Enhanced thermostability of a Rhizopus chinensis lipase by in vivo recombination in Pichia pastoris

ABSTRACT: BACKGROUND: Lipase from Rhizopus chinensis is a versatile biocatalyst for various bioconversions and has been expressed at high-level in Pichia pastoris. However, the use of R. chinensis lipase in industrial applications is restricted by its low thermostability. Directed evolution has been proven to be a powerful and efficient protein engineering tool for improvement of biocatalysts. The present work describes improvement of the thermostability of R. chinensis lipase by directed evolution using P. pastoris as the host. RESULTS: An efficient, fast and highly simplified method was developed to create a mutant gene library in P. pastoris based on in vivo recombination, whose recombination efficiency could reach 2.3 x 105 /mug DNA. The thermostability of r27RCL was improved significantly by two rounds of error-prone PCR and two rounds of DNA shuffling in P. pastoris. The S4-3 variant was found to be the most thermostable lipase, under the conditions tested. Compared with the parent, the optimum temperature of S4-3 was two degrees higher, Tm was 22 degrees higher and half-lives at 60degreesC and 65degreesC were 46- and 23- times longer. Moreover, the catalytic efficiency kcat/Km of S4-3 was comparable to the parent. Stabilizing mutations probably increased thermostability by increasing the hydrophilicity and polarity of the protein surface and creating hydrophobic contacts inside the protein. CONCLUSIONS: P. pastoris was shown to be a valuable cell factory to improve thermostability of enzymes by directed evolution and it also could be used for improving other properties of enzymes. In this study, by using P. pastoris as a host to build mutant pool, we succeeded in obtaining a thermostable variant S4-3 without compromising enzyme activity and making it a highly promising candidate for future applications at high temperatures.     

Molecular switch for the assembly of lipophilic drug incorporated plasma protein nanoparticles and in vivo image

A strategy to manipulate the disulfide bond breaking triggered unfolding, and subsequently assembly of human serum albumin (HSA) in a lipophilic drug-dependent manner is present. In this study, the hydrophobic region, a molecular switch of the HSA, was regulated to form HSA-paclitaxel (HSA-PTX) nanoparticles by a facile route. High-resolution transmission electron microscopy and fluorescence quenching indicate that HSA coassembled with PTX, which acts as a bridge to form core-shell nanoparticles about 50-240 nm in size, and that PTX might bind to the subdomain IIA sites of HSA. Change of ultraviolet absorption and circular dichroism spectra reveal the formation of HSA-PTX nanoparticles, which is a safety, injectable pharmaceutic nanocarrier system for tumor target. This method to prepare nanocarrier systems for hydrophobic guest molecules reveals a general principle of self-assembly for other plasma proteins and other pharmacologically active substances with poor water solubility. It also provides a basis for developing nanocarrier systems for a wide range of applications in nanomedicine, from drug delivery to bioimaging systems.     

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system^1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans^3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types^5,6, including neural progenitor cells (NPCs)^7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously^5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures^9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.     

Molecular characterization and immunohistochemical localization of a mitogen-activated protein kinase, Accp38b, from Apis cerana cerana

The p38 mitogen-activated protein kinase (MAPK) is involved in various processes, including stress responses, development, and differentiation. However, little information on p38 MAPK in insects is available. In this study, a p38 MAPK gene, Accp38b, was isolated from Apis cerana cerana and characterized. The quantitative real-time PCR (Q-PCR) analysis revealed that Accp38b was induced by multiple stressors. Notably, the expression of Accp38b was relatively higher in the pupae phase than in other developmental phases. During the pupae phase, Accp38b expression was higher in the thorax than in the head and abdomen and higher in the fat body than in the muscle and midgut. Immunohistochemisty showed significant positive staining of Accp38b in sections from the brain, eyes, fat body, and midgut of A. cerana cerana. These results suggest that Accp38b may play a crucial role in stress responses and have multiple aspects function during development.     

不同壳色福寿螺形态性状与体质量的关系

【背景】福寿螺因其食性杂、抗逆性和繁殖力强以及自然天敌少等不断扩散,侵害农作物,被列为我国首批外来入侵物种。国内外学者一直致力于研究对其的防治与监控。自然界中福寿螺存在2种壳色——黄色和黑色,壳色受遗传因素和环境因素的双重影响。广东省福寿螺多以黑色为主,福寿螺倾向于与不同壳色的螺交配。壳色在一定程度上影响其交配的选择性,但2种壳色的福寿螺繁殖力指标差异不显著。而关于这2种壳色的螺在形态学上的差异鲜有报道。【方法】利用生物统计软件和分析方法进行相关性分析、通径分析及多元回归分析,计算相关系数、通径系数和决定系数,研究2种壳色福寿螺形态性状与体质量的关系。【结果】2种壳色福寿螺的体质量、层高的变异系数较大,且黄色比黑色变异系数大。对黄色福寿螺体质量影响较大的依次为壳高、口宽;对黑色福寿螺体质量影响较大的依次为口宽、层高。【结论与意义】2种壳色福寿螺在形态性状方面差异显著,可以将壳色作为特征标记,为福寿螺的监测与灾害评估提供参考。     

High-level expression of a novel Penicillium endo-1,3(4)-β-d-glucanase with high specific activity in Pichia pastoris

A novel endo-1,3(4)-β-D-glucanase gene (bgl16C1) from Penicillium pinophilum C1 was cloned and sequenced. The 945-bp full-length gene encoded a 315-residue polypeptide consisting of a putative signal peptide of 18 residues and a catalytic domain belonging to glycosyl hydrolase family 16. The deduced amino acid sequence showed the highest identity (82%) with the putative endo-1,3(4)-β-glucanase from Talaromyces stipitatus ATCC 10500 and 60% identity with the characterized β-1,3(4)-glucanase from Paecilomyces sp. FLH30. The gene was successfully overexpressed in Pichia pastoris. Recombinant Bgl16C1 constituted 95% of total secreted proteins (2.61 g l?1) with activity of 28,721 U ml?1 in a 15-l fermentor. The purified recombinant Bgl16C1 had higher specific activity toward barley β-glucan (12,622 U mg?1) than all known glucanases and also showed activity against lichenan and laminarin. The enzyme was optimally active at pH 5.0 and 55°C and exhibited good stability over a broad acid and alkaline pH range (>85% activity at pH 3.0-7.0 and even 30% at pH 11.0). All these favorable enzymatic properties make it attractive for potential applications in various industries.