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PubMed2006

[Simultaneous control of microorganism, disinfection by-products and bio-stability by sequential chlorination disinfection].

A new safe chlorination disinfection process, the sequential disinfection process with short-term free chlorine plus chloramines was investigated its efficiency on control of indices of microorganism, disinfection by-products and bio-stability. This disinfection process may take the advantage of free chlorine's quick inactivation of microorganism and chloramines' low DBP yield and long-term residual. Thus, both control of microorganisms and DBPs can be achieved effectively and economically. The pilot test results show that safe chlorination disinfection process has a little better efficiency on total bacteria count, HPC and total coliform count than free chlorination disinfection, which indicate that free chlorine and chloramines might have the synergetic disinfection effect for their different attacking targets. It also had the same inactivation efficiency on polio virus and coliphage f2 as free chlorination in 120 minutes contact time. Sequential chlorination disinfection process could reduce 35.8% - 77.0% yield of trihalomethane and 36.6% - 54.8% yield of haloacetic acids. The poorer quality the water had, the more advantage the safe-chlorination disinfection had than free chlorination disinfection one. Moreover, sequential chlorination had better efficiency on bio-stability control than free chlorination.

Chloramines↗
PubMed2004

Effect of specific mutations in helix alpha7 of domain I on the stability and crystallization of Cry3A in Bacillus thuringiensis.

Insecticidal crystal (Cry) proteins of Bacillus thuringiensis crystallize after synthesis forming large inclusions that stabilize these toxins in the environment after cell lysis until eaten by an insect. Despite the biological importance of crystallization, little is known about the structural elements of Cry molecules that facilitate this process. We identified subdomains that affect Cry3A structure possibly through improper folding by chimeric-scanning mutagenesis, substituting short peptides of a truncated 70-kDa Cry1C molecule that does not crystallize into Cry3A, a wild-type 70-kDa molecule that crystallizes readily. Cry3A consists of three domains that contain five different blocks of conserved amino acids. Domain substitution and mutagenesis within these blocks suggested that the specific structure of block 2, which spans the junction between domains I and II, was important to the relative stability of Cry3A and subsequent crystallization. Amino acid sequences of particular importance to stability in Cry3A block 2 were identified using three substitution mutants, each spanning about a third of this block. One that consisted of Cry1C helix alpha7 yielded no detectable protein, whereas the other two produced characteristic Cry3A crystals. Specific mutations in this region showed tyrosine 268 was critical to normal stability of Cry3A and subsequent crystallization in that a mutant, Y268L, was less stable than wild-type Cry3A and failed to form a characteristic Cry3A crystal. Circular dichroism analysis showed a decrease in this mutant's alpha-helicity, indicating the importance of tyrosine 268 to the specific conformation of helix alpha7 that facilitates stability and normal crystallization.

Amino Acid Sequence↗
PubMed1990

The influence of fixation peg design on the shear stability of prosthetic implants.

The variety of fixation peg designs existing on prosthetic implants indicates uncertainty regarding the optimum design of fixation pegs for the reduction of stress and relative motion at the bone-implant interface. Fixation pegs have a number of important functions on a prosthesis, one of which is to reduce shear stress and shear displacement at the bone-implant interface. This is a parametric study intended to identify trends in the shear stability of prostheses incorporating a range of fixation peg designs. The parameters varied included the number of fixation pegs on a surface, the size of the pegs, and the aspect ratio (length/diameter) of the pegs. Mechanical tests were performed on urethane foam blocks with mechanical properties comparable to trabecular bone. The results indicated the following: (a) Fixation pegs act independently in resisting shearing force if they are spaced sufficiently far apart. (b) For any given shear displacement, smaller pegs generate a greater resistive shear force per unit of peg projected area in the direction of the applied load than larger pegs having the same aspect ratio. (c) Smaller diameter pegs cause the supporting material to yield at lower displacements. (d) Pegs with a high aspect ratio provide high shear stability with a minimum amount of bone removed, but may bend if the aspect ratio becomes excessive. (e) Smaller, slender pegs generate a greater resistive shear force at a given displacement per unit of peg volume than larger, lower aspect ratio pegs.(ABSTRACT TRUNCATED AT 250 WORDS)

Equipment Design↗
PubMed2005

Energizing cell-free protein synthesis with glucose metabolism.

