桔林油脂酵母的生长和淀粉酶产生

在模拟淀粉废水的连续培养中对桔林油脂酵母及突变体的生长和淀粉酶产生进行了研究。在低稀释率（０．０３～０．２５ｈ－１）、ｐＨ４．８、３２℃条件下出现最大淀粉酶产量。Ｄ＝０．１０ｈ－１时，突变体所产生的淀粉酶活性为２３ｕ／ｍｌ，最大淀粉酶产量为１．４５ｕ／ｍｌ·ｈ。细胞生物量的产量在Ｄ＝０．１５ｈ－１时最大，细胞生物量的产率取决于葡萄糖的消耗，其产率值为０．７９。培养液中的淀粉浓度平均减少８９％。     

橘林油脂酵母发酵条件的初步研究

以橘林油脂酵母（Lipomyces kononenkoae CICC1714）为实验材料,通过设计单因素实验,确定了摇瓶发酵培养的最佳产油条件为：氮源为硫酸铵,碳源为葡萄糖,培养温度28℃,接种量10%,初始pH值为7.0,最适碳氮比为99。优化后批次发酵的生物量为7.01g/L,油脂含量15.91%,油脂产量1.12g/L。经半连续发酵后最终的生物量为24.13g/L,油脂产量为3.06g/L。     

啤酒生产废水培养斯达油脂酵母发酵油脂培养基的优化

以啤酒生产废水及添加一定的营养因子为发酵基质,采用单因素和正交试验设计对斯达油脂酵母(Lipomyces starkeyi 1809)发酵产油脂和COD降解的培养基进行了优化,并利用气相色谱分析比较了菌体油脂的脂肪酸组成.结果表明:最佳培养基组成为在啤酒生产废水中添加碳源和氮源的C/N为55∶1(即木糖28.88 g/L和硫酸铵1.0 g/L),FeCl3·6H2O3 mg/L,色醇150 μmol/L(在发酵36 h时添加),初始pH 5.5;用此培养基在摇床转速120 r/min、温度28℃下培养斯达油脂酵母120 h,菌体生物量、油脂产量、油脂含量和废水的COD降解率分别达到12.28 g/L、4.40 g/L、35.80%和50.32%,生物量和油脂产量比优化前分别提高了137.98%和117.82%,所产菌体油脂的不饱和脂肪酸指数( IUFA)达到63.95%,其中油酸的相对含量高达56.29%,比优化前提高了5.45%,优化效果显著.     

Medium optimization for lipid production through co‐fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi

Co‐fermentation of lignocellulose‐based carbohydrates is a potential solution to improve the economics of microbial lipid production. In the present paper, experiments were performed to optimize the media composition for lipid production by the oleaginous yeast Lipomyces starkeyi AS 2.1560 through co‐fermentation of glucose and xylose (2 : 1 wt/wt). Statistical screening of nine media variables was performed by a Plackett–Burman design. Three factors, namely mixed sugar, yeast extract and FeSO₄, were found as significant components influencing cellular lipid accumulation. Further optimization was carried out using a Box–Behnken factorial design to study the effects of these three variables on lipid production. A mathematical model with the R² value at 96.66% was developed to show the effect of each medium composition and their interactions on the lipid production. The model estimated that a maximal lipid content of 61.0 wt‐% could be obtained when the concentrations of mixed sugar, yeast extract and FeSO₄ were at 73.3 g/L (glucose 48.9 g/L, xylose 24.4 g/L), 7.9 g/L and 4.0 mg/L, respectively. The predicted value was in good accordance with the experimental data of 61.5%. Compared with the initial media, the optimized media gave 1.59‐fold and 2.03‐fold increases for lipid content and lipid productivity, respectively.     

斯达氏油脂酵母产油脂培养基及产油条件的优化

为了提高斯达氏油脂酵母的细胞产量,考察了该菌在不同碳氮比条件下的产油脂情况以及添加无机盐对产油脂的影响,得知最佳培养基组成为:葡萄糖70g/L,硫酸铵1.0g/L,酵母粉0.5g/L,磷酸二氢钾9.0g/L,硫酸镁1.0g/L,pH 5.5,培养过程中每24h回调pH。摇瓶装液量为500mL三角瓶装培养基100mL,接种量为10%(种龄28h)。在上述条件下,30℃振荡(180r/min)培养96h,所得菌体干重、油脂含量分别达21.65g/L和43.11%。     

斯达油脂酵母菌(Lipomyces starkeyi 2.1608)在不同培养基中产胞外多糖的研究

通过对斯达油脂酵母菌（Lipomyces starkeyi 2.1608）在不同培养基中的摇瓶发酵培养,比较研究了该菌的增殖和产胞外多糖等发酵特性。结果表明,在高浓度Mn^2＋（79．0mg／LMnCl2·4H2O）和高浓度Zn^0＋（0．670mg／L ZnSO4·7H2O）的CG培养基中,总菌数和胞外多糖产量最高,总菌数分别是LS培养基的6．9倍、W培养基的2．4倍;胞外多糖产量分别是LS培养基的4．9倍、W培养基的1．9倍,但细胞油脂蓄积量最低。在缺Mn^2＋和高浓度Zn^2＋（0．0182mg／L ZnSO4·7H2O）的LS培养基中,该菌细胞增殖数量和胞外多糖产量最低,但细胞油脂蓄积量和油脂产量最高,分别为CG培养基的9．8倍和1．4倍,W培养基的6．9倍和2．4倍。在Mn^2＋和Zn^2＋浓度介于二者之间的W培养基中,该菌增殖数量和胞外多糖产量介于二者之间。在C／N一定的条件下,高浓度的Mn^2＋和Zn^2＋可促进细胞增殖和产胞外多糖,而抑制油脂合成;缺Mn^2＋和低浓度Zn^2＋可抑制细胞增殖和产胞外多糖,促进油脂合成。     

Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae

Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities.     

Cloning and characterization of a dextranase gene from Lipomyces starkeyi and its expression in Saccharomyces cerevisiae

A dextranase-encoding cDNA from L. starkeyi KSM22 was isolated and characterized. The 2052 bp cDNA fragment (lsd1) harbouring the dextranase gene exhibited one open reading frame (ORF) composed of 1824 bp flanked by a 41 bp 5'-UTR and a 184 bp 3'-UTR, including a 27 bp poly(A) tail. The lsd1 gene contains no introns. The open reading frame encodes a 608 amino acid polypeptide (LSD1) with a 67.6 kDa predicted molecular mass. There was a 77% deduced amino acid sequence identity between the LSD1 dextranase and the dextranase from Penicillium minioluteum. The primary structure of LSD1 dextranase exhibits distant similarity with the enzymes of the glycosyl hydrolase family 49 that comprises Penicillium dextranase. The optimum pH of LSD1 was 6.0 and the optimum temperature was 37 degrees C. LSD1 dextranase activity was substantially abolished by exposure to 1 mM Hg2+, Ag3+ and Mn2+. LSD1 exhibited high hydrolysing activity towards dextran (100%), soluble starch (22%) and mutan (8%).