Fu-Pang Lin

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing.

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.

Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E

The bifunctional activities of α-amylase and pullulanase are found in the cloned recombinant amylopullulanase. It was encoded in a 2.9-kb DNA fragment that was amplified using polymerase chain reaction from the chromosomal DNA of Thermoanaerobacter ethanolicus 39E. An estimated 109-kDa recombinant protein was obtained from the cloned gene under the prokaryotic expression system. The optimum pH of the recombinant amylopullulanase was 6.0. The most stable pH for the α-amylase and pullulanase activity was 5.5 and 5.0, respectively. The optimum temperature for the α-amylase activity was 90°C, while its most stable temperature was 80°C. Regarding pullulanase activity, the optimum temperature and its most stable temperature were found to be 80 and 75°C, respectively. Pullulan was found to be the best substrate for the enzyme. The enzyme was activated and stabilized by the presence of Ca2+, whereas EDTA, N-bromosuccinimide, and α-cyclodextrin inhibited its bifunctional activities. A malto-2–4-oligosac-charide was the major product obtained from the enzymatic reaction on soluble starch, amylose, amylopectin, and glycogen. A single maltotriose product was found in the pullulan hydrolysis reaction using this recombinant amylopullulanase. Kinetic analysis of the enzyme indicated that the Km values of α-amylase and pullulanase were 1.38 and 3.79 mg/mL, respectively, while the Vmax values were 39 and 98 µmol/(min · mg of protein), respectively.

Biochemical characteristics of C-terminal region of recombinant chitinase from Bacillus licheniformis - implication of necessity for enzyme properties

The functional and structural significance of the C‐terminal region of Bacillus licheniformis chitinase was explored using C‐terminal truncation mutagenesis. Comparative studies between full‐length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, kcat/Km, was slightly increased for the truncated enzymes toward the soluble 4‐methylumbelliferyl‐N‐N′‐diacetyl chitobiose or 4‐methylumbelliferyl‐N‐N″‐N‴‐triacetyl chitotriose or insoluble α‐chitin substrate. By contrast, changes to substrate affinity, Km, and turnover rate, kcat, varied considerably for both types of chitin substrates between the full‐length and truncated enzymes. Both truncated enzymes exhibited significantly higher thermostabilities than the full‐length enzyme. The truncated mutants retained similar substrate‐binding specificities and abilities against the insoluble substrate but only had approximately 75% of the hydrolyzing efficiency of the full‐length chitinase molecule. Fluorescence spectroscopy indicated that both C‐terminal deletion mutants retained an active folding conformation similar to the full‐length enzyme. However, a CD melting unfolding study was able to distinguish between the full‐length and truncated mutant molecules by the two phases of apparent transition temperatures in the mutants. These results indicate that up to 145 amino acid residues, including the putative C‐terminal chitin‐binding region and the fibronectin (III) motif of B. licheniformis chitinase, could be removed without causing a seriously aberrant change in structure and a dramatic decrease in insoluble chitin hydrolysis. The results of the present study provide evidence demonstrating that the binding and hydrolyzing of insoluble chitin substrate for B. licheniformis chitinase was not dependent solely on the putative C‐terminal chitin‐binding region and the fibronectin (III) motif.

Site‐Directed Mutagenesis of Asp313, Glu315, and Asp391 Residues in Chitinase of Aeromonas Caviae

Site‐directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae .Kinetic parameters for 4‐methylumbelliferyl‐N, N‐diacetylchitobiose or 4‐methylumbelliferyl‐N,N, N‐triacetylchitotriose hydrolysis were determined with wild‐type and mutant chitinases. Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat. However, the effect of the these mutations on the K m values were different. The function ofthe carboxylgroup of Asp313 was partially replaced by the amide ofAsn when the 4‐methylumbelliferyl‐N, N, N‐triacetylchitotriose substrate was used. Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.

Biochemical Characterization of Two Truncated Forms of Amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 to Identify Its Enzymatically Active Region

The enzymatically active region of amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 (TsaNTOU1Apu) was identified by truncation mutagenesis. Two truncated TsaNTOU1Apu enzymes, TsaNTOU1ApuM957 and TsaNTOU1ApuK885, were selected and characterized. Both TsaNTOU1ApuM957 and TsaNTOU1ApuK885 showed similar specific activities toward various substrates. The overall catalytic efficiency (kcat/apparent Km) for the soluble starch or pullulan substrate, however, was 20–25% lower in TsaNTOU1ApuK885 than in TsaNTOU1ApuM957. Both truncated enzymes exhibited similar thermostability and substrate-binding ability against the raw starch. The fluorescence and circular dichroism spectrometry studies indicated that TsaNTOU1ApuK885 retained an active folding conformation similar to that of TsaNTOU1ApuM957. These results indicate that a large part of the TsaNTOU1Apu, such as the C-terminal carbohydrate-binding module family 20, the second fibronectin type III, and a portion of the first FnIII motifs, could be removed without causing a serious aberrant structural change or a dramatic decrease in hydrolysis of soluble starch and pullulan.