In traditional cell-free protein synthesis reactions, the energy source (typically phosphoenolpyruvate (PEP) or creatine phosphate) is the most expensive substrate. However, for most biotechnology applications glucose is the preferred commercial substrate. Previous attempts to use glucose in cell-free protein synthesis reactions have been unsuccessful. We have now developed a cell-free protein synthesis reaction where PEP is replaced by either glucose or glucose-6-phosphate (G6P) as the energy source, thus allowing these reactions to compete more effectively with in vivo protein production technologies. We demonstrate high protein yields in a simple batch-format reaction through pH control and alleviation of phosphate limitation. G6P reactions can produce high protein levels ( approximately 700 microg/mL of chloramphenical acetyl transferase (CAT)) when pH is stabilized through replacement of the HEPES buffer with Bis-Tris. Protein synthesis with glucose as an energy source is also possible, and CAT yields of approximately 550 mug/mL are seen when both 10 mM phosphate is added to alleviate phosphate limitations and the Bis-Tris buffer concentration is increased to stabilize pH. By following radioactivity from [U-(14)C]-glucose, we find that glucose is primarily metabolized to the anaerobic products, acetate and lactate. The ability to use glucose as an energy source in cell-free reactions is important not only for inexpensive ATP generation during protein synthesis, but also as an example of how complex biological systems can be understood and exploited through cell-free biology.

Cell Culture Techniques↗
PubMed2001

A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold.

We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5x10(7) independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coli. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications.

Amino Acid Sequence↗
PubMed2001

Synthesis, characterization, solution stability, and X-ray crystal structure of the thiolatocobalamin gamma-glutamylcysteinylcobalamin, a dipeptide analogue of glutathionylcobalamin: insights into the enhanced Co-S bond stability of the natural product glutathionylcobalamin.

Glutathionylcobalamin (gamma-glutamylcysteinylglycinylcobalamin; gamma-GluCysGly-Cbl) is a natural product which functions as an intermediate in the biosynthesis of the active B(12) coenzymes adenosylcobalamin and methylcobalamin. Of interest to the present studies is glutathionylcobalamin's unique stability in comparison to other thiolatocobalamins, notably the > or =6 x 10(4) fold less stable cysteinylcobalamin, Cys-Cbl. In order to determine which parts of the glutathione tripeptide contribute to the overall stability of glutathionylcobalamin, two cysteine-containing dipeptides, which are truncated versions of glutathione, were used to synthesize their corresponding cobalamins, specifically gamma-glutamylcysteinylCbl (gamma-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl). As with glutathionylCbl, the dipeptide gamma-GluCys-Cbl forms a stable thiolatocobalamin. However and most interestingly, CysGly-Cbl is observed to be unstable much like Cys-Cbl. The results require that the extra stability of glutathionylcobalamin and its congeners, compared to cysteinylcobalamin and its analogues, must be derived from destabilization by the gamma-NH(3)(+) group in cysteinylcobalamin, or stabilization by the gamma-NHC(=O)- amide linkage in glutathionylcobalamin, or both. To probe any ground-state structural basis for the possible stabilization in gamma-GluCys-containing cobalamins, gamma-GluCys-Cbl was crystallized and yielded the first X-ray structural determination of a true thiolatocobalamin, and only the second structure of a cobalamin containing a Co-S bond, the first example being Randaccio and co-workers' 1999 structure of the thioketone complex, thioureacobalamin, (NH(2))(2)CSCbl. Key features of the structure of gamma-glutamylcysteinylcobalamin include (i) a normal Co-S bond length of 2.267(2) A, (ii) a Co-N(axial) bond length of 2.049(6) A, (iii) two alternate conformations of the gamma-glutamylcysteinyl moiety, and (iv) folding of the corrin ring upward by 24.2 degrees, the highest degree of folding yet observed for a cobalamin. These results do not show any strong stabilization (e.g., no shortened Co-S bond), although it is not clear for certain what the effect is (stabilizing or destabilizing) of the elongated Co-N(axial) bond; instead, the crystallographic results suggest that the metastable Cys-Cbl probably has a Co-S cleavage transition state that is stabilized (along with, possibly, any ground-state destabilization of the Co-S bond). Overall, the results strongly suggest that placing a positive charge on the gamma-NH(3)(+) stabilizes the Co-S bond cleavage transition state, thereby setting the stage for the needed full thermolysis product and kinetic studies-as a function of the axial-base on-off equilibrium-that will be required to understand in even greater detail the unique stability of glutathionyl- (gamma-glutamylcysteinylglycinyl-) and gamma-glutamylcysteinylcobalamins.

