米曲霉giF-10内切葡聚糖酶基因的克隆表达及酶学性质分析

根据NCBI(GenBank Accession No.:D83732.1)中注册的米曲霉内切葡聚糖酶CelB基因序列设计引物,以本实验室自行筛选的天然米曲霉基因组DNA为模板,PCR高保真扩增出内切葡聚糖酶基因eg,将其定向插入到酵母表达载体pPICZαA上,转化酵母宿主菌X33,刚果红水解圈筛选结果表明已成功表达。SDS-PAGE分析表明,表达产物分子量约为65 ku。对酵母表达工程菌X33-eg进行发酵条件优化,结果显示最适甲醇诱导浓度为0.75%,用1 L三角瓶诱导培养5天达到最高酶活120 U/mL。对重组酶的酶学性质分析表明,其最适反应pH值和温度分别为pH4.0和45℃,在30～45℃和pH3.4～pH6.9范围内可保持内切葡聚糖酶最高酶活力70%以上。     

Efficient production of cellulolytic and xylanolytic enzymes by the rumen anaerobic fungus, Neocallimastix frontalis, in a repeated batch culture

In a batch culture of Neocallimastix frontalis in a medium (pH 6.8) containing 8 g.l(-1) cellulose, the concentrations of the fermentation products and the cellulolytic and xylanolytic enzymes did not increase in comparison with those of cultures in a medium containing 4 g.l(-1) cellulose. Therefore, kinetic studies were performed to determine the effect that products such as acetate, formate, lactate and ethanol have in inhibiting the growth. The reduction of the specific growth rate by the fermentation products could be expressed by using a noncompetitive inhibition model, and it was found that at low concentrations, acetate was the strongest inhibitor among the inhibitory products studied. In order to reduce the inhibition by the fermentation products, a repeated batch culture was carried out whereby none of the fatty acids exceeded 50 mM. In this repeated batch culture, xylanase, endoglucanase, beta-glucosidase, and avicelase were continuously produced in the medium during cultivation, and 18200, 4550, 3790 and 129 IU g.l(-1) of these enzymes, respectively, were produced up to 20 d of culture.     

Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus

Co-immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. The concentration of ethanol produced by immobilized cells was higher than that by free cells of S. diastaticus and Z. mobilis in mixed-culture fermentation. In repeated-batch fermentation using co-immobilized cells, the ethanol concentration increased to 53.5 g/l. The co-immobilized gel beads were stable up to seven successive batches. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h.     

Production of bacterial endoglucanase from pretreated oil palm empty fruit bunch by bacillus pumilus EB3

In this study, endoglucanase was produced from oil palm empty fruit bunch (OPEFB) by a locally isolated aerobic bacterium, Bacillus pumilus EB3. The effects of the fermentation parameters such as initial pH, temperature, and nitrogen source on the endoglucanase production were studied using carboxymethyl cellulose (CMC) as the carbon source. Endoglucanase from B. pumilus EB3 was maximally secreted at 37 degrees C, initial pH 7.0 with 10 g/l of CMC as carbon source, and 2 g/l of yeast extract as organic nitrogen source. The activity recorded during the fermentation was 0.076 U/ml. The productivity of the enzyme increased twofold when 2 g/l of yeast extract was used as the organic nitrogen supplement as compared to the non-supplemented medium. An interesting finding from this study is that pretreated OPEFB medium showed comparable results to CMC medium in terms of enzyme production with an activity of 0.063 U/ml. As OPEFB is an abundant solid waste at palm oil mills, it has the potential of acting as a substrate in cellulase production.     

Ethanol production by a new pentose-fermenting yeast strain, Scheffersomyces stipitis UFMG-IMH 43.2, isolated from the Brazilian forest

The ability of a recently isolated Scheffersomyces stipitis strain (UFMG-IMH 43.2) to produce ethanol from xylose was evaluated. For the assays, a hemicellulosic hydrolysate produced by dilute acid hydrolysis of sugarcane bagasse was used as the fermentation medium. Initially, the necessity of adding nutrients (MgSO(4)·7H(2)O, yeast extract and/or urea) to this medium was verified, and the yeast extract supplementation favoured ethanol production by the yeast. Then, in a second stage, assays under different initial xylose and cell concentrations, supplemented or not with yeast extract, were performed. All these three variables showed significant (p < 0.05) influence on ethanol production. The best results (ethanol yield and productivity of 0.19 g/g and 0.13 g/l/h, respectively) were obtained using the hydrolysate containing an initial xylose concentration of 30 g/l, supplemented with 5.0 g/l yeast extract and inoculated with an initial cell concentration of 2.0 g/l. S. stipitis UFMG-IMH 43.2 was demonstrated to be a yeast strain with potential for use in xylose conversion to ethanol. The establishment of the best fermentation conditions was also proved to be of great importance to increasing the product formation by this yeast strain. These findings open up new perspectives for the establishment of a feasible technology for ethanol production from hemicellulosic hydrolysates.     

Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

响应面优化清酒发酵工艺研究

采用Plackett-Burman设计法和响应面分析法对清酒发酵工艺进行优化。先用Plackett-Burman设计从5个因素中筛选出对清酒品质有显著影响的因素,再用最陡爬坡实验及Box-behnken设计进一步优化。结果表明,酵母接种量,米曲接种量和发酵温度是影响清酒品质的显著因素,优化后的发酵工艺:酵母接种量1.5%(mL/g饭米),米曲接种量23%(g/g饭米),发酵温度13℃,发酵时间35d,加水量125%(g/g饭米)。在此发酵工艺下可制得品质优良的清酒。     

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.     

Effect of yeast culture and Aspergillus oryzae fermentation extract on ruminal characteristics and nutrient digestibility

Four nonpregnant and nonlactating Holstein cows fitted with ruminal fistulas were assigned to each of four diets in a 4 X 4 Latin square design. Dietary treatments were 1) basal diet containing 50% concentrate; 2) basal diet plus 90 g/d yeast culture; 3) basal diet plus 2.63 g/d Aspergillus oryzae fermentation extract; 4) basal diet plus 90 g/d of A. oryzae fermentation extract and yeast culture. Cows were fed diets at a rate of 86 g DM/kg BW.75 for 14 d adaptation followed by an 8-d collection period. Digestibility of dry matter was increased by A. oryzae and A. oryzae and yeast culture combination treatments. Digestibility of CP was increased regardless of fungal culture addition. Hemicellulose digestibility, percent ruminal cellulolytic organisms, and acetate to propionate ratio were increased by the addition of fungal supplements.     

乳酸菌和米根霉混合固态发酵对黑青稞生化成分的动态变化

本研究以黑青稞为原料,探究2株乳酸菌(R₁、R₅)、1株米根霉单独及混合发酵黑青稞过程中生化成分的动态变化。结果表明,3株菌株单独发酵过程中,R₅发酵的黑青稞其pH明显下降,最低达到4.32±0.01,且总酸含量显著高于其余两株菌,最终达到2.52%±0.12%;而米根霉发酵的黑青稞其还原糖含量及乙醇含量明显高于R₁、R₅,最高分别达到(6.94±0.22)、(9.19±0.49) mg/g;R₁、R₅发酵得到的氨基酸态氮含量较为接近且高于米根霉,最终分别达到(0.10±0.01)、(0.11±0.01) mg/g。混合发酵结果表明,混菌发酵能显著提高黑青稞发酵制品的营养品质、改善其风味,其中R₅+米根霉混合发酵的黑青稞制品在发酵过程中pH下降较快,且产酸较多,pH由降低5.65±0.08至4.16±0.08,总酸含量由0.48%±0.03%增加至7.16%±0.03%,乙醇含量较低,最终仅为(8.25±0.35) mg/g,氨基酸态氮含量较高,最终达到(0.11±0.01) mg/g,同时感官品质优于其他发酵组合,48 h评分最高,达到(86±0.89)分,适宜发酵制备低酒精、高营养型黑青稞制品。而R₁+米根霉混合发酵的黑青稞制品产酸较少,但其乙醇含量最高,最终达到(9.80±0.60) mg/g,适宜发酵低酸、高酒精黑青稞发酵制品。综合考虑,最终确定混合固态发酵48 h时其黑青稞制品营养品质及感官特性最佳。根据发酵黑青稞产品的品质及风味要求,可选择适宜的混合发酵菌株。该研究可为开发黑青稞产品工业化生产奠定基础。     

柑橘皮渣液态发酵制取曲酸工艺优化

以柑橘皮渣为原料,接种米曲霉,以液态摇瓶发酵的形式进行曲酸生产的研究。采用Box-Behnken中心组合设计,通过响应面法优化柑橘皮渣发酵的最佳工艺。结果表明,最佳发酵参数为米曲霉接种量4%、柑橘皮渣添加量13 g/100 mL、酵母膏添加量1.5 g/100 mL、MgSO4·7H2O添加量0.05 g/100 mL,在此条件下发酵制得的曲酸质量浓度最高,为（11.84±1.44） mg/mL。     

Malo-ethanolic fermentation in grape must by recombinant strains of Saccharomyces cerevisiae.

