响应面法优化超声微波联合提取杏鲍菇中麦角硫因工艺

为优化杏鲍菇中麦角硫因的提取工艺,以麦角硫因含量为指标,研究干燥方式、液料比、乙醇浓度、微波条件和超声条件等单因素对提取杏鲍菇中麦角硫因的影响,并在单因素实验的基础上使用响应面法优化杏鲍菇中麦角硫因的提取工艺。结果表明:真空冷冻干燥后杏鲍菇中麦角硫因含量为(0.20±0.02)mg/g湿重,显著高于热风干燥所得的(P<0.05),但与新鲜杏鲍菇的没有显著性差异(P>0.05),这表明真空冷冻干燥处理能很好地保留杏鲍菇中的麦角硫因;结合单因素试验和响应面分析,确定超声微波联合提取杏鲍菇中麦角硫因的最佳工艺条件为液料比48:1(mL/g)、53%乙醇、微波条件(500 W,65℃,5 min)、超声条件(450 W,5 min),在此优化条件下,获得麦角硫因的含量为(3.79±0.02)mg/g干重,与预测值误差在1%以内。本研究所得到的优化工艺条件具有一定的可行性,可为食用菌中麦角硫因的提取提供参考,具有广阔的应用前景。     

营养因子强化麦角硫因生物合成的研究

以糙皮侧耳CGMCC 6232菌丝体深层发酵制备麦角硫因,通过向发酵培养基中添加不同种类及浓度的无机盐、维生素、生长调节剂、有机酸、氨基酸等营养因子,考察对麦角硫因合成积累的影响,研究可能的前体氨基酸对麦角硫因生物合成的影响规律。结果表明:添加氯化铵、硝酸铵、叶酸、VB1、吲哚丁酸、丙酮酸、柠檬酸、谷氨酸、蛋氨酸、半胱氨酸、组氨酸和甜菜碱对麦角硫因的合成有一定的促进作用,其中以发酵第4天向发酵液中添加15 mmol/L蛋氨酸最利于麦角硫因的合成,发酵液中麦角硫因的含量达192.4 mg/L,比对照组(102 mg/L)提高了88.6%。     

正交设计优化平菇下脚料中麦角硫因的提取工艺

利用正交试验优化平菇下脚料中麦角硫因的回流提取工艺。通过高效液相色谱法,Kromasil100-5NH_2柱(250mm×4.60 mm,5μm),流动相乙腈-5 mmol/L醋酸铵(80∶20,体积比),254 nm下进行检测,建立麦角硫因的含量测定方法,以平菇下脚料中麦角硫因的含量及得率为评价指标。在单因素试验的基础上采用正交设计方法 L_9(3~4)考察提取时间、提取次数和料液比对麦角硫因提取效果的影响。结果表明平菇下脚料中麦角硫因的最佳提取工艺条件为:提取溶剂10%乙醇,料液比1∶6(g/m L),提取时间为2 h,提取2次。通过验证试验,麦角硫因的总提取率可达87.45%,含量可达0.5%。     

麦角硫因机体内的分布与代谢和其在疾病中的作用研究进展

麦角硫因是一种稀有的天然氨基酸类强抗氧化剂,由放线菌、蓝细菌、部分真菌（例如链霉菌、分枝杆菌等）和蘑菇等微生物合成。动物（包括人类）和植物自身不能在体内合成麦角硫因,植物可通过根系从土壤中吸收微生物合成的麦角硫因,而动物可从食物中吸收和积累麦角硫因。人体通过有机阳离子转运蛋白1型从食物中吸收麦角硫因,其广泛分布于人体的细胞和组织中。已有大量研究报道了麦角硫因的合成、自然界中的分布、化学性质、生理功能和其作为抗氧化剂的特性。尽管研究证明麦角硫因在体外具有明显的抗氧化活性和细胞保护作用,但对其在体内的生理功能研究仍有限。本文旨在综述麦角硫因的独特性质、生物合成以及预防疾病的潜力。     

