Significant abilities of titanium(IV)-activated glass fibre paper and its papain conjugates to chill-proof beer

It has been demonstrated that titanium(IV)-activated glass fibre paper, unlike untreated glass fibre paper, acts as a potent agent for chill-proofing beer: three treatments of beer with titanium(IV)-activated glass fibre paper (0.2m²/l) for 12 h were found to produce complete chill-proofing. Chill-proofed beer was found not to be contaminated with titanium(IV) species at a concentration above a detection limit of 2 p.p.m., so augering well for the safety of titanium(IV)-activated glass fibre paper as a chill-proofing agent. The physical form of titanium(IV)-activated glass fibre paper provides a major advantage over particulate chill-proofing agents in that it may be easily and rapidly separated from beer. The chill-proofing ability of titanium(IV)-activated glass fibre paper was found to decline with repeated use of the material. Moreover, all the observed chill-proofing effects were paralleled by reductions in the absorbance of beer across the ultraviolet and visible regions of the spectrum. These results demonstrate that titanium(IV)-activated glass fibre paper imitates hydrous titanium(IV) oxide in its effects upon the properties of beer and accord with other evidence that the surface of titanium(IV)-activated glass fibre paper is chemically similar to that of hydrous titanium(IV) oxide. The mechanism of the chill-proofing action of titanium(IV)-activated glass fibre paper is concluded to be analogous to that of hydrous titanium(IV) oxide and probably to involve the non-specific adsorption or ligand bonding of beer proteins and polyphenols to titanium(IV). Two papain (papain, EC 3.4.22.2) conjugates of titanium(IV)-activated glass fibre paper, of different catalytic activities, have been examined for chill-proofing ability. The chill-proofing behaviour observed for each papain conjugate was found not to differ from that observed for the corresponding catalytically-inactive S-carboxy-methyl derivative, prepared by treatment of the papain conjugate with bromoacetic acid, nor to differ greatly from that of titanium(IV)-activated glass fibre paper. On this basis the chill-proofing abilities of the papain conjugates are attributed solely to their abilities to adsorb beer constituents and not to their catalytic activities.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

A study of the beer chill-proofing behaviour of a water-insoluble papain conjugate of hydrous titanium(IV) oxide-use of hydrous titanium(IV) oxide as a novel chill-proofing agent

An active papain (EC 3.4.22.2) conjugate of hydrous titanium(IV) oxide has been found to exhibit substantial ability to chill-proof beer. However, this ability has been shown to be nearly identical to the chill-proofing abilities found to be exhibited by an inactive S-carboxymethyl derivative of the papain conjugate, which was prepared by treating the papain conjugate with bromoacetic acid, and by free hydrous titanium(IV) oxide. Further, the chill-proofing achieved by each material was observed to correlate with reductions in the absorbance of beer in both the ultraviolet and visible regions of the spectrum. On this basis, it is concluded that chill-proofing by these materials occurs solely by the adsorption of beer constituents and that this adsorption is probably non-specific. The rider to this conclusion is that the coupling of papain to hydrous titanium(IV) oxide, in order to give an active immobilized enzyme that acts as a reusable chill-proofing agent, is without efficacy. The broader significance of these observations in the development of enzymic chill-proofing agents is discussed. The potential of free hydrous titanium(IV) oxide as an adsorbent chill-proofing agent has been examined. It has been shown that the treatment of beer for 12 h at 4°C with fresh hydrous titanium(IV) oxide (200 g/hl) produces nearly complete chill-proofing. Chill-proofing is enhanced both by prolongation of treatment and by increased number of treatments. Further, beer that had been chill-proofed by hydrous titanium(IV) oxide was found to contain titanium(IV) at a concentration below the detection limit (2 p.p.m.) of the adopted colorimetric method of analysis. These results auger well for the safe commercial use of hydrous titanium(IV) oxide as a novel and effective chill-proofing agent, particularly as hydrous titanium(IV) oxide may be prepared conveniently on site from a readily available and inexpensive material, titanium(IV) chloride.     