Recombinant strains of Saccharomyces cerevisiae with the ability to reduce wine acidity could have a significant influence on the future production of quality wines, especially in cool climate regions. L-Malic acid and L-tartaric acid contribute largely to the acid content of grapes and wine. The wine yeast S. cerevisiae is unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of a malo-ethanolic fermentation. However, strains of Sz. pombe are not suitable for vinification due to the production of undesirable off-flavours. Heterologous expression of the Sz. pombe malate permease (mae1) and malic enzyme (mae2) genes on plasmids in S. cerevisiae resulted in a recombinant strain of S. cerevisiae that efficiently degraded up to 8 g/l L-malic acid in synthetic grape must and 6.75 g/l L-malic acid in Chardonnay grape must. Furthermore, a strain of S. cerevisiae containing the mae1 and mae2 genes integrated in the genome efficiently degraded 5 g/l of L-malic acid in synthetic and Chenin Blanc grape musts. Furthermore, the malo-alcoholic strains produced higher levels of ethanol during fermentation, which is important for the production of distilled beverages.     

米根霉高效利用玉米淀粉生产乳酸的发酵条件优化

研究米根霉HB12利用玉米淀粉生产乳酸的发酵条件优化。从土壤中新筛选得到一株以高浓度玉米淀粉为原料发酵生产乳酸的米根霉HB12。通过单因素及正交试验,得到最佳发酵培养基组成(g/L)为：玉米淀粉140、NH4Cl 2、KH2PO4 0.3、MgSO4·7H2O 0.3、ZnSO4·7H2O 0.05、CaCO3 80;最佳培养条件为：摇瓶装液量50mL/250mL,接种量为2.5×106个孢子,35℃、200r/min培养108h。该条件下,菌株最大产酸量为104.9g/L,产酸速率为0.97g/(L·h),对玉米淀粉的转化率达74.9%,产酸量提高了49.4%。此菌株能够直接高效利用价格低廉来源广泛的玉米淀粉发酵生产乳酸,具有很好的工业应用前景。     

Sake yeast strains have difficulty in entering a quiescent state after cell growth cessation

Sake yeast strains produce a high concentration of ethanol during sake brewing compared to laboratory yeast strains. As ethanol fermentation by yeast cells continues even after cell growth stops, analysis of the physiological state of the stationary phase cells is very important for understanding the mechanism of producing higher concentrations of ethanol. We compared the physiological characteristics of stationary phase cells of both sake and laboratory yeast strains in an aerobic batch culture and under sake brewing conditions. We unexpectedly found that sake yeast cells in the stationary phase had a lower buoyant density and stress tolerance than did the laboratory yeast cells under both experimental conditions. These results suggest that it is difficult for sake yeast cells to enter a quiescent state after cell growth has stopped, which may be one reason for the higher fermentation rate of sake yeast compared to laboratory yeast strains.     

Cell surface engineering of yeast: construction of arming yeast with biocatalyst

A cell surface engineering system of yeast Saccharomyces cerevisiae has been established and novel yeasts armed by biocatalysts (enzymes-glucoamylase, alpha-amylase, CM-cellulase, beta-glucosidase, and lipase), termed "arming yeasts", were constructed. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half of yeast alpha-agglutinin and expressed in S. cerevisiae. Glucoamylase was shown to be displayed on the cell surface in its active form and anchored covalently to the cell wall. S. cerevisiae itself is unable to utilize starch, while the surface-engineered yeast could grow on starch as the sole carbon source. For further improvement of the ability to directly ferment starchy materials by the cell surface-engineered yeast, engineered yeasts displaying two amylolytic enzymes on the cell surface were constructed. The gene encoding R. oryzae glucoamylase with its own secretion signal peptide and a truncated fragment of the alpha-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast alpha-factor were fused with the gene encoding the C-terminal half of the yeast alpha-agglutinin. The surface-engineered yeast co-displaying glucoamylase and alpha-amylase by the integration of their genes into the chromosomes could grow faster on starch as the sole carbon source than the engineered cells displaying only glucoamylase. The system was further applied to the construction of a novel cellulose-utilizing yeast by displaying cellulolytic enzymes in their active form on the cell surface of S. cerevisiae. Engineered yeasts co-displaying FI-carboxymethylcellulase (CM-cellulase), one of the endo-type cellulases, and beta-glucosidase from Aspergillus aculeatus on their cell surface were also constructed. The yeasts displaying these cellulases were given the ability to assimilate cellooligosaccharide, suggesting the possibility that the assimilation of cellulosic materials may be carried out by S. cerevisiae displaying heterologous cellulase proteins on the cell surface. The system has also been used for the cell surface display of R. oryzae lipase (ROL). Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance the lipase activity. The insertion of an appropriate length of a linker peptide as a spacer is effective in the display of ROL, having the active region at the C-terminal portion, on the cell surface. Thus, cell surface engineering will be capable of conferring novel additional abilities upon living cells and will herald a new era in the field of biotechnology.     

