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Chickpea (Cicer arietinum L.) seed is a potential source of protein ingredients with desirable nutritional and functional properties. Knowledge of molecular characteristics of a food protein is essential before a protein can gain widespread use as a food ingredient. The objectives of this study were to prepare chickpea proteins using different extraction methods and precipitation methods and to investigate molecular characteristics using polyacrylamide gel electrophoresis (PAGE; Native and SDS), reversed phase high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS) techniques. Proteins of ground chickpea seed were extracted with sodium hydroxide (NaOH) and with citric acid solutions and precipitated with addition of acid and by cryoprecipitation. The protein contents of the protein preparation ranged from 49% to 97%. The microstructures of chickpea protein isolates examined by scanning electron microscope (SEM) revealed the presence of starch grains in the cryoprecipitates from citric acid extraction but not in isoelectric precipitates. The globulins (legumins and vicilins), glutelins, and albumins from both citric acid and NaOH isolates were characterized by Native-PAGE. The cryoprecipitates contained mainly the globulin-rich proteins. With SDS-PAGE characterization, protein subunits were identified as follows: (i) legumin subunits: MW 40, 39, 26, 23, and 22 kDa, (ii) vicilin subunits: MW 50, 37, 33, 19, and 15 kDa, (iii) glutelin subunits: 58, 55, and 54 kDa, and (iv) albumin subunits: 10 kDa. Separation of fractions of isolated chickpea proteins by RP-HPLC showed that early eluting fractions (Rt 20-30 min) consisted of subunits of MW 6.5-31 kDa (SDS-PAGE). At elution time 30-36 min, the fractions obtained were composed mainly of mixtures of legumin and vicilin subunits (MW 14-45 kDa). The major subunits of chickpea protein fractions from both cryoprecipitates and isoelectric precipitates are legumin basic subunit (MW∼23 kDa) and vicilin-rich proteins (MW∼19, 17, 15 kDa). ESI-MS analysis of fractions separated by RP-HPLC showed MW ranging between 5.1 and 53.5 kDa. The subunits of MW 35366, 27626, 22864, 20531, 16092, and 15626 Da of fractions from ESI-MS corresponded to MW 35.3, 28.0, 24.1, 20.5, 16.1, and 15.3 kDa identified in SDS-PAGE. These fractions were identified as legumin-rich and vicilin-rich proteins.

Centrifugation of coconut milk resulted in cream, skim milk, and insoluble solids. Proteins were isolated from skim milk by the addition of acid, with or without heating. The separation and isolation gave the following coconut protein preparations: coconut milk, coconut skim milk, insoluble solids, acid precipitate, and acid-heat precipitate. / Trypsin inhibitory activity (TIA) of the coconut protein preparations was relatively low while tryptic digestibility of the isolated proteins was considerably lower than those of the coconut milk and skim milk, the digestibility of coconut protein preparations was lower than that of casein. In general, the emulsifying and farming properties of coconut protein preparations were lower than casein. The insoluble solids showed the highest viscosity when compared with the coconut protein preparations. In contrast to the whey protein concentrate (WPC), the apparent strain of gels from the acid precipitate increased as the pH increased. The gelation properties at pH 3 of the insoluble solids were better than WPC. / The estimated molecular weight by size-exclusion chromatography of coconut protein preparations gave 3 fractions with MW ranging from 6850 Da to 229402 Da. In native PAGE, coconut proteins were separated into at least 3 subunits and under SDS-denatured conditions, the major protein subunits showed MW of 54531 Da and 25008 Da, respectively. RP-HPLC separation of coconut milk, acid precipitate, and acid-heat precipitate gave 3 fractions containing several species of MW ranging between 35574 Da to 51209 Da when analyzed by mass spectometry.