Compositions and compositional-behavioural relationships of enzymes immobilized on porous inorganic supports via titanium(IV) species

The compositions and compositional-behavioural relationships of glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) immobilized on titanium(IV)-activated porous inorganic supports have been investigated for several transition metal activation techniques based on the metal-link/chelation method developed by our group. The highest activity (239 Ug^?1 matrix) of immobilized glucoamylase was obtained with the hydrous titanium(IV) oxide derivative of the support when this and a 15% w/v TiCl₄ solution were dried at 45°C in vacuum for 30 h. However, the immobilized enzyme preparation displayed a very unstable behaviour, as did also the preparation which was obtained by drying the mixture of support and transition metal solution at atmospheric pressure. This was mainly due to an enzyme deactivation by titanium inhibition instead of enzyme loss in substrate solution. When amination and carbonylation steps were included in the immobilization technique much more stable preparations were obtained, mainly when the support was activated by drying at 45°C with a 15% w/v TiCl₄ solution ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1495}\text{h}$) although with a lower initial activity (35.6 Ug^?1 matrix). The pure TiCl₄ support activation rather than TiCl₄/HCl solution support activation led to less stable immobilized enzyme preparations (washing and amination solvent chloroform, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 365}\text{h}$; washing and amination solvent water, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 276}\text{h}$) than the preparation obtained with the dried titanium(IV)-activated support. This was due to loss of enzyme-titanium(IV) complex in solution, as the interactions between the titanium(IV) and the silanol groups of the porous silica are weak. However, the amination (with 1,6-diaminohexane) and carbonylation (with glutaraldehyde) steps always led to immobilized enzyme preparations with constant specific activities and protein/titanium(IV) ratio. This suggests that the spacing effect introduced by these reactions removes the titanium(IV) inhibition of glucoamylase.     

Alkylimido complexes of titanium(IV)

TiCl₄ reacts with t-butylamine in benzene to give [Ti(NCMe₃)Cl₂(NH₂CMe₃)₂]_x and t-butylamine hydrochloride. The IR spectrum indicates both c/s and trans metal dichlorides (300; and 308, 208 cm⁻¹). In the ¹³C NMR spectrum the t-butylimido quaternary carbon resonance occurs at 72.1 ppm. A dimeric structure incorporating symmetric t-butylimido bridges is proposed. TiCl₄ in benzene react under reflux with two equivalents of Me₃SiNHCMe₃ to give [Ti(NCMe₃)Cl₂(NH₂CMe₃)]_x and with iso-propylamine and ethylamine to give complexes of the form [Ti(NR)Cl₂(NH₂R)₂]_x. Broad bands below 800 cm⁻¹ in the IR spectra suggest polymeric MNM bridges. For [Ti(NCHMe₂)Cl₂(NH₂CHMe₂)]_x the iso-propylimido CH resonance in the ¹³C NMR spectrum occurs at 67 ppm. [Ti(NCMe₃)Cl₂(NH₂CMe₃)₂]₂ reacts with L=bipy or tmed to give [Ti(NCMe₃)Cl₂(L)]₂, and TiCl₄ reacts with two equivalents of Me₃SiNHCMe₃ in benzene and then tmed to give [Ti(NCMe₃)Cl₂(tmed)]₂. The ¹³C NMR spectrum shows the t-butylimido quaternary carbon resonance at 73.5 ppm and the tmed resonances are chemically equivalent. A dimeric μ-NCMe₃ bridging structure is proposed for the complex.     

Photogeneration of titanium(III) from titanium(IV) citrate in aqueous solution

Current interest in the biochemistry of Ti(IV) arises from its widespread use in white pigments and its potential in therapeutic agents. Citrate is known to form strong complexes with Ti(IV). We show here that Ti(III) citrate is generated in a facile manner and in good yield by the action of UV radiation on Ti(IV) citrate in aqueous solution. The Ti(III)-citrate species formed was isolated and characterised by UV-Visible spectroscopy, showing an absorption at 547 nm (epsilon=100 M(-1)cm(-1)), and by electron paramagnetic resonance (EPR) spectroscopy giving a resonance at g=1.949 (linewidth=60G) . An X-ray structure of the parent Ti(IV) complex in the form [TiNa(3)(C(6)H(6)O(7))(2)(C(6)H(5)O(7))(H(2)O)(6.8)].2H(2)O is reported along with a study of the reaction of Ti(IV)-citrate with N,N-ethylenebis(o-hydroxytoluene)glycine (EHTG), which was more rapid than those of other related Ti(IV) complexes.     

