Strategies for engineering the thermo-stability of glycosidase / 生物工程学报

Glycosidases are widely used in food and pharmaceutical industries due to its ability to hydrolyze the glycosidic bonds of various sugar-containing compounds including glycosides, oligosaccharides and polysaccharides to generate derivatives with important physiological and pharmacological activity. While glycosidases often need to be used under high temperature to improve reaction efficiency and reduce contamination, most glycosidases are mesophilic enzymes with low activity under industrial production conditions. It is therefore critical to improve the thermo-stability of glycosidases. This review summarizes the recent advances achieved in engineering the thermo-stability of glycosidases using strategies such as directed evolution, rational design and semi-rational design. We also compared the pros and cons of various techniques and discussed the future prospects in this area.

Improvement of catalytic activity of Aspergillus terreus lipase by site-directed mutagenesis / 生物工程学报

The catalytic activity of Aspergillus terreus lipase (ATL) was improved by rational design. According to the sequence analysis and homologous modeling, several amino acids involved in the lid domain and substrate binding pocket domains of the acidic lipase ATL were mutated by site-directed mutagenesis, and eight mutants were constructed. These mutants and the wild type lipase ATL were expressed in Pichia pastoris GS115 and the enzymatic properties were characterized. The mutants ATLLid and ATLV218W exhibited higher hydrolytic activity than ATL towards p-nitrophenyl laurate. The kcat values of ATLLid and ATLV218W towards p-nitrophenyl laurate were 39.37- and 50.79-fold higher, and the kcat/Km values were 2.85- and 8.48-fold higher than the wild type, respectively. Although thermostability of these mutants decreased slightly, ATLLid and ATLV218W still exhibited the maximum activity at pH 5.0 and high stability in a broad range of pH (4.0-8.0), which were similar to the wild type. Using homologous modeling and molecular docking technology the mechanism for the improvement of catalytic activity was analyzed. These findings not only shed light on the relationship between the lid domain/substrate binding pocket domain and catalytic activity but also provided comprehensive scheme for further engineering to gain more efficient lipases.

Colorimetric Detection of Iodine Ion Based on Its Inhibition Effect on Peroxidase-Like Activity of Platinum Nanoparticles / 分析化学

As an important inorganic element, iodine not only plays an important role in human growth and metabolism, but also has an irreplaceable role in the chemical engineering, medicine, food and other fields.Thus the detection of iodine ion is of important significance.In this work, colorimetric recognition and sensing of iodine ion with high sensitivity was proposed based on target induced shielding of the peroxidase-like activity of bare platinum nanoparticles ( PtNPs ).This assay exhibited simplicity and cost-effectiveness.The recognition of iodine ion by bare PtNPs could be fulfilled in a few seconds and the assay could be accomplished within 10 min.The detection range for iodine ion was 20 nmol/L-5.0 μmol/L and the detection limit was 8 nmol/L ( S/N = 3 ).Furthermore, the new assay system did not require surface modification of nanoparticles, nor complex organic synthesis.This assay was successfully applied to detection of iodine concentration in real salt and water samples, exhibiting promising application prospects.

Preparation and Properties of Tremella-like Fe-N-C Composite Catalyst for Oxygen Reduction Reaction / 分析化学

The Fe-N-C composite catalyst was prepared by the thermal decomposition of the chelate precursors based on Fe central ions and o-phenylenediamine ligands.It was observed from the scanning electron microscopy that the crumpled carbon micro-and nano-sheets were intertwined to form a free-standing tremella-like 3D structure.The N2 adsorption/desorption experiments revealed that the composite contained ample micro-and meso-pores and had a specific surface area of 290 m2/g.Graphitic C and multi-crystal Fe3C as main components were confirmed by the X-ray diffraction, and N-doping in the general form of graphite N and pyridine N was also verified by X-ray photoelectron spectroscopy.The electrochemical measurement showed that the tremella-like Fe-N-C composite catalyzed oxygen reduction through a four-electron path in an alkaline solution, and its activity was comparable to the commercial Pt/C catalyst.After 2000 cycles, the limited current density of the Fe-N-C catalytic electrode only decreased less than 5%, and the half-wave potential shift negatively 5 mV, which suggested that the Fe-N-C composite catalyst had better catalytic stability than the commercially used Pt/C catalyst.

Catalytic Reduction of P-Nitro Phenolusing Green Synthesized Silver Nanoparticles and their Anti-Bacterial Activity.

In this work, we reporta simple and green approach method for the green synthesis of stable silver nano particles (AgNPs) using syzigium cumini leaf extract. Leaf extract acts as both reducing and stabilizing agent. The synthesized nanoparticles were characterized using UV-visible spectroscopy (UV-vis), Fourier transform electron microscopy(FTIR), X-ray powder diffraction (XRD), Scanning electron microscopy(SEM), zeta Potential and Transmission electron microscopy (TEM).The synthesized AgNPs was characterized by a peak of 439- 441nm.The influence of reaction time on the synthesis of nanoparticles were studied. Zeta potential value -14.1shows a moderate stable of silver nanoparticles with Syzigium cumini leaf extract. The TEM image clearly shows the synthesized silver nanoparticles shape is spherical and the average size is 13 nm. Synthesized nanoparticles had significant anti-bacterial activity and it also shows good catalytic activity on the reduction of para nitro phenol (4- NP) to para amino phenol (4-AP).

Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus; characterization of catalytic activity

OBJECTIVE@#To study characteristics of phospholipases C (PLCs), their importance for producing microorganisms as well as the potential of their use for industrial purposes.@*METHOD@#PLC from Bacillus cereus (B. cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomonas aeruginosa (P. aeruginosa) D183 of Gram-negative ones. Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis. Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method.@*RESULTS@#Maximum activity was at pH 7 and 8 and 40 °C for P. aeruginosa PLC, and pH 8-10 and 37 °C for B. cereus PLC. Both PLCs were inhibited by Pi at 5 mM or higher, whereas, PLC from B. cereus only was inhibited by EDTA. Activity of P. aeruginosa PLC was not affected by removing Zn(2+) ions from reaction mixture or their replacement with Ca(2+), Ba(2+), Mg(2+) or Mn(2+) ions. Vis-à-vis, activity of B. cereus PLC was found to be metal ion dependent. PLCs from both isolates were relatively thermostable and showed maximum affinity toward phosphatidylcholine. Sphingomyelin and phosphatidylethanolamine were not good substrates and phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and cardiolipin could be considered non-substrates.@*CONCLUSION@#Human body physiological conditions could favor activity of P. aeruginosa and B. cereus PLCs. These enzymes may participate in phosphate scavenging and virulence of producing isolates but not in autolysis. PLCs from both isolates are potential candidates for industrial use.

Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus; characterization of catalytic activity

Objective: To study characteristics of phospholipases C (PLCs), their importance for producing microorganisms as well as the potential of their use for industrial purposes. Method: PLC from Bacillus cereus (B. cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomonas aeruginosa (P. aeruginosa) D183 of Gram-negative ones. Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis. Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method. Results: Maximum activity was at pH 7 and 8 and 40 °C for P. aeruginosa PLC, and pH 8-10 and 37 °C for B. cereus PLC. Both PLCs were inhibited by Pi at 5 mM or higher, whereas, PLC from B. cereus only was inhibited by EDTA. Activity of P. aeruginosa PLC was not affected by removing Zn

Preparation of Ce Doped Pt/GAC Catalyst and Study on Its Catalytic Oxidation of Condensate Wastewater / 航天医学与医学工程

Objective To prepare the Pt/CeO2/GAC catalyst used to catalyze oxidation of condensate wastewater and to investigate whether the doping of Ce can increase catalytic activity for oxidizing condensate wastewater.Methods Impregnation glycol reduction method was adopted to prepare Pt/CeO2/GAC catalyst,and it was characterized by BET,XPS and XRD.The catalytic activity was determined by batch experiment.Results The experimental results revealed that more than 93.4% of ethanol in the condensate wastewater was oxidized to aldehyde and acetic acid with 13.33 g?L-1 Pt/CeO2/GAC catalyst dose,for 2 h reaction time,at 40 ℃ and normal pressure.In contrast,the oxidation efficiency was only 85.0% with Pt/GAC catalyst without doping Ce in the same reaction condition.Conclusion It is proven that the doping of Ce in Pt/GAC improves the catalytic activity.The characterization of Pt/CeO2/GAC catalyst shows that Ce is presented as Ce4+ that not only increases the content of chemisorbed oxygen on the surface of the catalyst,but also increases the content of Pt greatly.

The Expression of Human Carboxypeptidase Al and Its Active Center Gene / 中国肿瘤生物治疗杂志

Objective: To clone human carboxypeptidase A1 and its active center gene as well as construct recombinant vector for expression before analysis their activities. Methods: CPA1 and CPAlactive center gene were amplified by RT-PCR from pancreas tissue, and then sequencing was carried out. The correct target genes were cloned into prokaryotic vector pGEX-4T-1 and transformed into E. coli BL21 before sequence analysis. After induced by IPTG, gene products were analyzed by SDS-PAGE. Target expressed proteins were denaturated, renaturated, purified and evaluated through MTT and agar colony form test. Results: Human carboxypeptidase Al and its active center gene were cloned successfully. New expected protein band of Mr 66,000 and 46,000 appeared on SDS-PAGE after inducement. Both of the expressed proteins have catalytic activity in vitro, but the activity of the latter is inferior when applied to tumor cells. Conclusions: Human carboxypeptidase A1 and its active center gene were cloned successfully. Their prokaryotic expression products were obtained too. The expressed proteins have catalytic activity in. vitro. A new prosperous beginning of further improvement for CPA1 therapy system has been established based on CPA1 and its active center gene in terms of ADEPT against prostate cancer to clinical application.

L-asparaginase de soro de cutia (Dasyproctasp.). III - Anticorpos precipitantes antienzima / Serum L-asparaginase from agouti (Dasyproctasp.). III - Anti-enzyme precipitating antibodies

Dez imune-soros foram estudados quanto à presença de anticorpos precipitantes anti-L-asparaginase. Após reação de precipitação em meio líquido, usando como antígeno preparação enzimática parcialmente purifica da do soro de cutia, sobre na dantes e precipitados foram incubados em presença de substrato, a L-asparagina. A presença de enzima convertia a L-asparagina em ácido L-aspártico e ambos os aminoácidos eram posteriormente caracterizados por cromatografia em camada delgada de sílica-gel. Dentre os dez imune-soros, sete possuíam alto nível de anticorpos precipitantes antienzima que inibiam parcialmente a atividade catalítica da L-asparaginase. Três imune-soros possuíam anticorpos anti-L-asparaginase em baixo nível. Não foi encontrada correlação entre o conteúdo protéico dos imune-soros e o nível dos anticorpos antienzima (AU).