Role of antioxidant enzymatic defences against oxidative stress H(2)O(2) and the acquisition of oxidative tolerance in Candida albicans

In Candida albicans, trehalose plays an essential role as a protector of cell integrity against oxidative challenge. A double homozygous mutant, tps1/tps1, deficient in trehalose synthesis, displayed severe cell mortality when exposed to high H(2)O(2) concentrations, compared with its congenic parental (CAI-4) strain (Alvarez-Peral et al., 2002). We have examined the putative role of a set of well-known antioxidant enzymes as components of the defence mechanism against oxidative challenges. When exposed to mild non-lethal oxidative treatment (0.5 mM H(2)O(2)), a significant induction of catalase, glutathione reductase (GR), and Cu,Zn-superoxide dismutase (SOD) was recorded in tps1/tps1 exponential cultures. However, in CAI-4 cells, subjected to the same conditions, there was only a clear activation of catalase, Mn-SOD and Cu,Zn-SOD activities. The degree of activation was always much more pronounced in the trehalose-deficient mutant than in its wild-type counterpart, except for Mn-SOD activity. After exposure to severe oxidative stress (50 mM H(2)O(2)) only GR and catalase activities increased in tps1/tps1 cultures, whereas in CAI-4 cells GR but not catalase was induced. In both cell strains, 50 mM H(2)O(2) caused inhibition of the Mn- and Cu,Zn-SOD isozymes, this inhibition being more pronounced in tps1/tps1 cells. C. albicans is able to acquire adaptive oxidative tolerance by pretreatment with a low non-stressing concentration of H(2)O(2) before exposure to a drastic oxidative challenge. When these antioxidant activities were measured during the adaptive response, a greater degree of enzymatic antioxidant induction was consistently observed in the tps1/tps1 mutant with respect to the CAI-4 strain. Together with a higher intrinsic sensitivity of tps1/tps1 cells, we suggest that this unexpected increase might be explained in terms of a compensatory mechanism to overcome the lack of endogenous trehalose upon drastic oxidative exposure, although this induction was not sufficient to improve the percentage of cell viability.     

Characterization of a disulphide-bound Pir-cell wall protein (Pir-CWP) of Yarrowia lipolytica

In this work we have studied the disulphide-bound group of cell wall mannoproteins of Yarrowia lipolytica and Candida albicans. In the case of Y. lipolytica, SDS-PAGE analysis of the beta-mercaptoethanol-extracted material from the purified cell walls of the yeast form, showed the presence of a main polypeptide of 45 kDa and some minor bands in the 100-200 kDa range. This pattern of bands is similar to that obtained in identical extracts in Saccharomyces cerevisiae (Moukadiri et al., 1999), and besides, all these bands cross-react with an antibody raised against beta-mercaptoethanol-extracted material from the purified cell walls of S. cerevisiae, suggesting that the 45 kDa band could be the homologue of Pir4 of S. cerevisiae in Y. lipolytica. To confirm this possibility, the amino-terminal sequences of two internal regions of the 45 kDa protein were determined, and degenerate oligonucleotides were used to clone the gene. The gene isolated in this way codes a 286 amino acid polypeptide that shows homology with the Pir family of proteins of S. cerevisiae (Russo et al., 1992; Toh-e et al., 1993), accordingly we have named this gene YlPIR1. Disruption of YlPIR1 led to a slight increase in the resistance of the cells to calcofluor white, Congo red and zymolyase, but did not cause changes in cell morphology, growth rate or morphological transition.     

Cloning, characterization and functionality of the orotidine-5'-phosphate decarboxylase gene (URA3) of the glycolipid-producing yeast Candida bombicola

Candida bombicola is a yeast species known to synthesize glycolipids. Although these glycolipids find several industrial, cosmetic and pharmaceutical applications, very little is known about the genetics of C. bombicola. A basic tool for genetic study and modification is the availability of an efficient transformation and selection system. In order to develop such a system, the URA3 gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes for an enzyme of 262 amino acids and shows high homology with the known orotidine-5'-phosphate decarboxylases of several other yeast species. The functionality of the gene was proved by complementation of a URA3-negative Saccharomyces cerevisiae strain.     