Cobalt↗
PubMed1981

LDH Calcutta-1: a mutation of the B subunit of human lactate dehydrogenase.

The electrophoretic variant of human LDH, Calcutta-1, occurs at phenotypic frequencies of 0-4% throughout India. The variant was examined by various electrophoretic techniques and by heat stability studies. The LD1 (B4) isoenzyme was purified from normal and variant bloods by affinity chromatography and ion-exchange chromatography. A minimum of five Calcutta-1 LD1 bands was demonstrated by isoelectric focusing. Electrophoresis of variant LD1 in high-molar urea-acrylamide denaturing gels resulted in two Calcutta-1 B subunit bands, while normal gels yielded only a single band. Homozygote Calcutta-1 LDH from red cells demonstrated a decreased heat stability, while heterozygote variant LDH showed a normal heat stability. This effect was confirmed when purified LD1's were compared. Evidence is presented suggesting a B-subunit variant showing thermolability in the homozygous form.

Electrophoresis, Starch Gel↗
PubMed2005

Effect of GGC (glycine) repeat length polymorphism in the human androgen receptor on androgen action.

BACKGROUND: The human androgen receptor (AR) contains glutamine (CAG) and glycine (GGC) repeat length polymorphisms. Normal glutamine repeat length affects androgen action, but an effect of normal glycine repeat length has not been studied. METHODS: To determine whether glycine/GGC repeat length affects AR function, we constructed AR cDNA expression vectors with different GGC repeat lengths in the physiological range (13-17 GGCs). AR constructs were transfected into AR-negative DU145 human prostate cancer cells along with an androgen-responsive reporter plasmid (PSA-firefly luciferase) and a transfection efficiency control plasmid (Renilla luciferase). RESULTS: Glycine repeat length had no significant effect on androgen-dependent AR transactivation activity expressed as firefly luciferase per unit amount of AR protein. However, AR protein levels (normalized for transfection efficiency) were inversely affected by glycine repeat length (P < 0.001; r = -0.9; e.g., GGC13 yielded 2.7 times more AR protein than did GGC17). Therefore, the net amount of AR activity per cell would be higher in cells expressing AR with a short glycine repeat. Based on programs that predict structure from RNA sequence, the GGC repeat can form a hairpin structure, the free energy of which decreases (i.e., hairpin stability increases) as a function of increasing repeat length. This suggests that hairpin stability may interfere with translation, accounting for the inverse effect of GGC repeat length on AR protein yields. CONCLUSIONS: The ability of a short GGC repeat to enhance androgen action provides a biologically plausible mechanism to account for reports that a short GGC repeat in the AR gene is a risk factor for prostate cancer.

Androgens↗
PubMed1999

Immunoaffinity layering of enzymes. Stabilization and use in flow injection analysis of glucose and hydrogen peroxide.

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.