Effect of C-terminal truncation on enzyme properties of recombinant amylopullulanase from Thermoanaerobacter pseudoethanolicus

The smallest and enzymatically active molecule, TetApuQ818, was localized within the C-terminal Q818 amino acid residue after serial C-terminal truncation analysis of the recombinant amylopullulanase molecule (TetApuM955) from Thermoanaerobacter pseudoethanolicus. Kinetic analyses indicated that the overall catalytic efficiency, kcat/Km, of TetApuQ818 was 8–32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate affinity, Km, and the turnover rate, kcat, were decreased significantly in both enzymatic activities of TetApuQ818. TetApuQ818 exhibited less thermostability than TetApuM955 when the temperature was raised above 85°C, but it had similar substrate-binding ability and hydrolysis products toward various substrates as TetApuM955 did. Both enzymes showed similar spectroscopies of fluorescence and circular dichroism, suggesting the active folding conformation was maintained after this C-terminal Q818 deletion. This study suggested that the binding ability of insoluble starch by TetApuM955 did not rely on the putative C-terminal carbohydrate binding module family 20 (CBM20) and two FnIII regions of TetApu, though the integrity of the AamyC module of TetApuQ818 was required for the enzyme activity.

Effects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase

C-Terminal truncation mutagenesis was used to explore the functional and structural significance of the C-terminal region of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606) included initial rate kinetics, fluorescence and circular dichroism (CD) spectrometric properties, and substrate binding and hydrolysis abilities. The overall catalytic efficiency, kcat/KM, of AcD1ChiAK606 with the 4MU-(GlcNAc)2 and the 4MU-(GlcNAc)3 chitin substrates was 15–26% decreased. When compared with AcD1ChiA, the truncated mutant AcD1ChiAK606 maintained 80% relative substrate-binding ability and about 76% of the hydrolyzing efficiency against the insoluble α-chitin substrate. Both fluorescence and CD spectroscopy indicated that AcD1ChiAK606 retained the same conformation as AcD1ChiA. These results indicated that removal of the C-terminal 259 amino acid residues, including the putative chitin-binding motif and the A region (a motif of unknown function) of AcD1ChiA, did not seriously affect the enzyme structure integrity as well as activity. The present study provided evidences illustrating that the binding and hydrolyzing of insoluble chitin substrates by AcD1ChiA were not absolutely dependent on the putative C-terminal chitin-binding domain and the function-unknown A region.

Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways

Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways.

Cloning and Characterization of Manganese Superoxide Dismutase Gene from Vibrio parahaemolyticus and Application to Preliminary Identification of Vibrio Strains

The sodA gene coding for manganese superoxide dismutase (Mn‐SOD) from the marine microorganism Vibrio parahaemolyticus was cloned, sequenced, and overexpressed in Escherichia coli by use of the pET20b (+) expression vector. The full‐length gene consisted of a 588‐bp open reading frame and encoded a polypeptide of 196 amino acid residues, with a calculated molecular mass of 21 713 Da. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by metal ion affinity chromatography. The recombinant VPMn‐SOD resisted thermo‐denaturation up to 60 °C and was insensitive to such inhibitors as EDTA, NaN3 and diethyldithiocarbamic acid. The specificity of V. parahaemolyticus Mn‐SOD gene probe was analyzed by cross‐species polymerase chain reaction to provide information for Vibrio strain identification.

Cloning, sequencing, expression and characterization of the manganese superoxide dismutase gene from Vibrio alginolyticus

The sodA gene coding for manganese superoxide dismutase from the marine microorganism Vibrio alginolyticus was cloned, sequenced and over‐expressed in Escherichia coli using the pET20b (+) expression vector. The full‐length gene was consisted of 603bp open reading frame, which encoded a polypeptide of 201 amino acid residues, with a calculated molecular weight of 22672Da. The deduced amino acid sequence of the sodA showed considerable homology to other Mn‐SODs. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by the metal ion affinity chromatography. The recombinant VAMn‐SOD resisted thermo‐denaturation up to 60°C and was insensitive to inhibitors such as H2O2, NaN3 and diethyldithiocarbamic acid.