星形假丝酵母在强化赤霞珠葡萄酒玫瑰香气中的应用

采用顶空固相微萃取结合气质联用（HS-SPME-GC-MS）技术测定酿酒酵母（Saccharomyces cerevisiae）CEC01和星形假丝酵母（Candida astrulatum）X11单菌发酵、共同发酵及顺序发酵葡萄酒中的挥发性香气物质,并对结果进行主成分分析（PCA）及聚类分析（CA）,探讨星形假丝酵母X11强化赤霞珠葡萄酒玫瑰香气的条件。结果表明,菌株X11单菌发酵和混合发酵葡萄酒中苯乙醇含量显著高于酿酒酵母CEC01单菌发酵（P＜0.05）,其中,菌株X11和CEC01以1∶10的比例顺序接种发酵的赤霞珠葡萄酒中苯乙醇含量高达12.85 mg/L,赋予葡萄酒浓烈的玫瑰香味。此外,菌株X11在发酵过程中还产生了乳酸丙酯（3.64～7.08 mg/L）、丁酸乙酯（2.55～30.18 μg/L）、辛酸乙酯（476.52～1 997.43 μg/L）等挥发性风味物质,赋予葡萄酒果香和奶酪香。结果表明,星形假丝酵母X11可以显著改善葡萄酒风味,具有一定商业开发价值。     

Evaluation of a single and combined inoculation of a Lactobacillus pentosus starter for processing cv. Arbequina natural green olives

The production of Arbequina naturally green olives is a traditional and spontaneous process in which lactic acid bacteria (LAB) and yeasts are present. To better control the fermentation of olives, strains of LAB and yeasts that had been isolated from brines were used in this study.     

产朊假丝酵母生长条件的优化

通过安瓿管复壮的产朊假丝酵母,用YPD培养基进行生长曲线的测定,得出12h是此种酵母菌的生长最优条件。对产朊假丝酵母进行单因素条件筛选,包括培养温度、摇床转速、碳氮比,实验得出温度在30℃、转速180r/min、碳氮比(葡萄糖和蛋白胨的质量比)为3∶1时菌体密度最高。通过采用L9(34)正交实验设计确定各因素对酵母菌生长的影响,优化条件为培养时间13h、温度30℃、转速160r/min、碳氮比约3∶1,此时菌体密度最高。     

无溶剂体系中毕赤酵母表面展示CALB全细胞催化合成肉桂醇酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞(Pp-CALB)为全细胞催化剂,以不同脂肪酸作为酰基供体,在无溶剂体系中和肉桂醇发生酯化反应合成肉桂醇酯。探究了Pp-CALB对于不同酰基供体的选择性,并且对反应温度、摇床转速、酶加量、底物摩尔比、酶的水活度和反应体系等一系列影响酶催化反应的因素进行了优化,建立了无溶剂体系中肉桂醇酯的酶法合成工艺。结果表明,在底物摩尔比为2:1,摇床转速为120 r/min,反应温度为50℃的条件下,添加初始水活度为0.53的Pp-CALB 0.05 g,反应3h,肉桂醇的酯化率可达81.34%,在最适反应条件下,使用Pp-CALB连续反应8次后,肉桂醇的酯化率仍然能达到70.00%以上,具有较好的操作稳定性。     

扁平云假丝酵母与酿酒酵母混合发酵对葡萄酒乙醇含量及香气的影响

为降低葡萄酒中的乙醇含量,采用不同接种方式（同时接种和顺序接种）在两个温度下（13?℃和23?℃）进行扁平云假丝酵母（Candida humilis）与酿酒酵母（Saccharomyces cerevisiae）的发酵实验。结果表明：23 ℃时混合发酵除高产甘油外,还有效降低了乙醇含量,其中与S. cerevisiae单独发酵相比,顺序接种可降低乙醇体积分数约2.59%,降醇幅度达到17.56%。在香气方面,混合发酵能产生较高含量的酯类物质,其中顺序接种最为明显,如乙酯类物质总量和乙酸乙酯含量分别增加了18.30%和16.03%,丁酸乙酯含量提高了2.77?倍,明显增加了花香和果香;13?℃时,混合发酵显著提高了β-大马士酮的含量,其中同时接种发酵增加26.19%。结果表明C. humilis与S. cerevisiae进行混合发酵具有较好的降醇效果,可以为葡萄酒的降醇研究提供一种新的解决方法。     

真空冷冻干燥橄榄假丝酵母的制备及其对苹果青霉病的防治

选用真空冷冻干燥技术,糖醇类试剂包括海藻糖、蔗糖、乳糖、山梨糖醇,抗氧化类试剂谷氨酸钠,以及赋形剂包括脱脂奶粉和β-环糊精三大类试剂作为保护剂,通过保护剂的单因素筛选试验、正交试验得到真空冷冻干燥橄榄假丝酵母的最优保护剂配方为海藻糖添加量15%、谷氨酸钠添加量2%和脱脂奶粉添加量10%,所得酵母存活率为69.7%。此外,通过比较冻干橄榄假丝酵母和新鲜橄榄假丝酵母对苹果果实青霉病的发病率和病斑直径的研究,并测定冻干橄榄假丝酵母和新鲜橄榄假丝酵母接种处理后酵母在苹果果实伤口处的生长动态,以期制备性能良好的生物防治试剂。