Characterization of two fructosyl-amino acid oxidase homologs of Schizosaccharomyces pombe

Two putative fructosyl-amino acid oxidase genes, FAP1 and FAP2, found in the Schizosaccharomyces pombe genome were cloned and expressed. Both of the gene products (Fap1 and Fap2) were flavoproteins and have no activity for fructosyl-amino acids. It was suggested that Fap1 and Fap2 are an L-pipecolic acid oxidase and L-saccharopine oxidase, respectively.     

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.     

Alteration of substrate specificity of fructosyl-amino acid oxidase from Ulocladium sp. JS-103

We showed by random mutagenesis that one-amino-acid substitution at Arg94 in fructosyl-amino acid oxidase from Ulocladium sp. JS-103 enhanced substrate specificity toward fructosyl valine (FV), a model compound of hemoglobin A(1c). Kinetic analysis showed that the specificity of the R94W mutant enzyme toward FV was 14-fold that of the wild-type enzyme. The mutant enzyme obtained will be useful in developing an enzymatic measurement method for hemoglobin A(1c).     

不同类型丝状真菌发酵产酶性能比较及其混菌发酵性能研究

比较了实验室前期从福建红曲黄酒酿造用曲中分离及台湾生物资源研究及保存中心(BCRC)的不同丝状真菌菌株(黑曲霉AN19、黄曲霉AF20、米曲霉AO35、米根霉RO45、黑曲霉BCRC31494、黄曲霉BCRC31654、米曲霉BCRC30222和米根霉BCRC32229)的产酶性能,从中筛选出产液化酶、糖化酶和蛋白酶活力较高的4株菌株:黄曲霉AF20、米根霉RO45、黑曲霉BCRC31494和米曲霉BCRC30222。将此4株丝状真菌菌株分别进行单一和两两混合发酵糯米,研究其产酶及产糖性能。结果表明:接种纯种米根霉RO45到糯米培养基中,测得的液化酶活力和还原糖产量最高,然而整个发酵过程蛋白酶活力始终较低;米根霉RO45与米曲霉BCRC30222混合发酵,不仅保持较高的液化酶活力和还原糖产量,还能够显著提升发酵体系中的蛋白酶活力。本研究结果对于黄酒传统酿造工艺的改进和优良产酶菌株的筛选提供了重要的基础数据。     

Identification of genes from Aspergillus oryzae that are preferentially expressed in membrane-surface liquid culture

We identified 22 genes from Aspergillus oryzae that are preferentially expressed in membrane-surface liquid culture (MSLC), among which Ser/Thr protein kinase (aopk1) and phosphatase (aoppt) genes were cloned. We also revealed that aopk1 encodes a protein with an N-terminal sequence 150 amino acid residues longer than that predicted from the registered sequence in GenBank.     

米曲霉3042的液体培养特性及其产氨基酰化酶的特性研究

对液体培养的米曲霉所产生的氨基酰化酶胞内外分布及其酶的最适ＰＨ、温度和热稳定性进行了探讨。结果表明,米曲霉３０４２属于凝聚型菌株,当搅拌速度为９０－１２０ｒ／ｍｉｎ时可获得较好的菌球,氨基酰化酶属于人酶,存在于细胞内部或分布于细胞壁上;菌体酶最适Ｐ眯７．０,最适温度为５５℃。好的菌球;氨基铧酶属于胞内酶,存在于细胞内部或分布于细胞壁上,菌体酶活最适ＰＨ为７．０,最适温度为５５℃。     

米曲霉3.042尿苷/尿嘧啶营养缺陷型遗传转化体系的构建

米曲霉对潮霉素等多种抗生素不敏感,对其基因改造有一定困难.该研究以米曲霉3.042为出发菌株,建立pyrG筛选标记遗传转化体系.首先,分别运用紫外诱变法和左右臂同源重组法破坏米曲霉自身的pyrG基因,在含有5-氟乳清酸和尿苷/尿嘧啶的培养基平板进行筛选,最终两种方法分别获得性状稳定的尿苷/尿嘧啶营养缺陷型突变株米曲霉O...     

