Molecular breeding of Aspergillus kawachii overproducing cellulase and its application to brewing barley shochu

In order to improve fermentation of barley without addition of commercial cellulase, a white koji mold, Aspergillus kawachii IFO4308, was transformed with the egl1 gene encoding endoglucanase I (EGI) of Trichoderma viride and the endogenous cekA gene encoding endoglucanase (CekA). Transformants with egl1 under the control of the strong glaA promoter produced EGI in both submerged and solid-state cultures. However, the EGI produced in solid-state culture was unstable due to the acidic condition of this culture. A transformant N10 with two additional copies of the cekA gene exhibited endoglucanase activities against carboxymethyl-cellulose, which are 21- and 1.8-fold higher than that of the wild-type (wt) strain when the cells were cultivated in submerged and solid-state cultures, respectively. Cultivation of strain N10 in steamed barley for preparing koji followed by fermentation with Saccharomyces cerevisiae resulted in improved fermentation assessed based on higher productions of ethanol, amino acids, and organic acids, the reduction of residual sugar, and the low viscosity of barley mash. The overall fermentation result for the transformant carrying cekA was comparable with that for the wt strain using commercial cellulase. These results demonstrate that acquisition of only two-fold CekA activity by A. kawachii in the solid-state culture allows us to improve the brewing of barley shochu.     

高糖化力菌种的筛选及诱变育种

固体曲中精化酶能将淀粉水解为葡萄精,进而被微生物发酵生成酒精.以分离筛选出的1株产糖化酶的黑曲霉(酶活265.21U/g干曲)为出发菌,经紫外线及原生质体紫外线复合诱变育种,获得1株糖化酶活力较高、生产性能稳定的菌株,其酶活达到3367.79U/g干曲,比原始菌株提高1169.86%.     

Synthesis of Glucoamylase by Somatic Diploids of Aspergillus niger in Submerged Culture Conditions

Out of twelve forced heterocaryons of A. niger, in nine cases somatic diploids were obtained comprising a total of 107 strains. All were initially examined with respect to general amylase activity in test-tube microcultures. Over 21% of all diploid recombinants reached a higher coefficient of amylase activity than the parent haploid highly active in this respect. Four most active diploids selected from this group were examined with regard to glucoamylase synthesis. Also in this case diploids were characterized by a certain predominance in their activity over the initial prototrophic haploid strain (maximally about 16%).     

Amylase Synthesis by Forced Heterocaryons of Aspergillus niger in Submerged Culture Conditions

From 43 auxotrophic mutants A. niger found earlier 21 forced heterocaryons were obtained. All were examined with respect to total amylolytic activity by the method of test-tube microculture, whereas 12 of them (with the highest indices of this activity) were also tested with regard to glucoamylase synthesis. In most cases the amylase activity of hererocaryons was much higher than that of their component strains of lower and frequently of a higher activity. A distinct increase of this activity was also shown by heterocaryons formed from auxotrophs, originating from them in relation to prototrophic parent cultures with a weak amylase activity. However, none of the heterocaryons equalled the level of amylase synthesis, reached by a highly active prototroph A. niger C in this respect.     

Raw Starch Digestion by α-Amylase and Glucoamylase from Chalara paradoxa

Glucoamylase and α-amylase of Chalara paradoxa were separated by hydrophobic column chromatography using butyl-Toyopearl 650M. The α-amylase showed the highest activity at pH 5.5 and 45°C, and was stable in the pH range of 5.5–6.5 and at temperatures lower than 40°C. The glucoamylase showed the highest activity at pH 5.0 and 45°C, and was stable in the pH range of 4.0–7.5 and at temperatures lower than 45°C. The molecular mass of the α-amylase and glucoamylase estimated by SDS polyacrylamide gel electrophoresis was 80,000 and 68,000, respectively. Both glucoamylase and α-amylase could digest more effectively raw rice starch and raw corn starch than raw sago starch and raw potato starch. 2% raw rice starch in 10 ml solution was digested by more than 90% by 100 units of each amylase. When these amylases were used combined, raw corn starch was more effectively digested than they were used singly. This cooperative action in raw corn starch digestion was also observed when. C. paradoxa α-amylase and R. niveus glucoamylase were combined.     

