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Ming-chung Chang - One of the best experts on this subject based on the ideXlab platform.

  • a modified osmotic shock for periplasmic release of a recombinant Creatinase from escherichia coli
    Biochemical Engineering Journal, 2004
    Co-Authors: Yu Cheng Chen, Ming-chung Chang, Li An Chen, Shujen Chen, Tehliang Chen
    Abstract:

    Abstract A modified osmotic shock involving pretreatment of the cells with divalent cation (Ca 2+ or Mg 2+ ) for periplasmic release of a recombinant Creatinase from Escherichia coli was proposed. The pretreatment enhanced the release of lipopolysaccharide (LPS) when the cells were subsequently treated with ethylenediaminetetraacetate (EDTA), thus increased the permeability of the outer membrane. Based on the data of cell disruption by sonication, the standard osmotic shock resulted in a 60% recovery of Creatinase and a 3.9-fold purification. With Ca-pretreatment, the efficiency of osmotic shock could be improved to 75% recovery and 4.5-fold purification. The pretreatment involved incubation of the cells with 5 mM Ca 2+ for 5 min, which was accompanied by an increased EDTA concentration (from 0.5 to 5 mM) in the subsequent osmotic shock. Mg-pretreatment could function similarly, but was less effective. When the cells were pretreated simultaneously with Ca 2+ and Mg 2+ , no additive effect on the periplasmic release was found, indicating that the binding sites on LPS are common to both cations. A possible adsorption isotherm for the binding of the cations to LPS was postulated. In addition, a protocol capable of dealing with suspensions of increased cell concentration was suggested.

  • expression and export of pseudomonas putida ntu 8 Creatinase by escherichia coli using the chitinase signal sequence of aeromonas hydrophila
    Biochemical Genetics, 1998
    Co-Authors: Ming Chuan Hong, Jinq Chyi Chang, Ming-chung Chang
    Abstract:

    The gene for the Creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the Creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--Creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.

  • Use of colloid chitin and diatomaceous earth in continuous cake-filtration fermentation to produce Creatinase
    Process Biochemistry, 1998
    Co-Authors: Shiue-cheng Tang, Ming-chung Chang, Chu-yuan Cheng
    Abstract:

    Abstract A recombinant Escherichia coli M15(pQE3208) producing Creatinase was cultured in a cake-filtration fermentor containing colloid chitin and diatomaceous earth. The filter medium of this cake filtration was a hollow cylinder made of a 20 μm stainless steel sieve located in the centre of the fermentor. During filtration, colloid chitin, diatomaceous earth, and E. coli cells formed a film of filter cake on the 20 μm sieve. The filter cake impeded the outward flow of cells from the fermentor. By controlling the concentration of colloid chitin (3 g/litre), diatomaceous earth (6 g/litre), and the interval of air sparging (0·5 h), continuous cake-filtration fermentation achieved a cell density in the reactor three times higher and specific Creatinase activity 35% higher than in ordinary continuous fermentation. An operation mode has been proposed for continuous cake-filtration fermentation to implement the production of intracellular protein which is inversely related to the growth rate of microorganisms.

  • cloning of a Creatinase gene from pseudomonas putida in escherichia coli by using an indicator plate
    Applied and Environmental Microbiology, 1992
    Co-Authors: Ming-chung Chang, Chun Chin Chang, Jinq Chyi Chang
    Abstract:

    A genomic library of Pseudomonas putida DNA was constructed by using plasmid pBR322. Transformants of Escherichia coli in combination with Proteus mirabilis cells grown on Creatinase test plates were screened for Creatinase activity; transformants were considered positive for Creatinase activity if a red-pink zone appeared around the colonies. One Creatinase-positive clone was further analyzed, and the gene was reduced to a 2.7-kb DNA fragment. A unique protein band (with a molecular weight of approximately 50,000) was observed in recombinant E. coli by minicell analysis.

Chandra Shekhar Pundir - One of the best experts on this subject based on the ideXlab platform.

