Formation of chitin-based nanomaterials using a chitin-binding peptide selected by phage-display

Targeting polymers with peptides is an efficient strategy to functionalize biomaterials. Phage display technology is one of the most powerful techniques for selecting specific peptides for a wide variety of targets. A method to select a chitin-binding peptide from a 12-mer random peptide library was successfully performed against chitin immobilized in wells of microtiter plates. The synthetic chitin binding peptide (ChiBP) could bind to chitin beads and disrupt their structure. This selected peptide was successfully used to immobilize alkaline phosphatase on chitin. In addition, the peptide could induce colloidal chitin in water to form a chitin coat on the surface of plastic tubes. Scanning electron microscopy (SEM) revealed that the peptide could induce colloidal chitin and chitohexaose to form networks when the temperature was raised to 42°C.     

Expression, refolding and indirect immobilization of horseradish peroxidase (HRP) to cellulose via a phage-selected peptide and cellulose-binding domain (CBD).

We examined the potential immobilization of horseradish peroxidase (HRP) to cellulose with cellulose-binding domain (CBD) as a mediator, using a ligand selected from a phage-displayed random peptide library. A 15-mer random peptide library was panned on cellulose-coated plates covered with CBD in order to find a peptide that binds to CBD in its bound form. The sequence I/LHS, which was found to be an efficient binder of CBD, was fused to a synthetic gene of HRP as an affinity tag. The tagged enzyme (tHRP) was then immobilized on microcrystalline cellulose coated with CBD, thereby demonstrating the indirect immobilization of a protein to cellulose via three amino acids selected by phage display library and CBD.     

Chitosan scaffolds for biomolecular assembly: coupling nucleic acid probes for detecting hybridization

Chitosan, a naturally occurring biopolymer, was used as a scaffold for the covalent binding of single-stranded DNA oligonucleotide probes in a fluorescence-based nucleic acid hybridization assay. Chitosan's pH dependent chemical and electrostatic properties enable its deposition on electrodes and metal surfaces, as well as on the bottom of microtiter plates. A combinatorial 96-well microtiter plate format was used to optimize chemistries and reaction conditions leading to hybridization experiments. We found the coupling of oligonucleotides using relatively common glutaraldehyde chemistry was quite robust. Our hybridization results for complementary ssDNA oligonucleotides (E. coli dnaK sequences) demonstrated linear fluorescence intensity with concentration of E. coli dnaK-specific oligonucleotide from 0.73 microM to 6.6 microM. Moreover, hybridization assays were specific as there was minimal fluorescence associated with noncomplementary groEL oligonucleotide. Finally, these results demonstrate the portability of a DNA hybridization assay based on covalent coupling to chitosan, which, in turn, can be deposited onto various surfaces. More arduous surface preparation techniques involving silanizing agents and hazardous washing reagents are eliminated using this technique.     

A novel microtiter plate based method for identification of B‐cell epitopes

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of gas-liquid mass transfer phenomena in microtiter plates

Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method. The maximum oxygen transfer capacity (OTR(max)), the specific mass transfer area (a), and the mass transfer coefficient (k(L)) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system. The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters. To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter. The investigations revealed that the OTR(max) is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells. The mass transfer coefficient (k(L)) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors. The OTR(max) in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material. Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed. This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area. This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks. In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR(max), a, and k(L). An OTR(max) of up to 0.15 mol/L/h can be reached in square-shaped wells.     

Improved protocols for the isolation and in-situ cryopreservation of cell colonies

Stable transfection and cloning of cells often require physical separation of cell colonies. In order to conveniently isolate cell clones from petri dishes, we developed a protocol starting with a soft agar overlay of cells. This reduces the risk of cell diffusion between different colonies. Cells from individual colonies are mechanically removed, incubated with trypsin, and cell suspensions are seeded onto parallel microtiter plates. The cell clones on one microtiter plate can be cryopreserved in situ using the protocol described here which was tested for a variety of cell lines. Replica plates can be used for screening and further expansion of interesting clones. If screening can also be performed in situ, e.g., by immunocytochemistry, immunofluorescence, or the polymerase chain reaction, it is possible to perform most steps necessary in cell cloning experiments on microtiter plates.     

