Identification of archaeon-producing hyperthermophilic alpha-amylase and characterization of the alpha-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca2+ for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Combinational biosynthesis of a fluorescent cyanobacterial holo-alpha-allophycocyanin in Escherichia coli

Allophycocyanin ( APC) is a phycobiliprotein with various biological and pharmacological properties. An expression vector containing five essential genes in charge of biosynthesis of cyanobacterial APC holo-alpha subunit ( holo- ApcA) was constructed, resulting in over- expression of a fluorescent holo- ApcA in E. coli. After being cultured for 16 h, the dry cell density reached 22.5 gl(-1), and the expression of holo- HT- ApcA was up to 1 gl(-1) broth. The recombinant protein showed similar spectral features to native APC.

Molecular cloning and characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri

LPS-induced TNF-alpha factor (LITAF) is a novel transcriptional factor that was first discovered in LPS-stimulated human macrophage cell line THP-1. LITAF can bind to TNF-a promoter to regulate its expression. The first scallop LITAF (named as CfLITAF) was cloned from Zhikong scallop Chlamys farreri by Expressed Sequence Tag (EST) and Polymerase Chain Reaction (PCR) techniques. The cDNA of CfLITAF was of 1240 bp and consisted of a 5' untranslated region (UTR) of 112 bp, a 3' UTR of 678 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 16.08 kDa and theoretical isoelectric point of 6.77. A typical conserved LITAF-domain was identified in CfLITAF by SMART analysis. Homology analysis of the deduced amino acid sequence of CfLITAF with other known sequences by using the BLAST program revealed that CfLITAF was homologous to the LITAF from human and rat (Identity = 46%), cattle, horse, mouse and chicken (Identity = 48%), western clawed frog (Identity = 42%), and zebrafish (Identity = 50%). The mRNA expression of CfLITAF in different tissues including haemocytes, muscle, mantle, heart, gill and gonad, and the temporal expression in haemocytes challenged by LPS or peptidoglycan (PGN) were measured by Real-time RT-PCR. CfLITAF mRNA transcripts could be detected in all tissues examined and be up-regulated in haemocytes after LPS challenge. No significant changes were observed after PGN stimulation. All these data indicated the existence of LITAF in scallop and also provided clue on the presence of TNF-alpha-like molecules in invertebrates. (C) 2007 Elsevier Ltd. All rights reserved.

海带根中alpha-葡萄糖苷酶抑制剂的提取及其酶抑制作用动力学研究

alpha-葡萄糖苷酶是一类能够催化碳水化合物alpha-1,4-糖苷键水解的酶，是动物体内许多代谢途径中的关键酶，具有多方面的靶向药理活性。研究表明，alpha-葡萄糖苷酶与糖尿病，艾滋病，恶性肿瘤、溶酶体贮积症等疾病的发生机制有重要关系，已经成为治疗这些疾病的重要靶点。 海带（Laminaria japonica） 属褐藻门、海带属植物。自二十世纪四十年代从日本引入中国以来，已成为中国沿海重要的海洋经济作物。海带藻体叶片部分大都用来提炼碘、甘露醇和藻酸盐，但其假根部分一直被当作化工工业和食品工业的废料，而未得到有效的利用。在中国的沿海一带，海带根作为一种抗糖尿病的民间药物，有很长的历史。因此从海带根中提取分离降血糖成分并研究其作用机制将具有重要的意义。 本论文第一部分进行了海带根中具有alpha-葡萄糖苷酶抑制作用的活性组分的筛选。首先我们按照海带根中所含成分的极性不同进行了提取分离，即将海带根乙醇提取物依次用石油醚、乙酸乙酯、正丁醇萃取。结果乙醇粗体物、石油醚部位、乙酸乙酯部位、正丁醇部位与水部位的酶抑制IC50分别为1589µg/L、970µg/L、 380µg/L、1616µg/L、1106µg/L。乙酸乙酯部位的酶抑制活性最强。 本论文第二部分对海带根乙酸乙酯部位的提取物的体内降糖活性进行了研究，结果表明乙酸乙酯部位可以降低糖尿病大鼠餐后血糖，与阴性对照差异极显著（P<0.01）。小鼠服药后表现出的生命体征与伺服拜糖平后表现出的生命体征类似。 在以上工作基础上我们以alpha-葡萄糖苷酶的抑制活性为导向，用天然产物化学的方法对活性成分进行追踪，通过光谱、波谱与理化性质鉴定出了具有显著alpha-葡萄糖苷酶抑制活性的化合物：丁基-异丁基-邻苯酸二甲酯，并对其进行了酶抑制动力学研究。研究表明丁基-异丁基-邻苯酸二甲酯对alpha-葡萄糖苷酶表现为非竞争性抑制类型，IC50达到13.6μg/mL，且Km=2.39 g/L 本研究工作为进一步研究海带根降血糖成分的作用机理提供了研究基础。

Preparation, characterization and antifungal properties of 2-(alpha-arylamino phosphonate)-chitosan

In this paper, 20 kinds of different 2-(alpha-arylamino phosphonate)-chitosan (2-alpha-AAPCS) were prepared by different Schiff bases of chitosan (CS) reacted with di-alkyl phosphite in benzene solution. The structures of the derivatives (2-alpha-AAPCS) were characterized by FT-IR spectroscopy and elemental analysis. In addition, the antifungal activities of the derivatives against four kinds of fungi were evaluated in the experiment. The results indicated that all the prepared 2-alpha-AAPCS had a significant inhibiting effect on the investigated fungi when the derivatives concentration ranged from 50 to 500 mu g mL(-1). Furthermore, the antifungal activities of the derivatives increased with increasing the molecular weight and concentration. And the antifungal activities of the derivatives were affected by their dimensional effect and charge density. Besides, the rule and mechanism of the antifungal activities of them were discussed in this paper. (C) 2009 Elsevier B.V. All rights reserved.