Purification and Properties of the Raw Starch Digesting Amylase from Penicillium brunneum No. 24

Raw sago starch digesting amylase was obtained from Penicillium brunneum No. 24. The crude enzyme from this strain contains carboxymethylcellulase (CMC-ase), avicelase, α-amylase and α-glucosidase. Affinity chromatography (α-cyclodextrin-Sepharose 6B) of the enzyme after ammonium sulfate fractionation, Toyopearl HW-55F gel filtration, DEAE-Sephadex A-50 and DEAE-cellulose chromatographies fractionation steps, resulted in a homogeneous glucoamylase. SDS-polyacrylamide gel electrophoresis of purified enzyme showed a single band, and a molecular weight of 80,000 for the native glucoamylase from Penicillium brunneum No. 24 was observed. After modification of the native glucoamylase with subtilisin, the molecular weight was reduced to 76,000. It lost the ability to digest and adsorb onto raw starches. However, its ability to digest gelatinized starches was preserved.     

激活减曲酿造酱油的研究

研究了添加激活剂和减曲技术在酱油生产中的应用。结果表明,激活减曲法能提高米曲霉的孢子发芽率,增强抗菌能力。与对照相比,成曲中蛋白酶活力、淀粉酶、糖化酶活力都有提高,发酵10d时酱醅氨基氮的含最提高13．6％。激活减曲工艺降低物料损耗,提高酱油产量。     

PRODUCTION OF AMYLOLYTIC ENZYMES BY SEVERAL YEAST SPECIES

Several amylolytic yeast species, endomycopsis spp, schwanniomyces spp, pichia spp and saccharomyces spp have been compared for their ability to synthesise α-amylase (E.C. 3.2.1.1), glucoamylase (E.C. 3.2.1.3) and pullulanase (E.C. 3.2.1.9). Endomycopsis fibuligera strain 240 possessed the highest glucoamylase activity (208 nmoles glucose/min) and second highest α-amylase activity (128 nmoles maltose/min) and produced the largest amount of biomass. Schwanniomyces spp were the only yeast species studied which exhibited significant debranching activity. It was found that in endomycopsis spp, schwanniomyces spp and pichia spp, glucose, at a concentration above 3.0 × 10^?3 m , appeared to repress α-amylase activity. However, glucoamylase activity was not repressed at that glucose concentration.     

Fractionation of the Glucoamylase System from Black-koji Mold and the Effects of Adding Isoamylase and Alpha-amylase on Amylolysis by the Glucoamylase Fractions

The glucoamylase system of black koji mold was fractionated into four kinds of glucoamylase by using the corn starch adsorption technique supplement by DEAE-cellulose column chromatography. One of these fractions, referred to as glucoamylase I, had a strong debranching activity and is highly active in raw starch digestion. The raw waxy corn starch digestion by glucoamylase I was accelerated by adding α-amylase or isoamylase. The raw non-waxy corn starch digestion by glucoamylase I was accelerated by α-amylase but not so much by isoamylase as by α-amylase. On the other hand, another fraction, referred to as glucoamylase II, had very weak debranching activity and was most feeble in the digestion of raw starch. The raw waxy corn starch digestion by glucoamylase II was very weak, but was accelerated extremely by adding isoamylase, but not by α-amylase.     

不同菌种制备酱油大曲生产酶系特性及其耐盐性的比较

采用4株米曲霉菌种(j2、y2、x42、x72)与对照米曲霉菌株(沪酿3.042)制备大曲,探究其所产中性蛋白酶、酸性蛋白酶、α-淀粉酶以及糖化酶在盐浓度为0%、5%、10%、15%、20%下酶活力变化。实验结果表明,5株菌株所产酶系中,中性蛋白酶活随盐水浓度的增加显著(P<0.05)下降,酸性蛋白酶和糖化酶在5%盐浓度下活力显著下降(P<0.05),随后酶活力下降速率减慢,α-淀粉酶在5%的盐浓度下无显著下降(P>0.05),随后下降速率加快。此外,相同盐浓度下淀粉酶比蛋白酶更稳定,5株米曲霉菌株的α-淀粉酶和糖化酶在20%的盐浓度下残留率均达到50%。菌株y2、j2所产中性蛋白酶和菌株x72所产酸性蛋白酶的耐盐性优于对照菌株。     

A new promoter-probe vector for Saccharomyces cerevisiae using fungal glucoamylase cDNA as the reporter gene

A system is described for the selection of DNA sequences showing promoter activity in the yeast Saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp-type was constructed so as to carry multiple unique cloning sites at the 5′ end of the Aspergillus awamori glucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal-peptide is functional in S. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild-type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligating sau 3A digested S. cerevisiae genomic DNA to the BglII-linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.     

