Maximum volumetric production of β‐galactosidase by Kluyveromyces fragilis in fed‐batch culture with automatic control

The production of β‐galactosidase by Kluyveromyces fragilis was studied in different culture systems, with dissolved oxygen concentration control and using defined media. An operating strategy of fed‐batch culture with automatic control of substrate addition regulated by dissolved oxygen concentration, consisting of the replacement of variable volumes of broth by fresh medium (once the fed‐batch culture has finished), was designed. The volumetric enzyme productivity (Q_p, 13 600 UI dm^?3 h^?1) obtained was 38% higher than that reached in continuous culture of K fragilis with dissolved oxygen concentration control and far higher than that obtained by batch culture of K fragilis under the same aeration conditions. © 2002 Society of Chemical Industry     

Research note: Effect of dissolved oxygen level on lactase production by Kluyveromyces fragilis

The influence of dissolved oxygen (% DO) on lactase production by Kluyveromyces fragilis (NRRL-Y-1109) in chemostat culture using a defined medium was studied. The aim was to determine conditions for both high specific enzyme activity and high volumetric enzyme productivity. Significant differences in the specific enzyme activity and specific and volumetric enzyme productivity were found at the corresponding steady states when the DO was varied between 0 and 90%. Maximum lactase production was attained at 10% DO. Under this condition the best results were an enzyme activity of 5910 IU g⁻¹, specific production rate of 1810 IU g⁻¹ h⁻¹ and volumetric production rate of 1530 IU dm⁻³ h⁻¹. This seem to be due to the fact that at low aeration conditions the yeast metabolism is more reductive and as a consequence it verifies both higher specific lactose consumption rate and higher enzyme expression than in full aeration conditions. The results of this investigation are also compared with those of other studies of lactase production by Kluyveromyces sp. © 1998 Society of Chemical Industry     

EFFECTS OF OPERATING PARAMETERS ON THE GROWTH OF KLUVEROMYCES FRAG I LIS ON CHEESE WHEY

The fermentation of Kluveromycesfragitis on cheese whey was studied to assess the influence of temperature, pH, aeration rate, and lactose concentration on the biomass yield. The observations were analyzed statistically to determine the optimal operating conditions, A total of 17 experiments were conducted, 16 at the design points and 1 additional run to observe the growth of K.fragilis at the design's center point. The statistically significant factors were the temperature and lactose concentration. The optimal conditions for the growth of K.fragilis were: lactose concentration at 5%, temperature at 303 K, aeration rate at 2.0 vol/vol/min, and pH at 5,     

Production of an Alcoholic Beverage by Fermentation of Whey Permeate with Kluyveromyces fragilis II: Aroma Composition

Whey permeate was used for an alcoholic fermentation with Kluyveromyces fragilis (CECT 1123). The aim of the present study was the production of an alcoholic beverage of low alcoholic proof with acceptable concentrations of fusel alcohols. The effects of temperature and agitation on the concentration of volatile compounds was assayed in batch cultures. In addition, the production of acetaldehyde, ethyl acetate and fusel alcohols was studied in continuous culture because of interest in a continuous industrial process. Final concentrations of fusel alcohol were slightly lower at higher temperatures, while the specific production rate increased with temperature. Concentrations were suitable, in all cases, for a beverage. When agitation was assayed, an important increase in fusel alcohol concentrations was found at higher agitations. In continuous culture, the acetaldehyde, ethyl acetate and fusel alcohol concentrations were normal for an alcoholic beverage.     

Effect of some physico-chemical treatments on the kinetics of autolysed-yeast extract production from whey

In this work, the effects of salt and mechanical cell breaking on the overall activity of the endogenous enzymes of Kluyveromyces fragilis grown on whey-based media were studied at the optimal values of temperature (54°C) and initial concentration of yeast and whey protein (100 g dm^?3) previously selected. An unstructured kinetic model of first order was found to be capable of simulating quite satisfactorily the time course of residual suspended solids (Y) in the slurry, as well as Kjeldahl (KP) and Lowry (LP) protein and total carbohydrates (G) accumulation in the clear extract. The autolysis kinetic rate constant (k_a), and yield factors for Kjeldahl and Lowry protein and reducing sugars on total solids hydrolysed were found to be insensitive to the salt level and preliminary mechanical disruption of cells. Nevertheless, maximum production of yeast-whey protein extracts was achieved when the endogenous enzymes of ground cells are induced by 5% w/v salt addition. Except for their respective salt contents, both autolysates exhibited no apparent difference in chemical composition.     

人凝血因子IX在脆壁克鲁维酵母中的表达与分泌

通过改造人凝血因子IX基因(h-FIX)使其与乳酸克鲁维酵母(Kluyveromyces lactis)的分泌信号序列(Kss)融合，并置于脆壁克鲁维酵母(K．fragilis)LAC4基因调控型启动子的控制之下，构建成h-FIX基因表达盒。将此表达盒插入克鲁维酵母的整合质粒，得到了一个高稳定的h-FIX基因整合表达载体pHKB202-FP。经摇瓶培养并以半乳糖诱导此表达载体转化的酵母菌株，ELISA检测显示酵母上清液中的人凝血因子IX表达量可达到约200ng/ml。此结果表明人凝血因子IX蛋白首次成功地在脆壁克鲁维酵母中得到了表达和分泌。这对于促进人凝血因子IX的基因工程产业化具有实际意义。     

脆壁克鲁维酵母KfADE5,7基因的克隆及其5''''非编码区结构与功能研究

通过补偿乳酸克鲁维酵母(Kluyveromyces lactis)KlADE5,7基因突变,从脆壁克鲁维酵母(Kluyveromyces fragilis)基因组文库中克隆了KfADE5,7基因.该基因全长4975bp,编码793个氨基酸,氨基酸序列与已知的酿酒酵母(Saccharomyces cerevisiae)ScADE5,7基因编码区有68.3%的同源度.利用KfADE5,7-lacZ融合基因,对5'非编码区进行缺失分析,发现了具有完整启动子功能的区域,在启动子上游还发现了两个增强转录和两个减弱转录的区域.     

脆壁克鲁维酵母乳糖酶的提取与发酵优化

通过试验研究了一株脆壁克鲁维酵母胞内乳糖酶的破壁提取及其发酵培养基成分与培养条件优化,确定了该株酵母合适的破壁方法及最佳发酵培养条件。结果表明,采用球磨机对该酵母进行处理可以较好地释放胞内乳糖酶。其发酵生产乳糖酶的最佳培养基成分为：乳糖4%,酵母粉3%,MnCl21mol/L;最佳培养条件：接种量10%,温度28℃,初始pH7～7.5,摇床转数180r/min,培养时间36h。在此条件下培养,乳糖酶活力达1100U/mL,4140U/g干菌体。