Paecilomyces Lilacinus 中外切型葡萄糖胺苷水解酵素之研究暨生醫感測技術之開發

Abstract

Part 1 An entomopathogenic fungus, Paecilomyces lilacinus, was found to grow on chitosanase-detecting plates. Besides an endo-chitosanase, an exo-β-D-glucosaminidase was purified by cation-exchange chromatography from this microorganism cultivated in M9 minimal media containing 0.5% chitosan as the sole carbon source. The molecular weight of the enzyme is 95 kDa; the optimum pH and temperature for activity are 6.0 and 45°C, respectively. The purified exo-β-D-GlcNase promotes the hydrolysis of 95% deacetylated chitosan from its non-reducing end and liberates 2-amino-2-deoxy-D-glucopyranose (GlcN) as the sole product; however, 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) was not detected when chitin was used as the substrate. The cleavage pattern was confirmed by using real-time mass spectrometry shows that exo-β-D-glucosaminidase cleaves the glycosidic bonds between GlcN-GlcN and GlcN-GlcNAc but not between GlcNAc-GlcN or GlcNAc-GlcNAc. In the presence of a 10% solution of various alcohols, many alkyl-β-D-glucosaminides were obtained, indicating that exo-β-D-glucosaminidase is a retaining enzyme. Part 2 The study is separated into two sections. In the first section, we screened out the artificial antibody ─ PCTscFv from phage display library which was using procalcitonin (PCT) as antigen. PCT is an important bio-marker for diagnosing sepsis. After modifying fluorescein isothiocyanate (FITC) on PCTscFv and rhodamine on PCT, the recognition ability between PCTscFv and PCT was comfirmed by analyzing fluorescence spectra of fluorescent resonance energy transfer (FRET) phenomenon. And the equilibrium dissociation constant was calculated according to the fluorescenc spectra and Benesi-Hildebrand equation, KD = 16.4 (□M), respectively. After immobilized PCTscFv on quartz crystal microbalance (QCM) chip, the recognition ability between PCTscFv and PCT was comfirmed by QCM, and the detection limit of PCT concentration reached 10-10 g/mL. KD was calculated according to Langmuir isotherm equation, KD = 10.8 (nM). Furthermore, we successfully detected low concentration of PCT (10-10 g/mL) in human serum by adding anti-PCT antibody to amplify QCM signal. In the second section, we used VH (BPA) and VL (BPA) screened from phage display library, provided by Dr. Ueda, for detecting small molecule bisphenol A (BPA), which is one of the most common reagent for plastics producing. BPA may release into water or food plastic containers and cause different diseases. We fused maltose binding protein (MBP) to N terminal of VH or VL for better protein solubility, resulting in MBP_VH and MBP_VL. Then we used the open-sandwich immuno-assay (OSIA) technique to detect BPA on different platforms. After using OSIA technique combined with surface plasmon resonance spectrum (SPR) detection, we noticed that the 20% -COOH self-assembly monolayers (SAM) chip performenced the better detection range of BPA concentration, and the detection of BPA concentration could reach to 10-15 M, which is lower than other methods used nowadays. And this detection system also showed the specificity toward other molecules such as ethylphenol, tyramine and dopamine, which structures are similar to BPA. According to QCM combined with OSIA detections, the results comfirmed only when BPA exsisted that could form the sandwich structure MBP_VL-BPA-MBP_VH, and the detection limit of BPA is 10-10 M. This system also showed the specificity of recognizing BPA. Ultimatly, we immobilized MBP_VH and MBP_VL individually on gold nanoparticles (AuNP), forming AuNP-MBP_VH and AuNP-MBP_VL. After adding BPA into the mix solution of AuNP-MBP_VH and AuNP-MBP_VL, the color of AuNP was changed from red to dark purple according to the forming of sandwich structure leading AuNP to aggregate. AuNP method can detect BPA concentration to 10-9 ~ 10-10 M which is equal to 0.023 ~ 0.23 ppb. Besides, AuNP detection is a easy, quick and simple operation. The artificial antibody screened from phage display library can provide effiecient recognizations on either big molecule or small molecule antigens with high specificities. Furthermore, the phage display library screening and artificial antibody producing processes takes less time than traditional one. We firmly believe this system can provide a new direction for bio-recognization and bio-sensing.