8种食用菌营养全接触

检测单位：甘肃农业大学食品科学与工程系 样品种类：平菇、香菇、黑木耳、金针菇、双孢菇、茶树菇、杏鲍菇、白灵菇（木耳为干品,其他为鲜品）样品来源：茶树菇、杏鲍菇、白灵菇由甘肃某食用菌中心提供,其他均购于甘肃省兰州市农贸市场     

乙酰辅酶A含量对酿酒酵母乙酸乙酯合成的影响

通过缺失乙酰辅酶A水解酶（ACH）基因和过表达乙酰辅酶A合成酶（ACS）基因技术提高酿酒酵母合成乙酰辅酶A（acetyl- CoA）能力的同时,过表达醇酰基转移酶（ATF）基因,提高乙酸乙酯合成能力,并考察acetyl-CoA含量对酿酒酵母合成乙酸乙酯能力的影响。结果表明,敲除ACH1基因、且在敲除ACH1基因基础上过表达ACS1、ACS2基因均能提高酿酒酵母Acetyl-CoA含量,进而提高乙酸乙酯含量。较亲本菌株α5,缺失突变株α5ΔACH1、重组菌株α5-A1、α5-A2的Acetyl-CoA的含量均分别提高了52.5%、80.33%、52.79%,乙酸乙酯含量分别提高10.59%、26.12%、23.70%。在敲除ACH1基因、过表达ACS1和ACS2基因的基础上同时过表达ATF1基因,得到工程菌株A1-ATF1和A2-ATF1,较亲本菌株α5,乙酸乙酯含量分别提高226.09%、530.43%、289.57%,工程菌株A1-ATF1乙酸乙酯产量最高,为72.52 mg/L。研究表明,提高乙酰辅酶A含量能够促进乙酸乙酯的合成,为提高乙酸乙酯生成量提供了新思路。     

杏鲍菇麦角硫因的体外抗氧化力及环境因素对其稳定性的影响

研究杏鲍菇麦角硫因体外抗氧化力及环境因素对其稳定性的影响。分析杏鲍菇麦角硫因羟自由基清除力等体外抗氧化力和对鱼肉的保鲜效果,并测定不同环境下其DPPH自由基清除力的变化。在所设置条件下,杏鲍菇麦角硫因具有较强的羟自由基清除力、DPPH自由基清除力和ABTS自由基清除力,最高分别为64. 26%、96%和89. 28%,而FRAP抗氧化力较弱(接近0);对贮藏鱼肉较叔丁基苯二酚(tert-butyl hydroquinone,TBH)有更强的护色、维持p H、减缓蛋白质氧化及酸败腐变等效果; DPPH自由基清除力受酸性、K+/Mg2+等金属离子、氧化剂或还原剂等环境因素影响显著降低(P <0. 05),受温度影响不显著(P> 0. 05)。杏鲍菇麦角硫因体外抗氧化力总体较强,其稳定性易受部分环境因素影响。结果可为其在各领域的开发应用提供理论依据。     

优化前体供给与细胞膜通透性强化大肠杆菌合成麦角硫因

麦角硫因（Ergothioneine,ERG）具有抗氧化、抗炎、辐射保护等多种生理功能,广泛应用于食品、医药和化妆品领域。微生物发酵生产麦角硫因具有极大潜力,然而,由于工程菌株前体供应不充足等因素,麦角硫因的发酵质量浓度以及生产强度低下。作者旨在探究麦角硫因生产不同阶段中的前体限制,并通过优化前体供应解除限制,进而提高大肠杆菌麦角硫因合成能力。在证明组氨酸是限制麦角硫因合成的关键因素基础上,通过优化组氨酸供给,麦角硫因在摇瓶水平的发酵质量浓度达到175.81 mg/L;通过正交试验组合优化了半胱氨酸、柠檬酸铁胺和维生素B6的添加量,发酵质量浓度提高至182.61 mg/L;通过优化甲基供体的添加（1.5 g/L甲硫氨酸与0.6 g/L甜菜碱）以及通过添加0.2 g/dL的CaCl2提高细胞膜通透性,麦角硫因的发酵质量浓度最终提高至243.06 mg/L。采用分批补料发酵培养108 h后,麦角硫因在3 L发酵罐上的发酵水平达到2.01 g/L,生产强度为18.61 mg/（L·h）,为麦角硫因的规模化工业生产奠定了基础。     