Construction of a brewing yeast expressing the glucoamylase gene STA1 by mating

Standard brewing yeast cannot utilize larger oligomers or dextrins, which represent about 25% of wort sugars. A brewing yeast strain that could ferment these additional sugars to ethanol would be useful for producing low‐carbohydrate diabetic or low‐calorie beers. In this study, a brewing yeast strain that secretes glucoamylase was constructed by mating. The resulting Saccharomyces cerevisiae 278/113371 yeast was MAT a/α diploid, but expressed the glucoamylase gene STA1 . At the early phase of the fermentation test in malt extract medium, the fermentation rate of the diploid STA1 strain was slower than those of both the parent strain S. cerevisiae MAFF113371 and the reference strain bottom‐fermenting yeast Weihenstephan 34/70. At the later phase of the fermentation test, however, the fermentation rate of the STA1 yeast strain was faster than those of the other strains. The concentration of ethanol in the culture supernatant of the STA1 yeast strain after the fermentation test was higher than those of the others. The concentration of all maltooligosaccharides in the culture supernatant of the STA1 yeast strain after the fermentation test was lower than those of the parent and reference strains, whereas the concentrations of flavour compounds in the culture supernatant were higher. These effects are due to the glucoamylase secreted by the constructed STA1 yeast strain. In summary, a glucoamylase‐secreting diploid yeast has been constructed by mating that will be useful for producing novel types of beer owing to its different fermentation pattern and concentrations of ethanol and flavour compounds. Copyright © 2017 The Institute of Brewing & Distilling     

PROPERTIES OF A GENETICALLY-ENGINEERED DEXTRIN-FERMENTING STRAIN OF BREWERS' YEAST

A dextrin-fermenting strain of brewer's yeast was obtained by transformation with a recombinant plasmid (pLHCD6) containing a DEX gene. The transformant produced five-fold more extracellular amyloglucosidase (AMG) than the strain of Saccharomyces diastaticus from which DEX was cloned. The growth rate, however, was adversely affected and, consequently, the transformant fermented wort more slowly than the parental (untransformed) strain. A mixed culture was therefore used at pilot-scale, to maintain a rate of fermentation similar to that of the parental strain. The transformant by itself was capable of superattenuating wort (original gravity 1.040) to a specific gravity of 1.0023 (compared with 1.0046 for the parental strain). This represents the expected degree of dextrin breakdown by an AMG with no “debranching” activity which can only hydrolyse alpha-1,4 linkages. A high copy number for the plasmid pLHCD6 was maintained throughout fermentation. The beers produced using the Dex+ transformants were judged to be of sound quality.     

Characterisation and threshold measurement of aroma compounds contributing to the quality of Honkaku shochu and Awamori

Honkaku shochu and Awamori are traditional Japanese spirits. The detection and thresholds of 16 compounds in these spirits were determined so as to understand the compounds contributing to the aroma characteristics of Honkaku shochu and Awamori. The odour activity values calculated using detection thresholds suggested that 14 compounds affect the quality of Honkaku shochu and Awamori. The odour activity values of β-damascenone and rose oxide were much higher in sweet potato shochu and these two compounds are considered to contribute to the characteristics of sweet potato shochu. Similarly, the odour activity value of 1-octen-3-ol was higher in Awamori than in the other categories, suggesting that 1-octen-3-ol produces the characteristic aroma of Awamori. In addition, the sensory attribute terms of 16 compounds are summarised. © 2019 The Institute of Brewing & Distilling     

ISOLATION AND CHARACTERISATION OF THE AMYLOTIC SYSTEM OF SCHWANNIOMYCES CASTELLII

The amylolytic system of Schwanniomyces castellii has been isolated and purified by means of ultrafiltration followed by polyacrylamide gel electrophoresis. Both α-amylase and glucoamylase were purified. α-Amylase activity was stable from pH 5·5 to 6·5 and glucoamylase activity was stable at a more acidic range of pH 4·2 to 5·5. The optimal temperature of α-amylase activity was between 30 and 40°C with rapid deactivation at 70°C. The optimal temperature of glucoamylase was 40 to 50°C with rapid decline of activity at 60°C. The K_m of α-amylase with soluble starch as the substrate was 1·15 mg/ml and the K_m of glucoamylase with the same substrate was 10·31 mg/ml. Glucoamylase was able to hydrolyze α-1, 4 and α-1,6 glucosidic linkages, as demonstrated by its ability to hydrolyse maltose and isomaltose respectively, whereas α-amylase could hydrolyse α-1,4 glucosidic linkages only. α-Amylase was shown to be a glycoprotein, whereas no carbohydrates were associated with glucoamylase.     

