Purification and Characterization of Ovotransferrin from Crocodylus siamensis

Ovotransferrin (OTf) is the major glycoprotein in reptile egg whites. However, knowledge concerning its functional and biological properties remains limited. In this study, OTf from Crocodylus siamensis was purified and characterized. The proteins were precipitated with 80 % ammonium sulfate and then purified by anion exchange chromatography followed by hydrophobic interaction chromatography. The purified crocodile ovotransferrin (cOTf) had a molecular weight of 79 kDa. Analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) indicated multiple isoforms of cOTf, which had isoelectric points ranging from 6.0 to 6.8. cOTf was N-linked glycosylated protein identified by using PNGase F deglycosylation technique. Optimal autoproteolysis of cOTf occurred under acidic conditions and pH values more than 5, which differs from that of OTf.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc]₂ and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K_m and k_cat values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4–G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2–G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1–G5, and then the high-M_r intermediates (G3–G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.     

An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis

We provide evidence that chitinase A from Vibrio carchariae acts as an endochitinase. The chitinase A gene isolated from V. carchariae genome encodes 850 amino acids expressing a 95-kDa precursor. Peptide masses of the native enzyme identified from MALDI-TOF or nanoESIMS were identical with the putative amino acid sequence translated from the corresponding nucleotide sequence. The enzyme has a highly conserved catalytic TIM-barrel region as previously described for Serratia marcescens ChiA. The Mr of the native chitinase A was determined to be 62,698, suggesting that the C-terminal proteolytic cleavage site was located between R597 and K598. The DNA fragment that encodes the processed enzyme was subsequently cloned and expressed in Escherichia coli. The expressed protein exhibited chitinase activity on gel activity assay. Analysis of chitin hydrolysis using HPLC/ESI-MS confirmed the endo characteristics of the enzyme.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc](2) and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K(m) and k(cat) values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4-G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2-G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1-G5, and then the high-M(r) intermediates (G3-G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.     

Efficient E. coli expression systems for the production of recombinant β-mannanases and other bacterial extracellular enzymes

Two Escherichia coli expression systems based on T7 RNA polymerase promoter (pET system) and tac promoter (pFLAG system) have been used for the production and secretion of recombinant β-mannanases from Bacillus sp. Both E. coli OmpA signal peptide and native Bacillus signal peptide could be used efficiently for the secretion of recombinant enzymes into periplasmic space and culture media. The genes could be induced for over-expression with 0.1-1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) when the OD 600 of the culture broth reached 0.6-1.5. The recombinant enzymes could be harvested from whole cell lysate, perimplasmic extract, or culture broth after induction for 4-20 hours. Since the enzyme is C-terminally tagged with hexahistidine, the recombinant enzymes could be conveniently purified to apparent homogeneity by one-step immobilized-metal affinity chromatography (IMAC) using Ni-NTA resins. The characteristics of purified recombinant β-mannanases from B. licheniformis and B. subtilis, which share 78% amino acid identity, are slightly different. These systems should be applicable for the production of various recombinant bacterial extracellular enzymes.     

The Effects of Temperature and pH on Secondary Structure and Antioxidant Activity of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin (cHb) was purified by gel filtration chromatography and visualized by SDS-PAGE. Effects of temperature and pH on secondary structure and conformation changes of cHb were studied using circular dichroism spectropolarimeter and fourier transform infrared spectrophotometer. The secondary structure of intact cHb was mainly α-helices. cHb was not heat stable when heated at 65 °C and cooled down to original temperature, indicating the irreversible unfolding process. The stability of cHb at different pH ranging from 2.5 to 10.5 was determined. The maximum value of the α-helix content was found at pH 3.5 and tended to decrease at strong acid and strong base. The antioxidant activities of heat treated cHb and cHb in solution with pH range 2.5 to 10.5 were tested by DPPH radical scavenging assay. cHb at pH 4.5, having highest β-turn structure, showed highest radical scavenging activity. In contrast to pH, heat had no effect on antioxidant activity of cHb.     

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.     

Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism

This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (α/β)₈ TIM-barrel domain; and (iii) the small (α + β) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (−4)(−3)(−2)(−1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)₆-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F_o − F_c omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.     

Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS

The enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of alpha and beta anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated beta-anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher Mr chitooligomers, in the order of (GlcNAc)6 > (GlcNAc)4 > (GlcNAc)3, and showed no activity towards (GlcNAc)2 and pNP-GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues - Glu315 and Asp392 - by site-directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed approximately 20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC-MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild-type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP-glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild-type enzyme.     

Purification,Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P₅₀) and 1.96 (n value), and a small Bohr effect (δH⁺ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O₂ affinity at P₅₀ of 2.5 mmHg which may assure efficient utilization of the lung O₂ reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl₂·6H₂O] solution at a pH of 7.0–8.5.