Two leaves of amendments in pocket on front end paper. / Bibliography: leaves 177-185. / xix, 194 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes selective and nonselective procedures to extract recombinant protein from the cytoplasm of Escherichia coli. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1997

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Zhou, Xuechang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 51-53). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.ii / Acknowledgement --- p.iii / Table of contents --- p.iv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Protein crystallization --- p.1 / Chapter 1.1.1 --- The principle of protein crystallization --- p.3 / Chapter 1.1.2 --- Protein crystallization approaches --- p.4 / Chapter 1.1.3 --- Screening strategies and approaches --- p.6 / Chapter 1.2 --- Microfluidic systems for protein crystallization --- p.7 / Chapter 1.2.1 --- Integrated valve-controlled microfluidic system --- p.8 / Chapter 1.2.2 --- Droplet-based microfluidic system --- p.9 / Chapter 1.2.3 --- Objective of the research --- p.10 / Chapter 2 --- Nanoliter Liquid Dispensing Method --- p.12 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.2 --- Experimental --- p.14 / Chapter 2.2.1 --- The fabrication of SU-8 master --- p.14 / Chapter 2.2.2 --- The fabrication of PDMS microfluidic device --- p.16 / Chapter 2.2.3 --- The fabrication of glass and PMMA microwells --- p.17 / Chapter 2.2.4 --- The liquid dispensing into microwells --- p.17 / Chapter 2.3 --- Results and discussions --- p.20 / Chapter 2.3.1 --- The internal vacuum pumping source --- p.20 / Chapter 2.3.2 --- The efficiency of the pumping --- p.23 / Chapter 2.3.3 --- The removal of PDMS channel patch --- p.26 / Chapter 2.4 --- Conclusion --- p.29 / Chapter 3 --- The Screening of Protein Crystallization Conditions --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Experimental --- p.31 / Chapter 3.2.1 --- The design and fabrication of the screening chip --- p.31 / Chapter 3.2.2 --- The screening of protein crystallization conditions --- p.32 / Chapter 3.2.3 --- Crystallization and X-ray diffraction of an unknown protein --- p.33 / Chapter 3.3 --- Results and discussions --- p.33 / Chapter 3.3.1 --- Sparse matrix screening strategy in microwell arrays --- p.33 / Chapter 3.3.2 --- The results of the sparse matrix screening --- p.39 / Chapter 3.3.3 --- Crystal extraction and X-ray diffraction --- p.40 / Chapter 3.4 --- Conclusion --- p.41 / Chapter 4 --- Conclusion --- p.43 / Chapter 4.1 --- Summary --- p.43 / Chapter 4.2 --- Discussions and future directions --- p.44 / Appendix Information --- p.47 / References --- p.51