  • an improved amperometric creatinine biosensor based on nanoparticles of creatininase Creatinase and sarcosine oxidase
    Analytical Biochemistry, 2017
    Co-Authors: Parveen Kumar, Ranjana Jaiwal, Chandra Shekhar Pundir
    Abstract:

    Abstract An improved amperometric biosensor for detection of creatinine was developed based on immobilization of nanoparticles (NPs) of creatininase (CA), Creatinase (CI), and sarcosine oxidase (SOx) onto glassy carbon (GC) electrode. Transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR) were employed for characterization of enzyme nanoparticles (ENPs). The GC electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) at different stages of its amendment. The biosensor showed optimum response within 2s at pH 6.0 in 0.1 M sodium phosphate buffer and 25 °C, when operated at 1.0 V against Ag/AgCl. Biosensor exhibited wider linear range from 0.01 μM to 12 μM with a limit of detection (LOD) of 0.01 μM. The analytical recoveries of added creatinine in sera were 97.97 ± 0.1% for 0.1 mM and 98.76 ± 0.2% for 0.15 mM, within and between batch coefficients of variation (CV) were 2.06% and 3.09% respectively. A good correlation (R2 = 0.99) was observed between sera creatinine values obtained by standard enzymic colorimetric method and the present biosensor. This biosensor measured creatinine level in sera of apparently healthy subjects and persons suffering from renal and muscular dysfunction. The ENPs electrode lost 10% of its initial activity within 240 days of its regular uses, when stored at 4 °C.

  • immobilization of creatininase Creatinase and sarcosine oxidase on iron oxide nanoparticles chitosan g polyaniline modified pt electrode for detection of creatinine
    Enzyme and Microbial Technology, 2012
    Co-Authors: Sandeep Yadav, Sapna Singhla, Rooma Devi, Pratibha Bhar, Chandra Shekhar Pundir
    Abstract:

    Abstract Commercial enzymes, creatininase (CA) from Pseudomonas sp, Creatinase (CI) from Pseudomonas sp, sarcosine oxidase (SO) from Bacillus sp were co-immobilized onto iron oxide nanoparticles/chitosan-graft-polyaniline (Fe 3 O 4 -NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. Transmission electron microscopy (TEM) was used for characterization of Fe 3 O 4 -NPs. A creatinine biosensor was fabricated using Enzymes/Fe 3 O 4 -NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopic and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 2 s at pH 7.5 and 30 °C, when polarized at 0.4 V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on creatinine concentration ranging from 1 to 800 μM. The sensitivity of the biosensor was 3.9 μA μM −1  cm −2 , with a detection limit of 1 μM (S/N = 3). Apparent Michaelis–Menton ( K m ) value for creatinine was 0.17 mM. The biosensor showed only 10% loss in its initial response after 120 uses over 200 days, when stored at 4 °C. The biosensor measured creatinine in the serum of apparently healthy persons which correlated well with a standard colorimetric method ( r  = 0.99).

Lei Kai - One of the best experts on this subject based on the ideXlab platform.

  • study on the Creatinase from paracoccus sp strain wb1
    Process Biochemistry, 2006
    Co-Authors: Yuanyuan Wang, Weifeng Zhao, Xiaoming Jia, Lei Kai
    Abstract:

    Abstract A Creatinase-producing bacterium was isolated in this work. Based on its morphological and physiological characteristics, G + C mol% of DNA and 16S rDNA sequence, it was identified to be a Paracoccus sp. Creatinase produced from it was purified to electrophoretic homogeneity with recovery of 17.4% by ion exchange chromatography, hydrophobic chromatography and gel filtration. The molecular mass of the enzyme was estimated to be 48,000 Da by SDS-PAGE. The enzyme showed maximum activity at pH 7.5 and was stable at pH 5.5–9.5. The Km of the enzyme was 24.6 mM with creatine as substrate at 37 °C. Metal ions such as Cu2+, Hg2+ and Ag+ inactivated its activity completely. When the Creatinase was used to measure creatinine, the concentration of the creatinine and the absorbance of the dye at 500 nm were linearly related up to 2000 μmol/l. This study demonstrates that Paracoccus is a new source for producing creatinine-analyzing enzyme.

Jinq Chyi Chang - One of the best experts on this subject based on the ideXlab platform.