Animals↗
PubMed2001

Optimization of stereoselective ketone reduction by the white-rot fungus Merulius tremellosus ono991.

A recently isolated white-rot fungal strain, Merulius tremellosus ono991, displays high stereoselectivity during the reduction of arylketones. In order to increase the productivity and specific yield of the optically active alcohols, the culture conditions for the reduction of the model ketone compound 1'-acetonaphtone to alpha-methyl-1-naphtalenemethanol were optimized with respect to oxygen supply, choice of primary substrate and arylketone concentration. Alternative electron acceptors were also used to elucidate the role of reduction equivalents in the reduction process. The optimal yields of alpha-methyl-1-naphtalenemethanol were obtained in N2-flushed incubations with glycerol as primary substrate. The specific yield was increased from 57% to 98% compared to incubations under air with glucose. Most of the yield increase was due to N2-flushing and could be attributed to two factors. First, an increased stability of the product, alpha-methyl-1-naphtalenemethanol, in anaerobic compared to aerobic atmosphere was demonstrated. Second, fermentative metabolism increased reduced enzyme cofactors available for the reduction. Diverting reducing equivalents away from fermentation with alternative electron acceptors correlated with a decreased yield of alpha-methyl-1-naphtalenemethanol. Furthermore, the dependency of ketone reductase for common occurring metabolic reducing equivalents, NAD(P)H, was demonstrated by the reduction of 1'-acetonaphtone in cell extracts of M. tremellosus ono991.

Basidiomycota↗
PubMed1974

The chloroplast and cytoplasmic ribosomes of euglena: I. Stability of chloroplast ribosomes prepared by an improved procedure.

A new isolation procedure has resulted in an improved yield of stable 68S chloroplast ribosomes from Euglena gracilis var. bacillaris. Chloroplasts are isolated by suspending the cells in buffer I (sorbitol, 250 mm; sucrose, 250 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm) and passing through a French press at less than 1500 pounds per square inch. The crude chloroplasts are purified by three washings with buffer II (sorbitol, 150 mm; sucrose, 150 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm). Stable 68S chloroplast ribosomes are obtained when the isolated chloroplasts are resuspended in ribosome buffer (tris-HCI, pH 7.6, 10 mm; magnesium acetate, 12 mm; KCI, 60 mm) containing spermidine, 0.5 mm; mercaptoethanol, 14 mm; sucrose, 8% (w/w), passed through a French press at 4000 pounds per square inch and extracted with either 0.1% (w/v) sodium deoxycholate or 1.0% (v/v) Triton X-100. At 0 to 4 C in ribosome buffer, the purified 68S chloroplast monosome forms a 53S particle while the 35S particle, an expected product of monosome dissociation, cannot be detected. Spermidine and mercaptoethanol prevent the formation of 53S particles from 68S monosomes. The purified 53S particles derived from 68S monosomes contain 23S RNA as well as a significant amount of 16S RNA, suggesting that this particle may not be a true ribosomal subunit.

Journal Article↗
PubMed1967

Herpes simplex virus products in productive and abortive infection. I. Stabilization with formaldehyde and preliminary analyses by isopycnic centrifugation in CsCl.

Lysates of HEp-2 cells productively infected with herpes simplex virus yielded two bands on isopycnic centrifugation in CsCl gradients, ranging from 1.2 to 1.6 g/cm(3). One band, designated alpha, had a mean buoyant density of 1.27 g/cm(3) and contained herpes virions. Band beta had a mean density of 1.305 g/cm(3) and contained primarily complement-fixing viral antigens and little or no viral deoxyribonucleic acid (DNA). The products banding in the alpha and beta bands were unstable; fivefold or higher amounts were recovered by treating the cell extract with formaldehyde prior to centrifugation. Formaldehyde treatment increased the buoyant density of viral products in both the alpha and beta bands by about 0.015 g/cm(3). In addition, it stabilized hitherto inapparent products, forming a broad band gamma with a density range of 1.37 to 1.45 g/cm(3). The material in the gamma band was heterogeneous; it contained viral DNA, cellular DNA, and viral antigen. Formalinized lysates of DK cells abortively infected with herpes simplex virus yielded a beta band undifferentiated from that formed by extracts of productively infected cells. The gamma band was less dense and narrower. The alpha band was entirely missing.

