Production of two types of phytase from Aspergillus oryzae during industrial koji making

In our previous study, it was determined that phytase produced by Aspergillus oryzae plays an important role in supplying phosphate to yeast in the process of making sake. During koji making, two types of phytase (Phy-I and Phy-II) are produced. The purified phytases have high thermal and pH stability, in comparison to phytase purified from a submerged culture (ACP-II). In the present study, Phy-I and Phy-II retained their activities for 45 h. The NH2-terminal sequence of Phy-1, which is eight amino acids in length, was identical to that of ACP-II, but the molecular weights of these two forms, as estimated by SDS-PAGE, were quite different from each other (Phy-I, 120 kDa; ACP-II, 58 kDa). From the NH2-terminal amino acid sequence analysis of the predominant phytase (Phy-II), a molecular weight of 116 kDa was expected to reflect a new type of phytase produced only in koji culture. The substrate specificity of Phy-II was sufficiently broad that it hydrolyzed not only phytic acid and p-nitro phenyl phosphate, but also glucose 6-phosphate and glycerol 1-phosphate. In the process of making koji, Phy-I was produced at an early stage, followed by Phy-II; with both phytases being thought to function to hydrolyze phytic acid cooperatively.     

Production and some properties of salt-tolerant beta-xylosidases from a shoyu koji mold, Aspergillus oryzae in solid and liquid cultures

Beta-xylosidase production from a shoyu (soy sauce) koji mold, Aspergillus oryzae HL15, cultured in solid and liquid media was examined and some properties of the enzymes were studied. Three beta-xylosidases (Xy11, Xy12 and Xy13) were easily extracted with 0.5% NaCl from a solid medium and purified homogeneously on SDS-PAGE by chromatography. On the other hand, in a liquid medium, A. oryzae HL15 produced mainly cell-wall-bound beta-xylosidases which could not be extracted with 0.5% NaCl or any detergent. Cell-wall-bound beta-xylosidases, Xy11-CB and Xy12-CB, were liberated by digestion of mycelia with Yatalase and purified to a homogeneous state on SDS-PAGE by HPLC column chromatography. Four beta-xylosidases (Xy11, Xy12, Xy11-CB and Xy12-CB) exhibited not only high activity at high NaCl concentrations, but also similar properties; on the other hand, Xy13 differed in terms of thermostability and halophilic properties. The salt tolerance of beta-xylosidases in A. oryzae suggests that these enzymes are highly active and involved in releasing xylose in shoyu moromi mash.     

Molecular breeding of the Mureka-non-forming sake koji mold from Aspergillus oryzae by the disruption of the mreA gene

Mureka-non-forming sake koji molds were constructed from an Aspergillus oryzae industrial strain by the disruption of the mreA gene using a host-vector system with the ptrA gene as a dominant selectable marker. All of the mreA gene disruptants obtained retained the advantages of the host strain in terms of the brewing characteristics, while their isoamyl alcohol oxidase (IAAOD) activities were significantly lower than that of the host strain. Sake brewing was successfully carried out using the koji prepared with the disruptants, followed by storage of the resultant non-pasteurized sake (nama-shu). The isovaleraldehyde (i-Val) concentration in the sake brewed the host strain increased rapidly and reached the threshold values for mureka, 1.8 ppm and 2.6 ppm after storage at 20 degrees C for 42 d and 63 d, respectively, while those of the disruptants were less than 0.5 ppm even after storage at 20 degrees C or 30 degrees C for 63 d. In the sensory evaluation of the sake stored at 20 degrees C or 30 degrees C for 63 d, all members of the panel recognized the strong mureka flavor of the sake brewed with the host strain, while they did not detect this flavor in the sake brewed with the disruptants. Thus, we concluded that the mreA gene disruptants can be used for the production of sake in which mureka is not formed.     

纯种米曲霉酒曲的制备工艺优化研究

在单因素试验的基础上,选取影响纯种米曲霉酒曲糖化酶活力的3个主要因素(培养温度、培养时间、接种量),应用响应面试验对制曲的最佳工艺条件进行了研究,结果显示3个因素对米曲糖化酶活力的影响是显著的。利用Design-Expert软件对试验数据进行处理,得到制曲的最佳工艺条件为:培养温度38.70℃,接种量0.85‰,培养时间50.47 h。在此条件下米曲糖化酶活力的预测值为1 111.52 U。在最佳培养条件下对预测值进行验证,所测得的实际值与预测值基本一致。     

Synergistic degradation of arabinoxylan with alpha-L-arabinofuranosidase, xylanase and beta-xylosidase from soy sauce koji mold, Aspergillus oryzae, in high salt condition

This study addresses induction and some properties of alpha-L-arabinofuranosidase from a soy sauce koji mold, Aspergillus oryzae HL15, cultured on solid and liquid media. Alpha-L-arabinofuranosidase was induced by soybean polysaccharide and secreted into media on solid cultivation; the enzyme was associated with mycelium as a cell-wall-bound form in liquid cultivation. A major alpha-L-arabinofuranosidase, which was purified homogeneously on SDS-PAGE, showed highest activity in the presence of 10% of NaCl; also, somewhat higher activity was observed even in 25% NaCl than in the absence of NaCl. Arabinoxylan was synergistically degraded by xylanase, beta-xylosidase, and alpha-L-arabinofuranosidase from A. oryzae HL15 in the condition of imitative pH 5.0 and 15% NaCl concentration of the soy sauce moromi mash. These results suggest that arabinose and xylose, which are closely related to melanoidin formation, can be released by synergistic action of these enzymes in soy sauce moromi mash.     