Influence of coupling conditions on activity and operational stability of glucoamylase immobilized on titanium (IV)-activated controlled pore glass

Glucoamylase (EC 3.2.1.3) was coupled to controlled pore glass by using titanium(IV) chloride. The drying conditions used during the activation step were studied, and the highest activity (237 units/g of matrix) of immobilized enzyme was obtained when the support and the titanium(IV) chloride solution were dried at 45°C in vacuo for 16 h. After several washing cycles, the specific activity of the immobilized enzyme was ～13 units/mg of protein irrespective of the washing cycle used. However, this immobilized enzyme preparation was also the least stable ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1}\text{h}$). Investigation of the possibility of the stabilization of the linkage of the enzyme to the support by crosslinking with bifunctional reagents showed that the stabilization of the enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=100}\text{h}$) was achievable by treatment with a 5% glutaraldehyde solution at pH 7.0 for 2 h (product activity 67 units/g of matrix, specific activity 4 units/mg of protein); this product also showed no release of protein during use. A higher activity (296 units/g of matrix was achieved by stabilization by treatment with a 5% tannic acid solution at pH 7.0 for 2 h. The combined use of glutaraldehyde and tannic acid was effective in stabilizing the bound enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=80 to 120 h}$) with an initial activity of 116 units/g of matrix. When use was made of the same support in presilanized (3-aminopropyltriethoxy silane) form followed by glutaraldehyde coupling a similar initial activity (112 units/g of matrix) was obtained, but the operational stability was much better ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 640}\text{h}$.     

Poly(methoxygalacturonide) lyase immobilized via titanium onto solid supports

Poly(methoxygalacturonide) lyase (PMGL) (E.C. 4.2.2.10) was purified from a commercial preparation and immobilized by the metal link method. The properties of DEAE-cellulose-Ti-PMGL and of porous glass-Ti-PMGL were compared with those of the native enzyme; despite the presence of the metal and the heterogeneity of the substrate, pectin, typical substrate–enzyme-support interactions were demonstrated by shifts in pH optima and K_M values. The possible industrial application of DEAE-cellulose-Ti-PMGL is discussed.     

Formation of titanium(IV) transferrin by reaction of human serum apotransferrin with titanium complexes

The reaction of human serum apotransferrin with titanium(IV) citrate under physiological conditions results in the formation of a specific bis-titanium(IV) transferrin adduct (Ti₂Tf hereafter) with two titanium(IV) ions loaded at the iron binding sites. The same specific Ti₂Tf complex is formed by reacting apotransferrin with titanium(III) chloride and exposing the sample to air. The derivative thus obtained was characterized by spectroscopic techniques, including absorption, UV difference, circular dichroism and ¹³C NMR spectroscopies, and shown to be stable within the pH range 5.5–9.0. Surprisingly, the reaction of apoTf with titanium(IV) nitrilotriacetate (NTA) does not lead to formation of appreciable amounts of Ti₂Tf, even after long incubation times, although some weak interactions of Ti(IV)NTA with apoTf are spectroscopically detected. Implications of the present results for a role of transferrin in the uptake, transport and delivery of soluble titanium(IV) compounds under physiological conditions are discussed.     