太空酒曲中功能菌的生物酶活性研究

从太空酒曲中分离、选育出主要功能菌:根霉SKR_3、黑曲霉SKA_2、米曲霉SKO_3;与原生产中使用的曲药的主要功能菌——根霉SNR_1、黑曲霉SNA_4、米曲霉SNO_2的酶活性进行比较研究。结果表明:SKR_3的糖化酶、液化酶活性均比SNR_3高出两倍以上,SKR_3培养36hr后其糖化酶、液化酶活性达到SNR_1培养72hr后的两倍;SKA_2的糖化酶、液化酶活性比SNA_4的高出三倍,蛋白酶活性高出一至二倍,SKA_2培养48hr后糖化酶、液化酶活性达到SNA_1培养72hr的三倍;SKO_3与SNO_1的糖化酶、液化酶、蛋白酶活性基本无差异。     

植酸提高米曲霉酶活力的机理

米曲霉能分泌植酸酶,如制曲中添加一定量植酸,经植酸酶降解产生肌醇和磷酸,肌醇激活酶系,从而提高米曲霉的酶活力。（一平     

太空酒曲中功能菌的生物酶活性研究

从太空酒曲中分离、选育出主要功能菌：根霉SKR3、黑曲霉SKA2、米曲霉SKO3；与原生产中使用的曲药的主要功能菌-根霉SNR1、黑曲霉SNA4、米曲霉SNO2的酶活性进行比较研究。结果表明：SKR3的糖化酶、液化酶活性均比SNR1高出两倍以上，SKR3培养36h后其糖化酶、液化酶活性达到SNR1培养72h后的两倍；SKA2的糖化酶、液化酶活性比SNA4的高出三倍，蛋白酶活性高出一至二倍，SKA2培养48h后糖化酶、液化酶活性达到SNA4培养72h的三倍；SKO3与SNO1的糖化酶、液化酶、蛋白酶活性基本无差异。     

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".     

丝状真菌发酵对小麦营养成分及抗氧化活性的影响

研究两株丝状真菌(米曲霉和黑曲霉)发酵对小麦营养成分及抗氧化性的影响,通过测定发酵小麦中还原糖、氨基酸、总酚和总黄酮含量,发现两株丝状真菌均能显著提高小麦中还原糖、氨基酸、总酚和总黄酮含量,并且黑曲霉发酵小麦中可滴定酸含量也显著增加,而米曲霉发酵小麦中可滴定酸并没有显著变化。利用氧化自由基吸收能力(ORAC)方法对发酵过程中抗氧化性变化的研究发现,两株真菌发酵后,均能显著提升小麦的抗氧化性。因此发酵小麦相对未发酵小麦具有更好的营养价值。     

pH值及乳酸菌对米曲霉固态制曲过程的影响

考察了在固态培养条件下,不同的初始pH值及乳酸菌对米曲霉固态制曲过程的影响。以豆粕和麸皮(质量比为55∶45)为原料,1∶0.6的料水比,0.3%～0.5%的接种量接入沪酿3.042米曲霉种曲,33℃下制曲,定时取样测定固体曲的蛋白酶活力及孢子数。结果表明:适当偏酸性的环境有利于成曲蛋白酶的提高,培养基初始pH调节为6.5时米曲霉中性蛋白酶活力获得最大值(4 080±61)U/g干基,较对照组提高33%～37%;添加1.0%～2.0%乳酸菌,也同样起到改善米曲霉制曲过程的效果。     

高活力酸性蛋白酶酱油菌种的选育

本文通过对生产上使用的3.042米曲霉进行筛选，得到了酸性蛋白酶活力较高的菌株。将其与原始菌株混合制曲和发酵，可有效提高全氮利用率和氨基酸生成率。     

不同菌种发酵黄豆酱的氨基酸态氮的研究

通过对三种常用于黄豆酱制曲的菌种（即米曲霉、根霉和高大毛霉）制备黄豆酱,分别对三种菌的蛋白酶活性和蛋白组分以及酱醅的氨基酸态氮含量进行比较.结果表明,米曲霉的蛋白酶活力大于根霉和高大毛霉,蛋白质组分比高大毛霉和根霉的更为复杂,且米曲霉制得的黄豆酱氨基酸态氮含量较二者高.     