Rainer Jaenicke - One of the best experts on this subject based on the ideXlab platform.

  • stabilization of Creatinase from pseudomonas putida by random mutagenesis
    Protein Science, 1993
    Co-Authors: J Schumann, Gerald Bohm, Gunter Schumacher, Rainer Rudolph, Rainer Jaenicke
    Abstract:

    Creatinase (creatine amidinohydrolase, EC 3.5.3.3) from Pseudomonas putida is a homodimer of 45 kDa subunit molecular mass, the three-dimensional structure of which is known at 1.9 A resolution. Three point mutants, A109V, V355M, and V182I, as well as one double mutant combining A109V and V355M, and the triple mutant with all three replacements, were compared with wild-type Creatinase regarding their physical and enzymological properties. High-resolution crystal data for wild-type Creatinase and the first two mutants suggest isomorphism at least for these three proteins (R. Huber, pers. comm.). Physicochemical measurements confirm this prediction, showing that the mutations have no effect either on the quaternary structure and gross conformation or the catalytic properties as compared to wild-type Creatinase. The replacement of V182 (at the solvent-exposed end of the first helix of the C-terminal domain) does not cause significant differences in comparison with the wild-type enzyme. The other point mutations stabilize the first step in the biphasic denaturation transition without affecting the second one. In sum, the enhanced stability seems to reflect slight improvements in the local packing without creating new well-defined bonds. The increase in hydrophobicity generated by the introduction of additional methyl groups (A109V, V182I) must be compensated by minor readjustments of the global structure. Secondary or quaternary interactions are not affected. In going from single to double and triple mutants, to a first approximation, the increments of stabilization are additive.

  • Creatinase in its collapsed a state shows properties of a molten globule with dimeric quaternary structure
    FEBS Journal, 1993
    Co-Authors: J Schumann, Rainer Jaenicke
    Abstract:

    In the past, the molten globule state at acidic pH (A state) has mainly been observed for small single-domain proteins. For more complex proteins such as immunoglobulin, alternatively folded states, with certain characteristics of the molten globule but different thermodynamic properties, were observed. In the present work, the acid-induced structural characteristics of a homodimer, Creatinase from Pseudomonas putida, are described. The 91-kDa protein at pH 2 shows molten globule behavior in that there is (a) a high content of native-like secondary structure (monitored by far-ultraviolet circular dichroism), (b) changes in the solvent accessibility of intrinsic fluorophores (acrylamide quenching of protein fluorescence), (c) increased hydrophobic surface area (indicated by anomalous dye binding) and (d) a slight expansion of the hydrodynamic volume (calculated from S20,solv, obtained from analytical ultracentrifugation). The enzyme at pH 2 shows reversible cooperative transitions in guanidinium chloride or urea (at elevated ionic strength). Its quaternary structure remains unaltered, indicating that native-like subunit interactions are involved in the stabilization of the A state of the enzyme. Anions stabilize the compact conformation due to reduced intramolecular charge repulsion; for the same reason, the enzyme in its A state shows a strong tendency to aggregate at >0.3 M NaCl.

  • intrinsic stability and extrinsic stabilization of Creatinase from pseudomonas putida
    Biological chemistry Hoppe-Seyler, 1993
    Co-Authors: J Schumann, Hans Mollering, Rainer Jaenicke
    Abstract:

    Creatinase (creatine amidinohydrolase, EC 3.5.3.3), a homodimer of 45 kDa subunit molecular mass, shows only limited functional stability, and is inaccessible to reconstitution after preceding deactivation, denaturation and dissociation. The enzyme has been characterized regarding its native and denatured states. Studying its unfolding characteristics in the presence of "extrinsic factors", such as DTE, BSA and glycerol, it was possible to define solvent conditions where the stability of the enzyme is significantly improved. Apart from protecting essential thiol groups and charge screening effects, the stabilization is caused mainly by preferential solvation. In the presence of 20% (w/v) glycerol, the kinetic analysis of the time course of denaturation indicates that a partially active folding intermediate, rather than the whole molecule, is involved in the stabilization. The mixed solvent improves the thermal stability, as well as the stability toward GdmCl and urea.