Chemical nature of implant-derived titanium(IV) ions in synovial fluid

Previous investigations have indicated a deleterious leakage of Ti(III) and/or Ti(IV) species from Ti-Al-V alloy joint prostheses into adjacent tissue, synovium or synovial fluid (SF) in vivo. In view of the importance of the particular chemical nature of such complexes in determining their biological activity, we have employed high field proton (1H) NMR spectroscopy to "speciate" Ti(IV) in inflammatory SF. Treatment of osteoarthritic SF samples with increasing concentrations of Ti(IV) (0.10-1.03 mM [TiO(C2O4)2]2-) gave rise to a specific broadening of the citrate proton resonances, indicating that this bioavailable oxygen-donor ligand plays an important role in complexing implant-derived Ti(IV). 1H NMR analysis of Ti(IV)-loaded SF samples subsequently treated with a large excess of ascorbate (0.05 M) showed that this added Ti(IV) chelator was only poorly effective in removing this metal ion from Ti(IV)-citrate/Ti(IV)-oxycitrate complexes. The results obtained here provide evidence for complexation of the low-molecular-mass (non-protein-bound) fraction of implant-derived Ti(IV) by citrate in vivo.     

Synthesis and spectroscopic studies of mono(cyclopentadienyl)titanium(IV) derivatives with heterocyclic thioketones

The reactions of mono(cyclopentadienyl)titanium(IV) trichloride with two different classes of heterocyclic thioketones viz. 1-substituted-2-thiohydantoins and 1-substituted tetrazoline-5-thione were studied in anhydrous dichloromethane. The reaction products were characterized on the basis of elemental analyses electrical conductance, magnetic susceptibility and spectral (electronic, infrared and ¹H NMR) data.     

Irritancy and anti-inflammatory activity of bis(eta 5-cyclopentadienyl)titanium(IV) complexes in rats

Dichloro-bis(eta 5-cyclopentadienyl)titanium(IV) and some related complexes were compared with cis-dichlorodiammineplatinum(II) in rats for acute anti-inflammatory activity against carrageenan paw oedema, anti-arthritic activity against developing and established adjuvant-induced polyarthritis, immunosuppressant activity in a local graft-vs. host assay, irritant effects at sites of administration (paw, skin, peritoneum) and nephro- and gastro-toxicities. These titanium complexes, like cisplatin and its hydrolysis products, in vivo exhibited both anti-inflammatory and anti-arthritic activity as well as immunosuppressant effects. Nephro- and gastro-toxicity were much less severe than in rats given platinum complexes. In vitro they selectively inhibited [3H]thymidine incorporation by isolated thymocytes and prevented the germination of radish seeds. When given intraperitoneally, the anti-inflammatory activity may partly be due to a counter-irritant phenomenon since the titanium derivatives elicited an acute peritoneal effusion if they were injected towards the omentum. However, when injected subcutaneously or applied in dimethylformamide or dimethylsulfoxide to the skin, they manifested both anti-inflammatory and anti-arthritic activity without irritancy or much local skin damage. They might therefore have the potential of being useful drugs, especially if released slowly.     

Redox potentials of Ti(IV) and Fe(III) complexes provide insights into titanium biodistribution mechanisms

Transferrin, the human iron transport protein, binds Ti(IV) even more tightly than it binds Fe(III). However, the fate of titanium bound to transferrin is not well understood. Here we present results which address the fate of titanium once bound to transferrin. We have determined the redox potentials for a series of Ti(IV) complexes and have used these data to develop a linear free energy relationship (LFER) correlating Ti(IV) ? Ti(III) redox processes with Fe(III) ? Fe(II) redox processes. This LFER enables us to compare the redox potentials of Fe(III) complexes and Ti(IV) complexes that mimic the active site of transferrin and allows us to predict the redox potential of titanium-transferrin. Using cyclic voltammetry and discontinuous metalloprotein spectroelectrochemistry (dSEC) in conjunction with the LFER, we report that the redox potential of titanium-transferrin is lower than − 600 mV (lower than that of iron-transferrin) and is predicted to be ca. − 900 mV vs. NHE (normal hydrogen electrode). We conclude that Ti(IV)/Ti(III) reduction in titanium-transferrin is not accessible by biological reducing agents. This observation is discussed in the context of current hypotheses concerning the role of reduction in transferrin mediated iron transport.     

