Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris

The lipase genes of Yarrowia lipolytica, LIPY7 and LIPY8, fused with FLO-flocculation domain sequence from Saccharomyces cerevisiae at their N-termini, were expressed in Pichia pastoris KM71. Following the induction with methanol, the recombinant proteins were displayed on the cell surface of P. pastoris, as confirmed by the confocal laser scanning microscopy. The LipY7p and LipY8p were anchored on P. pastoris via the flocculation functional domain of Flo 1 p. The surface-displayed lipases were characterized for their application as the whole-cell biocatalyst. These lipases can also be cleaved off from their anchor by enterokinase treatment to yield functionally active proteins in the supernatant offering an alternative purification method for LipY7p and LipY8p. (c) 2007 Elsevier Inc. All rights reserved.

酵母在石油烃代谢过程中胞外乳化物质的形成及作用研究

本文研究了热带假丝酵母（C. tropicalis）和解脂假丝酵母（C. lipolytica）利用混和正烷烃（C_(10)-C_(18)）生长过程中胞外乳化物的产生及作用。结果表明胞外乳化物质的大量产出现于对数生长后期和生长平衡期，热水抽提菌体所得细胞表面物质具有乳化性能，其乳化性能与细胞生长密切相关，生长旺盛细胞表面具有较高乳化性能。以冷丙酮（3：1）沉淀发酵上清液，获得胞外主要乳化物质，经反复沉淀、透析及冷冻干燥，初步纯化乳化物产率为0.5 g/L，C. tropicslis胞外主要乳化物为脂蛋白-多糖复合物，其中，糖46%、脂40%、蛋白3%。脂由多种中性脂组成，糖单元为果糖及两种未知单糖，蛋白中80%为极性氨基酸，C. lipolytica胞外乳化物质主要是脂-糖蛋白复合物，主要组成：糖66%、脂17%、蛋白6%。脂由中性脂和极性脂组成，糖蛋白组成：糖95、蛋白5%。糖单元为甘露糖、果糖，蛋白中80%为极性氨基酸（主要为成糖氨基酸）。两种胞外乳化物均为水溶性大分子复合物，在低浓度（<10mg/L）即具有良好乳化性能。去除蛋白后的胞外乳化物对烷烃的乳化性能降低90%，pH在中性时，乳化性能最佳。CaCl_2(10-50 mM)，NaCl(1-20%)， EDTA(2.5-10 mM)对C. lipolytica胞外乳化物质的乳化性能影响较大，而对C. tropiclis胞外乳化物影响较小，两种胞外提取物对产生菌利用烃生长有刺激作用。槐糖脂、C. lipolytica胞包提取乳化物对C. tropicalis利用烃的生长有刺激作用，鼠李糖脂、吐温80则表现出抑制作用。胞外生物乳化剂对酵母菌利用烷烃的生长有着重要作用。

The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri

Cu, Zn superoxide dismutases (SODs) are rnetalloenzymes that represent one important line of defence against reactive oxygen species (ROS). A cytoplasmic Cu. Zn SOD cDNA sequence was cloned from scallop Chlamys farreri by the homology-based cloning technique. The full-length cDNA of scallop cytoplasmic Cu, Zn SOD (designated CfSOD) was 1022 bp with a 459 bp open reading frame encoding a polypeptide of 153 amino acids. The predicted amino acid sequence of CfSOD shared high identity with cytoplasmic Cu. Zn SOD in molluscs, insects, mammals and other animals, such as cytoplasmic Cu, Zn SOD in oyster Crassostrea sostrea gigas (CAD42722), mosquito Aedes aegypti (ABF18094), and cow Bos taurus (XP_584414). A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with Listonella anguillarum, Micrococcus luteus and Candida lipolytica respectively. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD dropped in the first 8-16 h and then recovered after challenge with L. anguillarum and M. litteus, but no change was induced by the C. lipolytica challenge. The results indicated that CfSOD was a constitutive and inducible acute-phase protein, and could play an important role in the immune responses against L. anguillarum and M. luteus infection. (C) 2007 Elsevier Ltd. All rights reserved.

Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri

C-type lectin is a family of Ca2+ dependent carbohydrate-recognition proteins which play crucial roles in the innate immunity of invertebrates by mediating the recognition of host cells to pathogens and clearing microinvaders as a pattern recognition protein (PRP). The cDNA of Zhikong scallop Chlamys farreri C-type lectin (designated CFLec-1) was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of CFLec-1 was 1785 bp, consisting of a 5'-terminal untranslated region (UTR) of 66 bp and an unusually long 3' UTR of 1040 bp with seven polyadenylation signal sequences AATAAA and a poly(A) tail. The CFLec-1 cDNA encoded a polypeptide of 221 amino acids with a putative signal peptide of 15 amino acid residues and a mature protein of 206 amino acids. Analysis of the protein domain features indicated a typical long-form carbohydrate-recognition domain (CRD) of 130 residues in the CFLec-1 deduced amino acid sequence. The expression pattern of CFLec-1 transcripts in healthy and bacterial challenged scallops was studied by semi-quantitative RT-PCR. mRNA transcripts of CFLec-1 could be mainly detected in the tissues of haemocytes, gill, gonad and mantle of unchallenged scallops, whereas the expression of CFLec-1 transcripts was increased in all the tested tissues after heat-killed Vibrio anguillarum challenge. The temporal expression of CFLec-1 mRNA in haemolymph challenged by Micrococcus luteus and V anguillarum was both up-regulated and reached the maximum level at 8 and 16 It post stimulation, respectively, and then dropped back to the original level. In order to investigate its immune functions, CFLec- I was recombined and expressed in Escherichia coli BL21(DE3)-pLysS as a fusion protein with thioredoxin. The recombinant CFLec-1 agglutinated bacteria E. coli JM109 in vitro, and the agglutination was Ca2+ dependent which could be inhibited by EDTA. But it did not agglutinate M. luteus, Candida lipolytica and animal erythrocytes including rabbit, rat, mouse, chicken, human group A, human group B, human group O. Meanwhile, the recombinant CFLec-1 could inhibit the growth of both E. coli JM 109 and M. luteus, but no inhibition activity against V anguillarum. These result indicated that CFLec-1 was a constitutive and inducible PRP which was involved in the reorganization and clearance of invaders in scallop. (c) 2006 Elsevier Ltd. All rights reserved.