Production of Yarrowia lipolytica lipase LIP2 in Pichia pastoris using the nitrogen source‐regulated FLD1 promoter

BACKGROUND: Yarrowia lipolytica lipase LIP2 (YlLIP2) is an important industrial enzyme that has many potential applications. Although it has been successfully expressed in Pichia pastoris under the control of the AOX1 promoter (pAOX1), there have been many efforts to develop new alternative promoters to pAOX1 in order to avoid using methanol in the fermentation. Investigation of YlLIP2 production in P. pastoris using the formaldehyde dehydrogenase 1 promoter (pFLD1) is especially attractive, since little is known about its application in methanol‐free culture strategies. RESULTS: Three fed‐batch cultivations were performed to investigate the production of YlLIP2 in a pFLD1‐based system. When methanol was used as the fed‐batch feeding substrate, the maximum YlLIP2 activity obtained in a 10‐L bioreactor was 30 000 U mL^?1 after 143 h of culture, whereas the maximum YlLIP2 activity was further increased to 35 000 U mL^?1 by adopting a co‐induction strategy with methanol and methylamine as a mixed fed‐batch substrate. Furthermore, the maximum YlLIP2 activity reached 13 000 U mL^?1 after 80 h of cultivation in a methanol‐free culture. CONCLUSION: The expression levels of YlLIP2 in the pFLD1‐based system were comparable with those in a pAOX1‐based system. The results suggest that pFLD1 is an attractive alternative to pAOX1, and may make it feasible to induce high yields of protein expression. Copyright © 2011 Society of Chemical Industry     

Cheese yeasts

Numerous traditionally aged cheeses are surface ripened and develop a biofilm, known as the cheese rind, on their surfaces. The rind of such cheeses comprises a complex community of bacterial and fungal species that are jointly responsible for the typical characteristics of the various cheese varieties. Surface ripening starts directly after brining with the rapid colonization of the cheese surface by yeasts. The initially dominant yeasts are acid and salt-tolerant and are capable of metabolizing the lactate produced by the starter lactic acid bacteria and of producing NH₃ from amino acids. Both processes cause the pH of the cheese surface to rise dramatically. This so-called deacidification process enables the establishment of a salt-tolerant, Gram-positive bacterial community that is less acid-tolerant. Over the past decade, knowledge of yeast diversity in cheeses has increased considerably. The yeast species with the highest prevalence on surface-ripened cheeses are Debaryomyces hansenii and Geotrichum candidum, but up to 30 species can be found. In the cheese core, only lactose-fermenting yeasts, such as Kluyveromyces marxianus, are expected to grow. Yeasts are recognized as having an indispensable impact on the development of cheese flavour and texture because of their deacidifying, proteolytic, and/or lipolytic activity. Yeasts are used not only in the production of surface-ripened cheeses but also as adjunct cultures in the vat milk in order to modify ripening behaviour and flavour of the cheese. However, yeasts may also be responsible for spoilage of cheese, causing early blowing, off-flavour, brown discolouration, and other visible alterations of cheese.     

Cloning of an epoxide hydrolase-encoding gene from Rhodotorula mucilaginosa and functional expression in Yarrowia lipolytica

Epoxide hydrolases (EHs), especially those of fungal origin, have the ability to catalyse the enantioselective hydrolysis of epoxides to their corresponding diols. Recombinant DNA technology has been used extensively to overproduce these catalysts for the efficient hydrolytic kinetic resolution of epoxides, which serve as high-value intermediates in the fine chemicals and pharmaceutical industries. Degenerate primers, based on data from available EH-encoding gene sequences, in conjunction with inverse PCR, were used to amplify the genomic EH-encoding gene from Rhodotorula mucilaginosa. The 2347 bp genomic sequence revealed a 1979 bp ORF containing nine introns. The cDNA sequence revealed an 1185 bp EH-encoding gene that translates into a 394 amino acid protein exhibiting low sequence homology towards the known EH proteins. The EH gene from R. mucilaginosa was functionally expressed in Yarrowia lipolytica using a constitutive integrative expression cassette. Whole-cell biotransformation of (2,3-epoxypropyl)benzene, using the recombinant EH, revealed activity and selectivity far superior to any other activity and selectivity reported in literature using wild-type organisms. The GenBank Accession No. for the R. mucilaginosa EH gene is AY627310.     

睡莲多酚提取工艺优化及其抗油脂氧化作用

探究睡莲多酚超声提取的最佳工艺条件,并在进一步纯化基础上,采用烘箱法评价其抗油脂氧化性能。以睡莲多酚提取率为指标,通过考察乙醇浓度、料液比、超声温度、超声时间对提取的影响,结合正交设计试验优化得出最佳提取工艺：乙醇浓度50 %,料液比1:50 g/ml,超声温度60 ℃,超声时间40 min,超声功率90 W,在此条件下睡莲多酚提取率达14.0 %以上。通过大孔树脂聚酰胺联用方法获得了纯化后的睡莲多酚,其多酚含量为79.29±0.75%以上。抗油脂氧化试验结果表明：睡莲多酚对葵花籽油、菜籽油和猪油的过氧化均有良好的抑制作用,且其抗氧化性与其添加剂量呈正相关。此外,睡莲多酚分别与Vit C、柠檬酸及TBHQ复合后对猪油的过氧化均具有一定的协同增效作用,其中Vit C+睡莲多酚复配组的活性最强。本试验优选的工艺提取效率高,且发现睡莲多酚具有较好的抗油脂氧化能力,可延长食用油脂的货架期,说明该有效部位是一种值得开发的天然抗氧化剂。     

Sphingosine as a New Antifungal Agent against Candida and Aspergillus spp.

