the levels ranged from 18.5 to 25 ng g(-1). Only 13% of the pig feed samples were contaminated with similar levels of toxins. Ninety-four black Aspergillus strains from feedstuffs were tested for OA production. Among these, the tested species were A. niger var. niger, A. niger var. awamori, A. japonicus var. japonicus, A. japonicus var. aculeatus and A. foetidus. For the detection of OA, three methodologies were applied: the two TLC methods used for the fast screening of the filamentous fungi for the production of OA were not sensitive enough to detect OA in any of the black Aspergillus strains. When an HPLC methodology was used, the results showed that 46% of the black Aspergillus strains were producers of OA, with levels ranging from 13 to 25 ng ml(-1) culture medium. The highest percentage of ochratoxicogenic strains was isolated from rabbit feeds with 100 and 78% of A. niger var. niger and A. niger var. awamori, with mean levels of 15.5 and 14.6 ng ml(-1), respectively. From pig feeds, 61% of the A. niger var. awamori were producers of this toxin with mean levels of 16 ng ml(-1). In poultry feeds, the lowest percentage of OA producer strains was detected. The results for the occurrence of OA in feeds from different sampling months depended on storage and humidity-temperature conditions. Therefore, a good storage practice becomes very important to prevent OA production     

黑曲霉在造纸白水循环中的应用

在造纸白水封闭循环过程中,回用白水含有大量的溶解和胶体物质,这些物质大量的聚集会给纸的性质以及纸机的运行产生不良影响。采用商品酶处理白水成本较高,而使用黑曲霉发酵产酶来处理白水能有效降低成本。黑曲霉可以根据培养基的不同而产不同的组合酶,     

Extracellular soluble polysaccharide (ESP) from Aspergillus kawachii improves the stability of extracellular beta-gluocosidases (EX-1 and EX-2) and is involved in their localization

Aspergillus kawachii produces two extracellular beta-glucosidases (EX-1 and EX-2) and one cell-wall-bound beta-glucosidase (CB-1), all of which are derived from the same bglA gene. Extracellular beta-glucosidases (EX-1 and EX-2) are stable in the crude solution form, but become unstable in the purified form under moderate conditions (pH 5.0 and 37 degrees C). Purified extracellular beta-glucosidases can bind to a mycelial cell wall fraction, even though these enzymes are released into the medium under solid culture conditions. A. kawachii produces an extracellular soluble the beta-glucosidases over the pH range of 3.0-7.0 and at temperatures below 50 degrees C. ESP directly interacted with the purified extracellular beta-glucosidases but did not affect the K(m) values of these enzymes. Moreover, ESP inhibited the adsorption of purified extracellular beta-glucosidases to the cell wall fraction and extracted them from it. These results that ESP plays important roles in the stability and localization of extracellular beta-glucosidases. ESP from A. kawachii directly binds to the enzymes and releases them to the medium from the cell wall layer and then stabilizes them.     

Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L

The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone was isolated and sequenced, within which an ORF of 1425 by encoding a protein of 475 as with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted as sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.     

Water activity and temperature effects on growth of Aspergillus niger, A. awamori and A. carbonarius isolated from different substrates in Argentina

The objectives of this study were to determine the effect of water activity, temperature, and their interactions on a) mycelial growth rate and b) the lag phase prior to grow of seven isolates of Aspergillus section Nigri isolated from peanuts, maize kernels, dried grapes and coffee cherries from Argentina. Three Aspergillus niger, three A. awamori and one A. carbonarius isolates examined showed optimum a(W) level for growth at 0.97 with optimal temperature of 30 degrees C. for most of the isolates and 25 degrees C for only one (A. awamori RCP176). Minimal a(W) for growth was 0.85 at the highest temperature tested. Overall growth was reduced up to 50% at 0.93 a(W). Growth was also to a large extend inhibited at 0.85 a(W) for most isolates even after 21 days of incubation at temperatures lower than 30 degrees C. The analysis of variance of the effect of single (isolate, a(W) and temperature), two- and three-way interaction showed that all factors alone and all interactions were statistically significant (P<0.001) in relation to growth rates and lag phase for A. niger, A. awamori and A. carbonarius isolates. These data are relevant since these species are isolated in high frequency on numerous substrates for human and animal consumption in Argentina.     

A second pectin lyase gene (pel2) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene products

A second pectin lyase gene, designated pel2, was isolated from a shoyu koji mold Aspergillus oryzae KBN616 and characterized. The structural gene comprised 1306 bp with three introns. The ORF encoded 375 amino acids with a signal peptide of 19 amino acids. The deduced amino acid sequence showed high similarity to those of A. oryzae Pel1, Aspergillus niger pectin lyases and Glomerella cingulata Pn1A. The pel2 gene was overexpressed under the control of the promoter of the A. oryzae TEF1 gene for purification and enzymatic characterization of its gene product. The gene product exhibited two molecular masses of 48 and 44 kDa due to different degrees of glycosylation. Both proteins had the same pH optimum of 6.0 and temperature optimum of 50 degrees C.     