Metaproteomics reveals protein composition of multiple saccharifying enzymes in nongxiangxing daqu and jiangxiangxing daqu under different thermophilic temperatures

Daqu provides hydrolytic enzymes that saccharifying the macromolecular carbohydrate in fermenting grains. The process of saccharification is the premise of Baijiu production. But little is known about the protein composition associated with the activity of saccharifying enzymes in daqu. This study investigated related proteins between nongxiangxing daqu and jiangxiangxing daqu by metaproteomics. We present that the amylase system (α-amylase, glucoamylase and α-glucosidase) and cellulase system (cellobiohydrolases, endoglucanase and β-glucosidase) are regarded as the main saccharifying enzymes. Combined with protein-based taxonomic annotation, the key proteins involving in amylase system were mainly from Kroppenstedtia, Lichtheimia, Byssochlamys and Thermomyces, while Thermoascu contributed the most to cellulase system. Moreover, it was found that the up-regulated amylase proteins at thermophilic fermentation period in jiangxiangxing daqu in comparison with nongxiangxing daqu were affiliated to Aspergillus, Rhizomucor, Byssochlamys and Thermomyces, along with Thermoascu contributing to cellulase system. These findings will provide clues for improving saccharification efficiency in Baijiu production.     

Intensification of Amylase Synthesis with Aspergillus niger by Way of Multistage Mutagenization

Conidia of amylolytically active Aspergillus niger C strain were subjected to four-stage mutagenization, using different combinations of mutagens in each stage (stage I – UV irradiation + ethyleneimine, stage II – UV irradiation + nitrosomethylurea, stage III – UV irradiation + N-methyl-N′-nitro-N-nitrosoguanidine, stage IV – acryflavine). In all, 378 strains were isolated after mutagenization, which were initially evaluated for the total amylolytic activity by the method of test-tube microculture. Nine most active mutants were then tested in submerged culture determining the activity of glucoamylase and α-amylase in post-culture liquid. The activity of α-amylase increased from over 7 to nearly 31%, and that of glucoamylase from 4 to over 61%.     

Analysis of Volatile Compounds in Shochu Koji,Sake Koji,and Steamed Rice by Gas Chromatography‐Mass Spectrometry

The volatile compounds in three types of koji (yellow, white, and black) and steamed rice were investigated using gas chromatography‐mass spectrometry. The objective of this study was to characterize each koji type based on its profile of volatile compounds. From the steamed rice and rice koji samples examined, a total of 29 volatile compounds were identified. Butanal, hexanal, dimethyl trisulfide and cyclohexyl isothiocyanate were detected only in steamed rice. The volatile profile of yellow koji (sake koji) was distinct from those of white and black kojis (shochu koji). On the basis of relative peak areas, alcohols and aldehydes were identified as being more abundant in yellow koji. White and black kojis had similar volatile profiles, and had a greater abundance of furans than yellow koji. We assume that the different suites of volatile compounds identified in shochu and sake kojis impart the respective characteristic flavours to shochu and sake.     

Properties of Residual Starches of Sugary-2 and Sugary-2 Opaque-2 Maize Following Amylase Hydrolysis

Properties of residual starches of sugary-2 opaque-2 and sugary-2 maize starch granules hydrolyzed with glucoamylases were investigated. A crude and two crystalline glucoamylases were used. The amylopectin fractions of both starches hydrolyzed easier than that of amylose with all enzymes. Residual starches hydrolyzed by the crude glucoamylase accumulated low-molecular weight materials, which was not observed in residual starches attacked by crystalline glucoamylase. It was suggested that in the crude enzyme the contaminating α-amylase caused the accumulation of the minified fraction. It is also suggested that the crystalline region of sugary-2 opaque-2 starch may consist of a mixture of A-type and B-type patterns. Evidence for this was from observation of the changes in X-ray diffraction patterns of residual starch following amylase and acid hydrolysis.     

Suppression of Fusarium graminearium growth by differently structured starch types

We investigated the growth behavior and amylolytic enzymes of Fusarium graminearum cultivated on different types of native starch granules including barley (A‐type crystalline polymorph), potato and Curcuma zedoaria (B‐type crystalline polymorph), cassava (C‐type crystalline polymorph), and high AM maize (A + Vh‐type crystalline polymorphs). F. graminearum grew poorly on B‐type starches and the accumulation of biomass was similar to that obtained for fungi cultivated under carbohydrate starvation conditions. In comparison, three‐ to fivefold higher accumulation of fungal biomass was observed for growth on the A‐, C‐ and A + Vh‐type starches. Fungal glucoamylase and α‐amylase activity increased over time in the presence of native starch granules. Interestingly, resistant B‐type starches induced the highest amylolytic activity indicating that F. graminearum interacts with B‐type granules although only limited degradation occur. Starch degradation products maltose and malto‐oligosacharides was found to increase glucoamylase and α‐amylase activity, whereas glucose acted as a catabolite repressor.