This study investigated whether sphingosine is effective as prophylaxis against Aspergillus spp. and Candida spp. In vitro experiments showed that sphingosine is very efficacious against A. fumigatus and Nakeomyces glabrataa (formerly named C. glabrata). A mouse model of invasive aspergillosis showed that sphingosine exerts a prophylactic effect and that sphingosine-treated animals exhibit a strong survival advantage after infection. Furthermore, mechanistic studies showed that treatment with sphingosine leads to the early depolarization of the mitochondrial membrane potential (Δψm) and the generation of mitochondrial reactive oxygen species and to a release of cytochrome C within minutes, thereby presumably initiating apoptosis. Because of its very good tolerability and ease of application, inhaled sphingosine should be further developed as a possible prophylactic agent against pulmonary aspergillosis among severely immunocompromised patients.     

Effect of Oils Extracted from Plant Seeds on the Growth and Lipolytic Activity of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Yeast

This study was aimed at evaluating the capability of Yarrowia lipolytica W29 for the synthesis of lipolytic enzymes in a medium containing plant oils from non‐conventional sources with some components displaying bioactivity. Oils from almond, hazelnut, and coriander seeds were obtained by using n‐hexane (Soxhlet method) and a chloroform/methanol mixture of solvents (Folch method), and their effect on the growth and lipolytic activity of Y. lipolytica was compared. A comparison of these two extraction methods showed that the extraction with n‐hexane was less effective regarding the oil extraction yields than the extraction conducted according to Folch's procedure. The lipolytic activity of the studied yeast was higher in the culture media containing oils extracted with the Soxhlet method than the Folch method but it was lower compared to olive oil medium. Among all oils tested, almond oil extracted with n‐hexane was the best inducer of extracellular lipases synthesized by Y. lipolytica. Its lipolytic activity achieved the maximum value of 2.33 U/mL after 48 h of culture. After 24 h of culture, it was close to the value obtained for the medium containing olive oil. Almond oil was a source of oleic and linoleic acids, which may determine differences in the lipolytic activity. The linoleic acid content in almond oil was higher than that found in other oils. When n‐hexane was used for extraction, the resultant oils were characterized by lower contents of polyphenols and poorer antioxidative activity.     

双菌法处理味精废水的工艺研究

采用假丝酵母和酿酒酵母双菌混合培养方法处理味精废水，其COD去除率明显高于单菌培养方法。对混合培养工艺进行研究，得到该双菌培养的最佳工艺条件为：接种龄为假丝酵母24h，酿酒酵母20h；接种比例为假丝酵母：酿酒酵母=4：1；总接种量为20％；接种顺序为酿酒酵母接种滞后于假丝酵母20h；培养时间为64h。     

十二碳二元酸生产菌培养基碳源与氮源的优化

以热带假丝酵母菌(Candida tropicalis)为试验菌株,在摇瓶发酵培养基础上,对种子培养基及发酵产酸培养基的碳源、氮源进行优化。结果表明:种子培养基最适碳源为葡萄糖,最适氮源为酵母浸粉FM902;菌体产酸培养基最适碳源为蔗糖(w)2%、葡萄糖(w)0.5%,比仅有2%蔗糖的培养基产酸量提高14.5%;最适氮源为0.08%(w)酵母浸粉FM902,比只含酵母浸膏LM902的培养基产酸量提高35.3%。     

大孔/介孔多级孔SiO2的制备及其在固定化脂肪酶中的应用

以具有三维骨架结构的环氧树脂大孔聚合物为模板,制备具有毫米级尺寸的大孔/介孔多级孔SiO_2。应用SEM、MIP、FTIR和N_2吸附-脱附法对材料孔道结构和表面性质进行表征。采用吸附法固定褶皱假丝酵母脂肪酶(CRL),研究CRL初始浓度、pH值及固定化时间对脂肪酶固定化的影响,对比研究了游离脂肪酶和固定化脂肪酶的酶学性质。结果表明,大孔/介孔SiO_2具有三维连续贯通的大孔孔道,孔壁由连续的SiO_2纳米薄膜构筑而成且表面存在丰富的介孔,比表面积为75.1m~2/g,孔隙率为92.3%;在CRL浓度为0.6mg/mL、pH值为8.0、固定化时间为10h时,固定化酶酶活达到4 825U/g。与游离脂肪酶相比,固定化脂肪酶的pH稳定性、热稳定性和储存稳定性明显提高,连续使用8次后的酶活为初始酶活的68%。利用环氧树脂大孔聚合物模板制备的大孔/介孔多级孔SiO_2在固定化酶方面具有良好的应用前景。     

Impact of Lipase-Mediated Hydrolysis of Castor Oil on γ-Decalactone Production by Yarrowia lipolytica

γ‐Decalactone is an industrially interesting peach‐like aroma compound that can be produced biotechnologically through the biotransformation of ricinoleic acid. Castor oil (CO) is the raw material most used as the ricinoleic acid source. The effect of different CO concentrations on the γ‐decalactone production by Yarrowia lipolytica was investigated in batch processing, and 30 g L^?1 was found to be the optimal oil concentration. Under these conditions, cells were able to produce lipase but at low activity levels, which might limit ricinoleic acid release and consequently, the γ‐decalactone production rate. Thus, the enzymatic hydrolysis of CO by commercial lipases was studied under different operating conditions. Lipozyme TL IM was found to be the most efficient and the optimal hydrolysis conditions were pH 8 and 27 °C. The use of hydrolyzed CO in the aroma production allowed a decrease in the lag phase for γ‐decalactone secretion.