蛋白内含子介导纯化的表达载体的构建及应用

将抗膀胱癌单链抗体(PG)融合在蛋白内含子(intein)的C末端,利用intein的N末端具有的chitin binding domain(CBD)对几丁质柱的吸附特性及intein的自切割活性对PG进行一次性纯化。同时,引入大肠杆菌分子伴侣肽基脯氨酰顺反异构酶(FkpA)与目的蛋白进行共表达及融合表达,以使目的蛋白以可溶形式存在。结果显示,PG融合蛋白对几丁质柱的吸附效率很高,intein的自切割释放PG的效率也较高,但PG仅出现在SDS洗脱液中。FM与PG共表达能显著地提高目的蛋白的可溶性。Intein介导的纯化系统前景远大。     

Growth of the salt-tolerant yeast Zygosaccharomyces rouxii in microtiter plates: effects of NaCl,pH and temperature on growth and fusel alcohol production from branched-chain amino acids

Zygosaccharomyces rouxii, a salt-tolerant yeast isolated from the soy sauce process, produces fusel alcohols (isoamyl alcohol, active amyl alcohol and isobutyl alcohol) from branched-chain amino acids (leucine, isoleucine and valine, respectively) via the Ehrlich pathway. Using a high-throughput screening approach in microtiter plates, we have studied the effects of pH, temperature and salt concentration on growth of Z. rouxii and formation of fusel alcohols from branched-chain amino acids. Application of minor variations in pH (range 3-7) and NaCl concentrations (range 0-20%) per microtiter plate well allowed a rapid and detailed evaluation of fermentation conditions for optimal growth and metabolite production. Conditions yielding the highest cell densities were not optimal for fusel alcohol production. Maximal fusel alcohol production occurred at low pH (3.0-4.0) and low NaCl concentrations (0-4%) at 25 degrees C. At pH 4.0-6.0 and 0-18% NaCl, considerable amounts of alpha-keto acids, the deaminated products from the branched-chain amino acids, accumulated extracellularly. The highest cell densities were obtained in plates incubated at 30 degrees C. The results obtained under various incubation conditions with (deep-well) microtiter plates were validated in Erlenmeyer shake-flask cultures.     

Directed evolution of a Bacillus chitinase

Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito-oligosaccharide-based value-added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error-prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose at various pH in microtiter plates. From a medium-throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose as substrate, the overall catalytic efficiency, k_cat/K_m of the improved chitinase was 2.7- and 2.3-fold higher than the average k_cat/K_m of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications.     

Development of a Micromethod for Identification of Anaerobic Bacteria

A microprocedure was described for determining the carbohydrate fermentation patterns of 48 anaerobic bacteria at one time in microtiter plates. The cultures were transferred into agar-filled wells of microtiter plates with a replicator inside an anaerobic glove box. Fermentation was measured both with a colorimetric indicator and with a small pH electrode. The method was approximately 97% accurate. It would be most useful for laboratories that need to identify large numbers of anaerobes at one time.     

Microtiter plates as mini-bioreactors: miniaturization of fermentation methods

In the past decade, the use of microtiter plates for microbial growth has become widespread, particularly in industry. In parallel, research in academia has provided a thorough insight into the complex relation between well dimensions, culture volumes, orbital shaking conditions and surface tension on the one hand, and oxygen-transfer rates and degrees of mixing on the other. In this review, I will discuss these fundamental issues and describe the current applications of microtiter plates in microbiology. Microtiter plates can now be considered a mature alternative to Erlenmeyer shake flasks.     

Glucose-containing polymer rings enable fed-batch operation in microtiter plates with parallel online measurement of scattered light,fluorescence, dissolved oxygen tension,and pH

Bioprocesses operated in batch mode can induce adverse effects like overflow metabolism, substrate inhibition, osmotic inhibition, oxygen limitation, and catabolite repression. To avoid these adverse effects, fed-batch is the predominant operation mode in industrial production. Nevertheless, screening for optimal production strains is usually performed in microtiter plates and shake flasks operated in batch mode without any online monitoring. Recently, a polymer-based controlled-release fed-batch microtiter plate with stable glucose release characteristics was described. In this study, a glucose-containing polymer matrix was used to manufacture polymer rings that were placed at the bottom of a 48-well microtiter plate. Thereby, the liquid content of the well became accessible for optical measurement by the BioLector device. Reflections caused by the polymer ring were minimized by adjusting the scattered-light measurement position. Influences on the measurement of the dissolved oxygen tension and pH could be avoided by choosing appropriate polymer-ring geometries. These adjustments enabled parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH of Escherichia coli BL21 (DE3) fed-batch cultivations. The online monitoring and fed-batch operation capabilities of the fed-batch microtiter plate presented in this study finds optimal application in screenings and initial process development.     