Carcinoma↗
PubMed2005

[Cloning and expression of the alpha-amylase gene from a Bacillus sp. WS06, and characterization of the enzyme].

A Bacillus sp. WS06, which produces an extracellular alpha-amylase, was isolated from the cecum in a piglet. An amyF gene from this Bacillus strain was cloned and its nucleotide sequence was determined. An open reading frame composed of 1581 bases, which encodes 526 amino acid residues was found. The amyF gene shows high sequence homologies with other microbial amylase genes, such as Bacillus megaterium and Bacillus polymyxa (93% and 53% identity). The deduced amino acid sequence revealed that four highly conserved regions of the alpha-amylase family. The amyF gene was overepressed using the pET21a vector and Escherichia coli BL21 (DE3). The recombinant enzyme was purified 22.2 fold to electrophoretic homogeneity and had a molecular mass of 57kD (by SDS-PAGE). The enzyme was optimally active at pH 7 and 55 approximately 60 degrees C and showed stability at the temperature below 55 degrees C. This enzyme efficiently hydrolyzed various types of starch to yield a series of malto-oligosaccharides by endo-cleavage mode.

Animals↗
PubMed1994

SSS1 encodes a stabilizing component of the Sec61 subcomplex of the yeast protein translocation apparatus.

The yeast SSS1 gene has been isolated as an extragenic high copy suppressor of sec61, a mutant displaying defects in protein translocation into the endoplasmic reticulum (ER). We found that SSS1 is an essential gene required for transfer of secretory precursors through the ER membrane. Here we demonstrate that the SSS1 product (Sss1p) is firmly bound to the ER membrane and exposes its amino-terminal half on the cytosolic side. Only detergent, or an alkali treatment, is effective at extracting Sss1p from the membrane. Coimmunoprecipitation experiments revealed that Sss1p and Sec61p participate in the same multisubunit complex. Cross-linking followed by immunoprecipitation specifically yielded an additional polypeptide of molecular mass 73 kDa. Moreover, Sss1p and Sec61p show mutually stabilizing interactions: Sss1p is destabilized in a sec61 mutant context, and mutated Sec61p is stabilized by Sss1p overproduction. These observations account for the isolation of SSS1 as a dosage-dependent suppressor of sec61. Since the polytopic integral membrane protein Sec61p is adjacent to translocating precursors and to ribosomes, and given the comparable translocation deficiencies of sss1 or sec61 mutants, we propose that Sss1p belongs to the "Sec61 subcomplex" that constitutes the pore of the membrane-bound translocation apparatus.

Biological Transport↗
PubMed1999

The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo.

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.

Bacterial Proteins↗
PubMed1993

Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli.

The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.

Cold Temperature↗
PubMed1987

Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex.

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.

Acetylation↗
PubMedDate not supplied

Immobilization as a tool for the stabilization of lignin peroxidase produced by Phanerochaete chrysosporium INA-12.

Lignin peroxidase immobilization was achieved by covalent coupling on CNBr-Sepharose 4B. Protein immobilization yield was around 80%. For veratryl alcohol oxidation, in the presence of hydrogen peroxide, both soluble and bound enzymes exhibited the same pH profile with an optimum near 2.5. Catalytic parameters (kc and Km) were seriously affected by immobilization. On the other hand, immobilization provided a noticeable stabilization of the enzyme against acidic pH and high temperatures. A 15-20 increase in the half-inactivation times at pH 2.2 and 2.7, respectively, could be observed. Bound enzyme was also much more thermostable than soluble.

Basidiomycota↗