Production optimization,purification, and characterization of a novel acid protease from a fusant by Aspergillus oryzae and Aspergillus niger

The production of a novel acid protease was enhanced by 44 % through statistical optimization. The cultural parameters, such as inoculum size, temperature, moisture content, and incubation time, were 8.59 × 10⁵ g^?1 dry koji, 31 °C, 57 %, and 86 h, respectively. This novel acid protease was purified by 17 folds with a recovery yield of 33.56 % and a specific activity of 4,105.49 U mg^?1. Far-UV circular dichroic spectra revealed that this purified protease contained 7.1 % α-helix, 64.1 % β-sheet, and 32 % aperiodic coil. This novel acid protease was active over the temperature range of 35–55 °C with optimum temperature of 40 °C and was stable in the pH range of 2.5–6.5 with optimum pH of 3.5. Mn²⁺ enhanced its activity while Co²⁺ showed inhibitory effect. With casein as substrate, the kinetic parameters of K _m, V _max, energy of activation (E _a), and attenuation index of inactivation velocity by heat inducing (λ) were 0.96 mg mL^?1, 135.14 μmol min^?1 mg^?1, 64.11 kJ mol^?1, and 0.59, respectively.     

Secretome of Aspergillus oryzae in Shaoxing rice wine koji

Shaoxing rice wine is the most famous and representative Chinese rice wine. Aspergillus oryzae SU16 is used in the manufacture of koji, the Shaoxing rice wine starter culture. In the current study, a comprehensive analysis of the secretome profile of A. oryzae SU16 in Shaoxing rice wine koji was performed for the first time. The proteomic analysis for the identification of the secretory proteins was done using two-dimensional electrophoresis combined with matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry based on the annotated A. oryzae genome sequence. A total of 41 unique proteins were identified from the secretome. These proteins included 17 extracellular proteins following the classical secretory pathway, and 10 extracellular proteins putatively secreted by the non-classical secretory pathway. The present secretome profile greatly differed from previous reports on A. oryzae growing in other solid-state nutrient sources. Several new secretory or putative secretory proteins were also found. These proteomic data will significantly aid the advancement of research on the secretome of A. oryzae, especially in solid-state cultures, and in elucidating the production process mechanism of Shaoxing rice wine koji. The findings may promote the technological development and innovation of the Shaoxing rice wine industry.     

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.     

Production of cellulose- and xylan-degrading enzymes by a koji mold, aspergillus oryzae, and their contribution to the maceration of rice endosperm cell wall

The production of cellulose- (CEL), xylan- (XYL), and pectin-degrading enzymes (PEC) by a koji mold, Aspergillus oryzae, was studied, and their contributions to the maceration of the rice endosperm cell wall were investigated with regard to the utilization of available rice in the sake mash. The sake koji mold showed higher CEL and XYL productivities, whereas the miso and soy sauce koji molds showed higher PEC productivity. Statistical analyses indicated that CEL and XYL contribute predominantly and synergistically to the maceration of the rice endosperm cell wall. A. oryzae produced at least three kinds of CEL (Cel-1, 2, 3) and two kinds of XYL (Xyl-1, 2) when cultured in a wheat bran medium. In the solid-state culture, the production of Cel-3 and Xyl-2 was markedly stimulated by decreasing the moisture content of the solid substrate, although the production levels of Cel-1 and Xyl-1 were almost the same. These data suggest that the production of Cel-3 and Xyl-2 is strongly influenced by culture conditions, and that water activity is one of the dominant factors in the regulation of their production.     

Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.     

Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.     

米曲霉ASP-m21产果胶酶及其酶学性质

以产果胶酶PG为主的米曲霉(Aspergillus oryzae)为出发菌株进行固态发酵研究.确定最适合发酵条件为:以30 g/dL麸皮,8 g/dL橘皮粉,添加2 g/dL的泡沫材料作为透气支撑载体,每千克原料添加8 g硫酸铵,10 g葡萄糖,接种体积分数为10%,经过42 h发酵,最终果胶酶酶活可达807 U/g(干曲计).果胶酶的最适反应温度为50 ℃,最适pH范围在3.0～3.6之间.试验结果表明:果胶酶在3.0～7.0的pH的范围内,适合产物生成速度最大的pH点在3.6左右,产物生成速度为0.3 mmol/(Lmin);50 ℃是酶促反应的最适温度,在30 min条件下生成的产物最高可达9 mmol/L.在桔子澄清实验中,向含有体积分数30%的橙汁中添加酶活为1 U/mL的果胶酶,分解果胶为半乳糖醛酸,并且随着半乳糖醛酸量的增加,橙子浆的黏度降低,透光率增加.反应7 h后,透光率可高达91.1%.     