酿酒酵母和大肠杆菌鸟嘌呤脱氨酶基因的克隆、原核表达及活性测定

与植物体内合成路径不同,微生物体内合成咖啡碱存在一条以黄嘌呤为底物,利用鸟嘌呤脱氨酶催化鸟嘌呤生成黄嘌呤有效合成咖啡碱的新途径。为克隆鸟嘌呤脱氨酶的基因,构建可高效合成黄嘌呤的原核表达载体并对外源蛋白活性进行检测,分别以酿酒酵母和大肠杆菌为研究材料,根据GenBank中酿酒酵母和大肠杆菌中鸟嘌呤脱氨酶基因gud1和egud序列设计引物,聚合酶链式反应特异扩增其基因片段,将目的基因连接至pMAL-c5X载体,转入大肠杆菌BL21（DE3）中诱导蛋白表达,并用高效液相色谱法鉴定其目的蛋白的催化活性。结果表明重组载体pMAL-gud1、pMAL-egud均可用来合成黄嘌呤,且GUD1比EGUD合成黄嘌呤的效率更高。研究结果将进一步丰富黑茶加工技术理论,同时为体外构建高效咖啡碱生物工程菌提供理论支持。     

酿酒酵母转氨酶Ⅰ基因的克隆及序列分析

利用酿酒酵母YT0801的总DNA为模板,采用PCR技术克隆出转氨酶I基因的DNA序列,利用生物信息学工具对其核酸序列和蛋白序列进行分析。测序结果表明DNA序列含有一个1503bp的开放阅读框,编码500个氨基酸,推测等电点为5.81,相对分子质量为56.2ku。同源性比对结果显示,该基因及推测的氨基酸序列与已报道的ARO8基因(Gen Bank Accession No:NM 001181067.1)的同源性均为100%。此外,对其理化性质、信号肽、疏水性、亲水性、二级结构和三级结构进行了分析。该基因的成功克隆,为其功能研究和生物合成2-苯乙醇提供了分子基础。     

中国被毛孢分离鉴定及其PKS、NRPS功能基因的多样性

探究中国被毛孢(Ophiocordyceps sinensis)遗传、聚酮合成酶(Polyketide synthase,PKS)、非核糖体多肽合成酶(Non-ribosomal peptide synthase,NRPS)的多样性,为寻找合成生物活性物质的潜在菌株奠定基础。采用组织分离法从冬虫夏草中分离获得一株具有较强抑菌活性的真菌ZG4-23,采用多基因系统分析的方法对该菌株进行鉴定;利用兼并性引物对菌株ZG4-23的PKS基因和NRPS基因进行PCR扩增及序列测定分析,构建系统发育树,明确菌株ZG4-23的PKS基因序列和NRPS基因序列的系统进化地位。结果表明,多基因序列分析显示,菌株ZG4-23与多个中国被毛孢菌株同源性高达99%,因此确定该菌株为中国被毛孢(O. sinensis)。菌株基因组含有PKS I、NRPS 基因,经比对,PKS I片段与已知Ophiocordyceps sinensis PKS序列相似度为99%,NRPS片段与Trichophyton rubrum NRPS序列相似度为76%。中国被毛孢具有较强合成生物活性次生代谢产物的潜在能力,值得进一步开发研究和应用。     

Molecular cloning and characterization of a glucan synthase gene from the human pathogenic fungus Paracoccidioides brasiliensis