Effective onion vinegar production by a two-step fermentation system

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/l total sugar, was smoothly converted to onion alcohol containing 30.6 g/l ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain IR-2. Stable operation was possible and the maximum productivity was about 8.0 g/l/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/l/h and 37.9 g/l, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.     

米曲霉Y6产中性蛋白酶和内切葡聚糖酶的研究

以提高中性蛋白酶和内切葡聚糖酶酶活为目的,优化了米曲霉Y6的制曲工艺。在单因素的试验基础上,采用正交试验优化的米曲霉Y6中性蛋白酶的最佳制曲工艺参数为温度30℃,接种量1.0×107个孢子/g干基,培养时间42h,含水量80%,其酶活达到3637U/g;内切葡聚糖酶的最佳制曲工艺参数为温度为30℃,接种量0.25×107个孢子/g干基,培养时间48h,含水量100%,其酶活达到810U/g。     

Beer brewing using a fusant between a sake yeast and a brewer's yeast

Beer brewing using a fusant between a sake yeast (a lysine auxotrophic mutant of sake yeast K-14) and a brewer's yeast (a respiratory-deficient mutant of the top fermentation yeast NCYC1333) was performed to take advantage of the beneficial characteristics of sake yeasts, i.e., the high productivity of esters, high tolerance to ethanol, and high osmotolerance. The fusant (F-32) obtained was different from the parental yeasts regarding, for example, the assimilation of carbon sources and tolerance to ethanol. A brewing trial with the fusant was carried out using a 100-l pilot-scale plant. The fusant fermented wort more rapidly than the parental brewer's yeast. However, the sedimentation capacity of the fusant was relatively low. The beer brewed using the fusant contained more ethanol and esters compared to that brewed using the parental brewer's yeast. The fusant also obtained osmotolerance in the fermentation of maltose and fermented high-gravity wort well.     

ETHYL CARBAMATE FORMATION IN GRAIN-BASED SPIRITS: PART III. THE PRIMARY SOURCE

Low concentrations of ethyl carbamate, which may occur in distilled spirits are shown to originate from traces of cyanide, produced in distillates by thermal decomposition of the cyanohydrin of isobutyraldehyde (IBAC) which is present in fermented wash. This compound arises during fermentation by the hydrolytic action of yeast beta-glucosidase on a naturally-occurring cyanogenic glycoside, identified as epiheterodendrin (EPH). EPH, which is a heat-stable precursor of IBAC is located in the acrospires of malted barley and is readily transferred to worts during mashing. Although many barley varieties display the ability to produce volatile cyanide, some low-yielders have been identified. EPH is absent from unmalted barley and its development is strongly influenced by malting conditions. Conversion of measurable cyanide (MC) into ethyl carbamate takes place during- or after distillation but, apparently, not before.     

两种非酿酒酵母与米曲霉互作特性研究

微生物互作在白酒酿造中扮演重要角色。费比恩塞伯林德纳氏酵母（Cyberlindnera fabianii）和葡萄牙棒孢酵母（Clavispora lusitaniae）是白酒酿造中常见的非酿酒酵母,但它们与霉菌的互作关系尚不清晰。本研究围绕感官评价、挥发性风味物质含量、酵母菌生物量、酶活力和代谢产物等方面,探讨了费比恩塞伯林德纳氏酵母HY-11和葡萄牙棒孢酵母HY-21与米曲霉（Aspergillus oryzae）M-08在酿造过程中的相互作用。结果表明,两种非酿酒酵母分别与米曲霉M-08混菌培养,费比恩塞伯林德纳氏酵母HY-11和葡萄牙棒孢酵母HY-21的生物量分别比单一酵母菌发酵提高4.96倍和0.97倍,α-淀粉酶活力分别提高70.31倍和107.73倍,表明两酵母菌对米曲霉M-08有依赖性。相比葡萄牙棒孢酵母HY-21,费比恩塞伯林德纳氏酵母HY-11与米曲霉M-08混菌发酵风味更好,发酵6 d具有浓郁哈密瓜香、酒香和酯香味,挥发性风味物质总量最高1408.97 μg/g,比单一条件下发酵提升381.87倍。米曲霉M-08与费比恩塞伯林德纳氏酵母HY-11和葡萄牙棒孢酵母HY-21有较好的相互作用,混菌发酵比单一酵母菌发酵体系游离氨基酸水平分别提高105.02和3837.29 μg/g,酯化酶活力分别增加27.67和387.82 U/g,从而促进挥发性风味物质的代谢。本研究阐明了两种非酿酒酵母与米曲霉M-08的相互关系和其风味特色,为其合理应用提供了理论依据。