Subcellular localization of fructosyl amino acid oxidases in peroxisomes of Aspergillus terreus and Penicillium janthinellum

Fructosyl amino acid oxidase (FAOD) is the enzyme catalyzing the oxidative deglycation of Amadori compounds, such as fructosyl amino acids, yielding the corresponding amino acids, glucosone, and H(2)O(2). In a previous report, we determined the primary structures of cDNAs coding for FAODs from two fungal strains Aspergillus terreus AP1 and Penicillium janthinellum and we found that both fungal FAODs included the putative peroxisome targeting signal 1 (PTS1) at the carboxyl terminal (Yoshida, N. et al., Eur. J. Biochem., 242, 499-505, 1996). In this study, we determined the intracellular localization of FAODs in these two fungi. Subcellular fractionation experiments and immuno-electronmicroscopic observations, together with the previous findings indicated that the FAODs were localized in peroxisomes of A. terreus AP1 and P. janthinellum. These FAODs were also found to belong to a new member of "peroxisomal sarcosine oxidase family protein" in eucaryotic cells.     

Effect of fermentation on the antioxidant activity of red beans (Phaseolus radiatus L. var. Aurea) ethanolic extract

The antioxidant activities of 50% ethanol extracts from red bean non-fermented and fermented by Bacillus subtilis or Aspergillus oryzae were determined using Sprague–Dawley rats as a testing model. Oral administration of all the extracts decreased the malondialdehyde (MDA) level in the liver but not in the brain tissue. Bacillus subtilis fermented extract increased the levels of ascorbic acid, α-tocopherol and glutathione (GSH) and the superoxide dismutase (SOD) activity in the liver tissue, while it increased only the ascorbic acid level in the brain tissue. Aspergillus oryzae fermented extract increased the levels of GSH and the SOD activity in the liver tissue, and it also increased GSH and ascorbic acid in the brain tissue. In general, the extracts from fermented red bean were more effective than the non-fermented extract in raising the antioxidant levels in the liver tissue.     

清酒酒曲生产用最适霉菌的筛选

对最适宜制作清酒酒曲的霉菌进行了筛选。对米曲霉3.5232、米曲霉3.800、黑曲霉3.4309、黑曲霉3.1858、根霉3.866分别进行耐酒精试验、耐温试验、耐酸试验,初筛得到米曲霉3.5232、黑曲霉3.1858、根霉3.866三株菌株,分别制成酒曲,通过酒曲α-淀粉酶活力、蛋白酶活力和糖化酶活力的测定最终筛选出米曲霉3.5232是最适宜制作酒曲的霉菌菌株。     

不同菌种发酵制备蚕豆酱的研究

本试验研究米曲霉、黑曲霉、黑根霉、高大毛霉4种不同单一菌种分别发酵制得的蚕豆酱中蛋白酶、淀粉酶活力及主要成分、酱色OD420nm值等指标在不同发酵时期的变化,并对发酵42d的蚕豆酱进行感官评价。结果表明,米曲霉的中性及碱性蛋白酶活力较其他三种菌强,分别高达546.84U和188.79U。米曲霉发酵的蚕豆酱氨基酸态氮含量最高,可达1.381 g/100g,根霉、黑曲霉次之,毛霉最少。米曲霉和黑曲霉制得酱的颜色深,酱色OD420nm值高,另两种霉制得酱的颜色较浅。米曲霉发酵制得蚕豆酱在色泽、气味和口感上都达到一级标准,是制作蚕豆酱的良好发酵菌种。     

米曲霉发酵所制大豆肽的分离纯化与抗氧化性研究

利用辐照诱发米曲霉孢子突变,筛选得到酶活力强且特异性高的米曲霉诱变菌株。利用米曲霉诱变菌株对豆腐皮加工后的剩余豆浆(下浆水)进行发酵,制备大豆肽的发酵液,发酵液经超滤和凝胶过滤进行分离纯化,采用铁离子还原抗氧化剂能力测定法(FRAP法)和二苯基苦基苯肼法(DPPH法)研究不同相对分子质量大豆肽的抗氧化能力。结果表明,相对分子质量在1 000～10 000范围内的大豆肽具有很好的抗氧化活性,其中抗氧化性和稳定性最好的大豆肽组分的相对分子质量在1 200～1 400之间。