1,3-beta-D-glucan is a fungal cell wall polymer synthesized by the multi-subunit enzyme 1,3-beta-D-glucan synthase. A subunit of this integral membrane protein was first described as the product of the FKS1 gene from Saccharomyces cerevisiae using echinocandin mutants. Other FKS1 genes were also reported for Candida albicans, Aspergillus nidulans and Cryptococcus neoformans. Here, we report the nucleotide sequence of the first homologous FKS gene cloned from the pathogenic fungus Paracoccidioides brasiliensis. An open reading frame of 5942 bp was identified in the complete sequence, interrupted by two putative introns, the first close to the 5' end and the second close to the 3' end of the gene. A promoter region is also described containing consensus sequences such as canonical TATA and CAAT boxes and, possibly, multiple sites for glucose regulation by creA protein. The deduced sequence of 1926 amino acid show more than 85% similarity to FksAp from A. nidulans, and 71% to Fks1p and Fks2p from S. cerevisiae. Computational analysis of P. brasiliensis Fks1p suggests a similar structure to transmembrane proteins, such as FksAp, with the presence of two domains composed by hydrophobic helices that limit the putative highly hydrophilic catalytic domain within the cytoplasm.     

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.     

Two genes encoding putative mitochondrial alcohol dehydrogenases are present in the yeast Kluyveromyces lactis

Four structural genes encoding isozymes of the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis have been identified by hybridization to ADH2 DNA probes from Saccharomyces cerevisiae. In this paper we report on the isolation of KlADH4 and the complete sequencing of KlADH3 and KlADH4, two genes which show high homology to KlADH1, the ADH gene previously isolated in K. lactis, and to the ADH genes of S. cerevisiae. When compared with KlADH1, both KlADH3 and KlADH4 encode amino-terminal extensions which show the characteristics of the mitochondrial targeting sequences. These extensions are poorly conserved both at the nucleotide and the amino acid level. Surprisingly, the KlADH4 extension shows a higher identity at the amino acid level to the one encoded by ADH3 of S. cerevisiae than to the KlADH3 presequence. KlADH3 and KlADH4, in contrast to the ADH3 gene of S. cerevisiae, show a strong bias in the choice of codons.     

Studies of yeast Kluyveromyces lactis mutations conferring super-secretion of recombinant proteins

We have isolated mutants responsible for a super-secretion phenotype in Kluyveromyces lactis using the gene coding for a Bacillus amyloliquefaciens alpha-amylase as a marker for secretion. These mutations defined two groups, dominant and recessive. The recessive mutant strain, which secreted the heterologous protein in five-fold excess compared to the wild-type strain, was used for the cloning of genes, restraining the super-secreting phenotype. In screening for genes affecting super-secreting phenotype, we found that multiple copies of 10 different independently isolated DNA sequences suppressed the super-secreting phenotype. The first among the genes characterized, named KlSEL1 ('secretion lowering') showed homology to Saccharomyces cerevisiae ORF YML013w. The KlSEL1 gene is predicted to encode a polypeptide of 620 amino acid residues containing a putative transmembrane domain and UBX domain, characteristic for the ubiquitin-regulatory proteins. We demonstrated that the disruption of the SEL1 orthologues in K. lactis and S. cerevisiae conferred the super-secreting phenotype. SEL1 isolated from S. cerevisiae suppressed the super-secretion phenotype in K. lactis klsel1 strain, likewise homologous KlSEL1. No other phenotypic features for strains lacking the SEL1 gene were noticed except for the S. cerevisiae mutant growth being notably slower than in a wt strain. No growth changes were observed in the K. lactis klsel1 mutant. The set of genes (suppressors of over-secreting phenotype) could be attractive for further analysis of gene functions, super-secreting mechanisms and construction of new strains. This collection could be useful for the expedient construction of reduced yeast genomes, optimized for heterologous protein secretion.     