Temperature regulation of microtiter plates for enzyme assays

To facilitate the use of microtiter plates as vessels for enzyme assays, an incubator has been designed to maintain the wells of the microtiter plates at the appropriate temperature. The temperature variation within a single well was +/- 0.02 degrees (standard error of the mean) at 25 degrees C and +/- 0.02 degrees at 37 degrees C. The temperature variation was the same for internal and peripheral wells within the plates, although the internal wells were approximately 0.14 degrees C warmer than the outer wells at 25 degrees C and 0.68 degrees C cooler at 37 degrees C. The overall uncertainty (95% confidence interval) of the well temperature in a typical plate was +/- 0.4 degrees C at 25 degrees C and +/- 0.7 degrees C at 37 degrees C. This uncertainty is realistic for routine enzyme determinations, as opposed to precise studies. The incubator was designed to allow access to the plates from above so that additions could be made during the incubation. To demonstrate the suitability of microtiter plates and the incubator for enzyme determinations, this method was used to measure the activity of myeloperoxidase and alpha-naphthylbutyryl esterase.     

A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85 degrees C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.     

利用内蛋白子剪切功能一步纯化重组人神经营养因子-3

将人神经营养因子 - 3(h NT3)基因插入含内蛋白子 -几丁质结合区 (Intein- CBD)片段的质粒p TXB1的多克隆位点 ,构建成重组子 p TXB- h NT3,随后转化入 E.coli 2 566并进行融合表达 .表达产物包涵体经 8mol/ L脲变性 ,并在 GSH,GSSG存在下复性 .复性后的融合蛋白经几丁质珠亲和柱吸附 .待洗涤杂蛋白后 ,加入 50 mmol/ L DTT在 4℃或 2 5℃进行剪切反应 48h,再用缓冲液洗脱 ,即得 h NT3.SDS- PAGE分析表明 ,h NT3达电泳纯 .其分子量约为 1 4 k D     

Use of recombinant cellulose-binding domains of Trichoderma reesei cellulase as a selective immunocytochemical marker for cellulose in protozoa

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.     

Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain.

The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 831 amino acids with a deduced molecular weight of 90,020. The deduced ChiB is a modular enzyme composed of a family 18 catalytic domain responsible for chitinase activity, two reiterated domains of unknown function, and a chitin-binding domain (CBD). The reiterated domains are similar to the repeating units of cadherin proteins but not to fibronectin type III domains, and therefore they are referred to as cadherin-like domains. ChiB was purified from the periplasm fraction of Escherichia coli harboring the chiB gene. The molecular weight of the purified ChiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. The pH and temperature optima of the enzyme were 6.0 and 45 degrees C, respectively. The Km and Vmax values for 4-MU-(GlcNAc)2 were estimated to be 6.3 microM and 46 micromol/min/mg, respectively. SDS-PAGE, zymogram, and Western blot analyses using antiserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrificum. Deletion analysis showed clearly that the CBD of ChiB plays an important role in hydrolysis of native chitin but not processed chitin such as colloidal chitin.     

Use of Recombinant Cellulose-Binding Domains of Trichoderma reesei Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent β-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.     

Immobilization of small molecules and proteins by radio-derivatized polystyrene

When molded polystyrene (PS) products (e.g., microtiter plates) or latex particles are irradiated with high-energy (1-10 Mrads) gamma rays in the presence of nonpolymerizable small molecules such as aromatic amines, some of these molecules incorporate into PS, which leads to the formation of radio-derivatized PS (RDPS). Two classes of RDPS can be identified regarding their ability for immobilization of biologically important molecules: 1) reactive RDPS that are able to form covalent bonds with molecules such as proteins without the help of cross-linkers, and 2) functionalized RDPS that can be used for the immobilization of molecules with activators (e.g., carbodiimides) or cross-linkers. The method can be used for the production of low-noise supports for binding assays. Most of the RDPS can be produced without impairment of the optical quality of PS, making derivatized microtiter plates suitable for colorimetric assays. The principle can be applied for the preparation of affinity sorbents, e.g., for high-performance affinity chromatography and for the immobilization of enzymes using latex PS particles.