亮氨酸氨肽酶LapA在无孢黑曲霉中的重组表达优化及酶学性质

针对目前国产食品级亮氨酸氨肽酶的工业生产产量不高的现状,将米曲霉、黑曲霉和酱油曲霉的5 个亮氨酸氨肽酶基因（lapA、lap1O、lap2、lap1S、lap1N）在低蛋白背景的无孢黑曲霉HL-1中进行重组表达,通过信号肽替换对其编码区进行改造,利用杂合启动子PnaII及营养缺陷标记pyrG构建表达载体,并基于规律间隔成簇短回文重复序列（clustered regularly interspaced short palindromic repeats,CRISPR）/Cas9工具设计亮氨酸氨肽酶的基因组定点整合策略,获得高活力的亮氨酸氨肽酶表达菌株,重组菌株LapA-C的亮氨酸氨肽酶活力达到11 701.2 U/mL,比未利用CRISPR工具的LapA-T重组表达菌株的酶活力（2 476.0 U/mL）提高了约3.7 倍。此外,通过融合6×His标签实现了重组亮氨酸氨肽酶LapA的纯化,并对其进行了酶学性质研究：蛋白质大小约为35.0 kDa,重组亮氨酸氨肽酶LapA最适pH值为8.5,最适温度为65 ℃。综上所述,本研究基于CRISPR策略成功实现了亮氨酸氨肽酶在黑曲霉中的高效重组表达。     

Cloning of a gene encoding a highly stable endo-beta-1,4-glucanase from Aspergillus niger and its expression in yeast

A gene encoding an endo-beta-1,4-glucanase, which is highly resistant to high temperature, protease and surfactant treatment, was isolated from Aspergillus niger IFO31125 and designated as eng1. The deduced amino acid sequence encoded by eng1 showed high homology with the sequence of a not-well-characterized cellulase encoded by eglB which has not yet been shown to be a stable enzyme. To confirm the sequence of the gene encoding the highly stable endo-beta-1,4-glucanase, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The enzyme retained 56% of the initial activity after 1 h of incubation at 80 degrees C and was stable in the range of pH 3.0-10.0. The optimal temperature for enzyme activity was 70 degrees C and the optimal pH was 6.0. The enzyme was highly protease-resistant and retained more than 80% of the initial activity after protease treatment for 3 d at 40 degrees C. The enzyme was also resistant to various surfactants. From these results, eng1 was confirmed to encode a very stable endo-beta-1,4-glucanase.     

白曲霉用于食醋糖化过程的研究

通过正交试验确定了白曲霉SICC3.917三角瓶与糖化曲的最佳培养基成分及最佳培养条件、糖化醪液的最佳制备工艺。结果表明白曲霉的三角瓶最佳培养条件为：麸皮添加量8.5g,玉米粉添加量1.5g,水添加量10.5g,培养温度30℃。白曲霉SICC3.917糖化曲的最佳培养条件为种曲接种量0.5%,熟料含水量52%,培养温度32℃,空气湿度90%。糖化醪液的最佳制备工艺为白曲霉糖化曲的添加量1.3%,大米粉浓度20.7%,糖化温度65℃,糖化时间3h。     

黑曲霉转化系统的优化及重组菌株高效筛选

黑曲霉(Aspergillus niger)是食品工业中重要的生产菌株,广泛应用于酶和有机酸生产.为提高黑曲霉遗传操作效率,对黑曲霉转化及重组菌株筛选策略进行优化.基于已报道的最佳转化条件对黑曲霉AG11进行电转化、农杆菌介导和聚乙二醇(polyethylene glycol,PEG)介导转化(Agrobacteriu...     

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.     

Some distinguishable properties between acid-stable and neutral types of alpha-amylases from acid-producing koji

The highly humid climate of Japan facilitates the growth of various molds. Among these molds, Aspergillus oryzae is the most important and popular in Japan, and has been used as yellow-koji in producing many traditional fermented beverages and foods, such as Japanese sake, and soy sauce. Taka-amylase A (TAA), a major enzyme produced by the mold, is well known worldwide to be a leading enzyme for industrial utilization and academic study, since many extensive studies have been carried out with TAA. In southern Kyushu, the other koji's of citric acid-producing molds have often been used, such as in the production of a traditional distilled liquor of shochu. The koji molds black-koji and white-koji produce two types of alpha-amylase, namely, acid-stable (AA) and common neutral (NA). The latter enzyme is enzymatically and genetically similar to TAA. In this review, we investigate AA from three molds, Aspergillus niger, A. kawachii and A. awamori, and the yeast Cryptococcus sp. regarding the distinguishable properties between AA and NA. (i) The N-terminus amino acid sequences of AA determined by molecular cloning started with the sequence of L-S-A-, whereas those of NA started with A-T-P-. (ii) Most of the full sequences of AA were composed of, besides a core catalytic domain, an extra domain of a hinge region and a carbohydrate binding domain, which could be responsible for raw-starch-digestibility. The AA from A. niger has no exceptionally extra domain, similarly to NA. (iii) Simple methods for distinguishing AA from NA using CNP-alpha-G3 and G5 as substrates were developed by our group. (iv) The number of subsite in AA on the basis of its cleavage pattern of maltooligosaccharides was estimated to be five, which differs from that of TAA, 7-9. AA has many advantages in industrial applications, such as its acid-stability, thermostability, and raw-starch digesting properties.     

Polymerase chain reaction-mediated characterization of molds belonging to the Aspergillus flavus group and detection of Aspergillus parasiticus in peanut kernels by a multiplex polymerase chain reaction

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.