Cloning of a fatty acid synthase component FAS1 gene from Saccharomyces kluyveri and its functional complementation of S. cerevisiae fas1 mutant

A gene encoding a fatty acid synthase component, FAS1, has been cloned from a genomic library of the polyunsaturated fatty acid (PUFA)-producing yeast Saccharomyces kluyveri. This gene (named Sk-FAS1) was found to contain an open reading frame of 6150 bp, coding for 2049 amino acids. The deduced Sk-FAS1 protein showed significant (75-59%) homology with FAS proteins from the other yeasts, including S. cerevisiae, Candida albicans and Yarrowia lipolytica. The substrate-binding sites of the acetyl transferase and malonyl/palmitoyl transferase domains, and the FMN- and NADPH-binding sites of the enoyl reductase domain, were all highly conserved. Expression of the Sk-FAS1 gene in S. cerevisiae complemented genetic disruption of the S. cerevisiae FAS1 gene (Sc-FAS1), suggesting the formation of a heterogeneous complex of Sk-FAS1 (beta) and Sc-FAS2 (alpha), which is able to function to synthesize fatty acids. Compared with the isogenic wild-type of S. cerevisiae, as well as S. kluyveri, the S. cerevisiae fas1 mutant carrying the Sk-FAS1 gene showed an increase in the relative amount of 16-carbon fatty acids and a decrease in 18-carbon fatty acids.     

Identification of bottom-fermenting yeast genes expressed during lager beer fermentation

It has been proposed that bottom-fermenting yeast strains of Saccharomyces pastorianus possess at least two types of genomes. Sequences of genes of one genome [S. cerevisiae (Sc)-type] have been found to be highly homologous (more than 90% identity) to S. cerevisiae S288C sequences, while those of the other [Lager (Lg)-type] are less so. To identify and discriminate Lg-type from Sc-type genes expressed during lager beer fermentation, normalized cDNA libraries were constructed and analysed. From approximately 22 000 ESTs, 3892 Sc-type and 2695 Lg-type ORFs were identified. Expression patterns of Sc- and Lg-type genes did not correlate with particular cell functions in KEGG classification system. Moreover, 405 independent clones were isolated that have no significant homology with sequences in the S288C database, suggesting that they include the bottom-fermenting yeast-specific (BFY) genes. Most of BFY genes have significant homology with the S. bayanus genome.     

Identification and characterization of amidase- homologous AMI1 genes of bottom-fermenting yeast

It has been proposed that a bottom-fermenting yeast strain of Saccharomyces pastorianus is a natural hybrid between S. cerevisiae and S. bayanus and possesses at least two types of genome. In the process of conducting expressed sequence tag (EST) analysis, we isolated bottom-fermenting yeast-specific (BFY) genes that have no significant homology with sequences in the S288C database. One of the BFY genes, AMI1, encodes a protein with homology to an amidase conserved among plants, Bacillus subtilis, Neurospora crassa, Schizosaccharomyces pombe and Saccharomyces species, with the exception of S. cerevisiae S288C. In the bottom-fermenting yeast, three alleles of AMI1 (one AMI1-A and two AMI1-B alleles) were found on different chromosomes. AMI1-A on chromosome XIII is most homologous to the S. bayanus AMI1 gene, while AMI1-B on chromosome X is most homologous to the Saccharomyces paradoxus AMI1 gene. Overproduction of AMI1 in S. cerevisiae resulted in a slow-growth phenotype. Although a hydropathy plot shows that Ami1p has a putative signal sequence, it was located in the cell, not secreted into the medium. By metabolome analysis of intracellular compounds, the amount of histidine and arginine is increased, and the amount of threonine, lysine and nicotinic acid is decreased in the Ami1p-overproducing strain as compared with the control, suggesting that Ami1p may hydrolyse some amides related to amino acid and niacin metabolism in the cell.     

Cloning and sequencing of the gene for apocytochrome b of the yeast Kluyveromyces lactis strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140)

The apocytochrome b genes from two strains of the yeast Kluyveromyces lactis, have been isolated and sequenced. The coding sequences in strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140) were identical but the upstream noncoding regions were slightly different. The sequences demonstrated the presence of a continuous open reading frame with no introns. The amino acid sequence, derived from the coding strand, showed 82% homology to the apocytochrome b of Saccharomyces cerevisiae strain D273-10B and only 58% homology to the protein from Schizosaccharomyces pombe strain 50. CUN and CGN codon families were absent from the K. lactis gene. Codon usage was very similar to that of other mitochondrial genomes with mostly U or A in the third position. There were two unusual features. All threonines were coded by ACA(U) and all arginines by AGA.