Cloning,expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35–37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg²⁺, Mn²⁺, Li⁺, Ca²⁺, Ni²⁺, CHAPS, DTT, and EDTA and inhibited by Hg²⁺, Ag⁺, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.     

添加剂对大孔吸附树脂固定化脂肪酶的影响

利用大孔吸附树脂DA-201为载体对海洋脂肪酶固定化,并探寻添加剂对固定化过程的影响。分别以NH_4Cl、甘露糖和甘氨酸为添加剂,采用单因素和正交实验相结合的方法优化条件。结果显示,以NH_4Cl为添加剂的最优条件:柠檬酸-柠檬酸钠缓冲液pH 6. 0,固定化温度30℃,载体投放量0. 5g,NH_4Cl浓度25mmol/L,固定化时间3. 0h,酶活力达到115. 27U/g;比不含有添加剂的固定化酶固定化效率提高47. 42%。以甘露糖为添加剂最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度35℃,载体投放量0. 5g,甘露糖浓度10mmol/L,固定化时间4. 5h;酶活力达到122. 75U/g,比不含有添加剂的固定化酶固定化效率提高6. 50%。以甘氨酸为添加剂的最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度20℃,载体投放量0. 5g,甘氨酸浓度为25mmol/L,固定化时间7. 5h;酶活力达到141. 69U/g,比不含有添加剂的固定化酶固定化效率提高26. 12%。采用不同添加剂对大孔吸附树脂DA-201的吸附固定化过程有较大影响,可以极大地提高吸附效率;同时发现缓冲液类型、pH、温度、添加剂浓度和固定化时间等对DA-201树脂吸附脂肪酶有很大影响,对后续吸附固定化工业酶研究有较好的参考价值。     

The aspartimide problem persists: Fluorenylmethyloxycarbonyl‐solid‐phase peptide synthesis (Fmoc‐SPPS) chain termination due to formation of N‐terminal piperazine‐2,5‐diones

Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc‐SPPS while synthesizing the N‐terminal Xaa‐Asp‐Yaa motif. This termination was caused by the formation of piperazine‐2,5‐diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine‐2,5‐dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six‐membered ring is formed whereas the theoretically conceivable seven‐membered 1,4‐diazepine‐2,5‐dione is not found. We propose a mechanism where nucleophilic attack of the N‐terminal amino function takes place at the α‐carbon of the carbonyl group of the corresponding Asi intermediate. In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 improves diabetic retinopathy

Diabetes induced a serious of complications including diabetic retinopathy. Our study aimed to investigate the role of Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 in diabetic retinopathy. A mice model of diabetic retinopathy was established, and expression of SDF-1 and CXCR4 in retina was examined by Real-time quantitative PCR (qRT-PCR). Cells of human retinal pigment epithelial cell line ARPE-19 were treated with CXCR4 siRNAs and expression vector, and cell viability was detected by MTT assay. We found that expression of SDF-1 and CXCR4 in retina was significantly downregulated in mice with diabetic retinopathy than in normal healthy mice. High glucose treatment downregulated the expression of SDF-1 and CXCR4 in ARPE-19 cells at both mRNA and protein levels. Transfection with CXCR4 siRNAs decreased, while transfection with CXCR4 expression vector increased cell viability under high glucose treatment. We concluded that SDF-1/CXCR4 pathway improved diabetic retinopathy possibly by increasing cell viability.

Abbreviations: SDF-1: Stromal cell-derived factor 1; CXCL12: C-X-C motif chemokine 12; qRT-PCR: Real-time quantitative PCR