Production of phytase by Aspergillus ficuum and reduction of phytic acid content in canola meal

Production of the phytase (EC 3.1.3.8) from Aspergillus ficuum in a submerged batch process was inhibited by high concentrations of glucose. The inhibition was overcome by applying a fed batch technique in the production of the enzyme. Tests carried out at different oxygen concentrations revealed that aeration had a beneficial effect on the production of the enzyme. The enzyme showed an optimum pH and temperature of 5·0 and 60°C, respectively. Preincubation of the enzyme preparation at 60°C resulted in relatively fast denaturation of the enzyme. Upon storage at 4°C it lost only 15% of its activity in 5 weeks. Aspergillus ficuum also produced phytase when grown on canola meal by a solid state technique. The enzyme catalysed degradation of the phytic acid present in the meal and completely eliminated it, rendering the commodity more suitable for animal feed. An apparent 10% increase in protein content of the canola meal was noted as a result of the growth of the microorganism.     

米曲霉KFRI 888产中性蛋白酶、a-淀粉酶酶学性质的研究

通过对米曲霉KFRI888产中性蛋白酶、a-淀粉酶的酶学特性进行了研究,实验发现,当温度高于40℃时,中性蛋白酶、a-淀粉酶的酶活都随着温度的升高而下降。而中性蛋白酶的最适作用pH为7.0～8.0,淀粉酶的最适作用pH为6.0～7.0,中性蛋白酶、a-淀粉酶的酶活都随着NaCl浓度的升高而下降,但a-淀粉酶耐盐性能比中性蛋白酶强。金属离子Ca2 、K 、Mg2 对a-淀粉酶有激活作用,且基本不影响蛋白酶的酶活。     

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。      

Properties of cellulose-degrading enzymes from Aspergillus oryzae and their contribution to material utilization and alcohol yield in sake mash fermentation

Four cellulose-degrading enzymes were identified in a solid-state culture of Aspergillus oryzae. The three major enzymes were purified and named Cel-1, Cel-2, and Cel-3, respectively. The molecular weights were determined to be 62, 120, and 34 kDa, respectively. The optimum temperature of Cel-3 activity was higher than that of the other enzymes. An acidic pH was found to be more suitable for Cel-1 activity than for the other enzymes, and Cel-3 was more stable under acidic conditions than the other two. These properties and the results of a protein homology search for N-terminal amino acid sequences suggest that Cel-1 and Cel-3 correspond to the previously isolated endo-1,4-beta-glucanase CelB and CelA, respectively. The analysis of substrate specificity suggested that Cel-2 is likely to be beta-glucosidase. The effect of Cel-1, Cel-2, and Cel-3 on the sake mash fermentation was determined and it was found that Cel-2 markedly improved material utilization and alcohol yield in sake mash fermentation.     

米曲霉GX0011β-果糖基转移酶的性质研究

β-果糖基转移酶是酶法由蔗糖生产低聚果糖所必需的酶,本文对从米曲霉GX0011分离纯化得到的β-果糖基转移酶性质进行了研究。结果表明,该酶催化蔗糖转化成蔗果三糖的Km和Vmax分别为0.319mol/L和0.713mol/min/L,最适pH和最适温度分别为5.0～6.0和45℃。葡萄糖是该酶的竞争性抑制剂,其抑制常数Ki=0.608mol/L。经苯甲磺酸氟(PMSF)、N-溴代二酰亚胺(NBS)、1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDC)和间硝基苯磺酸钠(TNBS)对酶进行化学修饰及底物保护实验,推测该酶活性中心可能包括丝氨酸/苏氨酸、色氨酸、天冬氨酸(谷氨酸)残基,而赖氨酸残基可能与维系酶活性中心构象有关,但不是酶活性必需基团。实验还表明,超声波能在一定程度上提高酶活力,在300W功率下作用5min可提高酶活12%左右。低浓度乙醇对酶活影响并不十分显著,乙醇浓度达20%时,酶活仅损失19%。     

Mapping haze-komi on rice koji grains using β-glucuronidase expressing Aspergillus oryzae and mass spectrometry imaging

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米曲霉形态对L-苹果酸生产的影响

L-苹果酸是一种重要的C_4化合物,广泛应用于食品、化工和医药行业。本文中以米曲霉为出发菌株,研究氮源种类、CaCO_3质量浓度、搅拌转速和通气量等关键营养条件和环境条件对Aspergillus oryzae形态和产L-苹果酸的影响。最优发酵条件为:胰蛋白胨为氮源、CaCO_3质量浓度为80 g/L、搅拌转速为600 r/min、通气量为2 vvm。进一步分析菌体形态与L-苹果酸产量的关系,得出当单位体积发酵液菌球总体积(V值)为76.4 mm3/mL时,L-苹果酸产量最高达109.9 g/L。