This thesis describes a novel microfluidic platform to control the macromolecular concentrations and their applications in constructing the phase diagram of polymer aqueous solutions and in the high-throughput screening of protein crystallization conditions at the nanoliter scale. The microfluidic platform was fabricated using the soft-lithography method and based on poly(dimethylsiloxane) (PDMS) material, which is widely used in microfluidic device. PDMS is gas and water permeable elastomer. By exploiting the permeability of the gas and water in PDMS, we developed the degassed-PDMS nanoliter liquid dispensing system, the controlled microevaporation method in constructing the phase diagram of polymer aqueous solution, and the screening platform of protein crystallization conditions. / This thesis describes two types of degassed-PDMS nanoliter liquid dispensing system. One is dispensing without the microvalve, in which various liquids are dispensed through the degassed PDMS microchannel. It involves two steps: in the first step, the PDMS microchannel patch (or the entire microchip) is placed in a vacuum chamber for a certain time; in the second step, the target liquid is deposited at the inlet of the PDMS channel and dispensed into the PDMS microchamber. The other method is dispensing with the aid of PDMS microvalve. This method combines the valve control and degassed PDMS pumping source, which provides more control over on the liquid dispensing, such as isolating, mixing, etc. / Zhou, Xuechang. / Adviser: Bo Zheng. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 53-56). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In the first part of this study, a non-ferric method for selective elimination of β-lactoglobulin from cheese whey was investigated. A new method was developed based on hexametaphosphate treatment of cheese whey. When Cheddar cheese whey was treated under the optimized conditions, i.e., 1.33 mg/mL sodium hexametaphosphate at 22°C and pH 4.07 for 1 hr, more than 80% of β-lactoglobulin was removed by precipitation. Almost all of the immunoglobulins and the major portion of α-lactalbumin were retained in the supernatant as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemical assays. By dialysis against distilled water 72.2% of the phosphorus was removed from the supernatant. In the second and the third part of the thesis, chromatographic methods were used for isolation of immunoglobulins and lactoferrin from whey proteins. By using gel filtration on Sephacryl S-300, 99, 83.3 and 92.1% biologically active immunoglobulin G were obtained for colostral whey, acid and Cheddar cheese whey, respectively. Lactoferrin, selectively adsorbed to the heparin-attached Sepharose, was eluted with 5 mM Veronal-HC1 containing 0.5M NaC1, at pH 7.2. 1,4-Butanediol diglycidyl ether-iminodiacetic acid on Sepharose 6B, or so-called metal chelate-interaction chromatography (MCIC), was loaded with copper ion and used for the same purpose. Of the two peaks obtained, the first yellowish peak was rich in lactoferrin, while the second peak was rich in immunoglobulins. Some of the physical and chemical properties of the proteins in these peaks, including immunochemical properties, isoelectric points, binding to bacterial lipopolysaccharides, and the mechanism of protein-metal interaction via histidine modification, and the capacity of the method were studied. The possibility of isolating immunoglobulins and lactoferrin from electrodialyzed whey was also investigated. In the fourth, fifth and sixth parts of the thesis, the method developed for isolation of immunoglobulins and lactoferrin from whey protein was applied to isolate these biologically important proteins directly from skimmilk, blood and egg white. The casein in skimmilk was found to compete with immunoglobulins for binding to copper ion in MCIC column when skimmilk was loaded in presence of 0.05 M Tris-acetate buffer containing 0.5 M NaC1, pH 8.2; however, this problem was solved by changing the equilibrating buffer to 0.02 M phosphate buffer containing 0.5 M NaC1, pH 7.0. When blood was directly applied to MCIC column, the yield of biologically active IgG was more than 95%. Ovotransferrin, strongly adsorbed to the MCIC column, was eluted with two-step elution protocols which suggests it exists in two forms. The histidine residues in immunoglobulins, caseins, transferrin and ovotransferrin were found to be involved in the mechanism of the interaction with the MCIC column. / Land and Food Systems, Faculty of / Graduate

In our previous studies, uncross-linked large diameter cellulose beads were optimized for solids content, bead size, pressure-flow limits, molecular accessibility and performance as an immunosorbent. Here, anion exchange (DEAE) cellulose beads were derivatized by two different procedures (defined as A and B) and the changes in bead morphology were correlated with transport and sorption kinetics. The kinetic characteristics clearly defined a minimum of two different types of protein binding site architecture. DEAE cellulose beads exhibited molecular exclusion of BSA near the edge of the bead in contrast to greater permeability seen in underivatized beads. Thus, accessible BSA binding sites are present only on the surface of the derivatized beads. DEAE cellulose beads derivatized by procedure B gave higher density of DEAE ligand as compared to beads derivatized by procedure A, as well as higher static and dynamic capacity for BSA. Even though DEAE cellulose beads (DP 2070, 450 μm diameter derivatized by procedure B) have lower small ion capacity than DEAE cross-linked agarose beads, as well as 1/4 the surface area, they exhibit equivalent binding capacity for BSA per volume of support. Thus, DEAE cellulose beads possess more sites per surface area as well as have lower ligand density per BSA site. Furthermore, BSA adsorption sites on DEAE cellulose beads derivatized by procedure B exhibit slow binding kinetics as compared to those derivatized by procedure A and also compared to DEAE crosslinked agarose beads. Thus, the rate limiting step for the adsorption of BSA on DEAE cellulose beads was not diffusion as suggested by the large diameter of the bead. Feasibility studies were performed for process scale applications to fixed and expanded bed anion exchange purification. The large diameter DEAE cellulose beads of this study maybe useful for process scale anion exchange as evident from purification of immunoglobulins from hybridoma cell culture in fixed bed. The balance of large diameter and density of these DEAE cellulose beads enable stable expanded bed purification of proteins such as recombinant human protein C from transgenic porcine whey. / Ph. D.