Nano-sized titanium(IV) ternary and quaternary complexes with electron-rich oxygen-based bidentate ligands

Some novel ternary and quaternary complexes of titanium(IV) of general formula [Ti(acac)Cl_3−n(OOCR)_n] (R = C₁₅H₃₁ or C₁₇H₃₅ and n = 1-3) have been synthesized by stepwise substitution of chloride ions of [Ti(acac)Cl₃] by straight chain carboxylic acid anions. The complexes are characterized by their elemental analyses, spectral (infrared, FAB mass, ¹H NMR and powder XRD) studies, molecular weight determination and molar conductance measurements. Infrared spectra suggested bidentate chelating nature of both acetylacetonate and carboxylate anions in the complexes. Monomeric nature of the complexes was confirmed by their molecular weight determination and FAB mass spectra. Molar conductance values indicated the complexes to be non-electrolytes in DMF. The complexes exhibited high resistance to hydrolysis. Their powder XRD data indicated the nano-size for the complexes. The coordination number of titanium(IV) in these complexes were found to be six, seven and eight which has been discussed in detail.     

The use of titanium (IV) oxide for the immobilisation of carbohydrate-directed enzymes.

The use of particles of porous titanium (IV) oxide as a suitable matrix for enzyme immobilisation has been investigated with dextranase. Treatment of the particles with enzyme in the presence and absence of ammonium ions showed that the presence of ammonia induced a greater coupling of protein, whereas a greater retention of enzyme specific activity was achieved in the absence of ammonia. Properties of the immobilised enzyme include a pH-dependence and reversibility of the coupling between enzyme and matrix. The immobilised dextranase was most stable at pH 5.0. Automated analytical techniques for measuring the activity of dextranase and other polysaccharidases in soluble and insoluble forms are also reported.     

Immobilization of yeasts on titanium activated inorganic supports

Summary A study of the immobilization of yeast cells with invertase activity by the metal link method was performed. Baker's yeast cells were immobilized on titanium activated porous silica support and on its alkylamine and aldehyde derivatives, their initial activities being 19.6, 39.9 and 10.6 U/ml of reactor respectively. When crosslinking of the immobilized cells was performed, an initial activity of 48.2 U/ml was achieved on the titanium activated support. Batch long-term stability tests were car ried out for 400 hours and the crosslinked preparations showed an unsta ble behaviour compared with the very stable preparations obtained with the simple metal-link method.A higher activity (56.2 U/ml) was obtained when a titanium activated macroporous support, pumice stone, was used as cell carrier, which compared favourably with calcium alginate entrapped cells (17.7 – 31.3 U/ml)     

Acid activation of titanium alkoxide systems - Structural characterisation of Ti(IV) sulfonyl-imide complexes

Treatment of [Ti(OⁱPr)₄] with the sulfonyl-imine systems Tos₂NH ([(p-Me-C₆H₄SO₂)₂NH]) and Tf₂NH ([(CF₃SO₂)₂NH]) results in the formation of the new Lewis acidic titanium sulfonyl-imide complexes [Ti(OⁱPr)₂(O,O′-Tos₂N)₂] (1) and [Ti(OⁱPr)₂(HOⁱPr)₂(O-Tf₂N)₂] (2), respectively. The molecular structures of the complexes have been determined by single crystal X-ray diffraction. The reaction of [Ti(OⁱPr)₃(OAr)] (Ar = 2,6-di-tert-butyl-4-methyl phenyl) with Tf₂NH results in the formation of the dimeric complex [Ti(OⁱPr)₃(O,O′-Tf₂N)]₂ (3), which has also been structurally characterised. The ability of the complexes to catalyst the Friedel-Crafts acylation of anisole has also been assessed.     

Active water-insoluble derivatives of papain and other enzymes based on preformed diazonium-type supports.

Papain (EC 3.4.22.2) has been coupled to supports of titanium (IV) oxide and cellulose, which are particulate and pre-coated with diazotised 1,3-diaminobenzene, giving water-insoluble and stable derivatives which possess low proteolytic activity but high esterolytic activity. In addition the reversible binding of zinc (II) at the active site of papain has been exploited to inhibit protectively the enzyme during its linkage to the aforementioned supports, thereby yielding water-insoluble derivatives of papain having superior activity upon reactivation with EDTA. Application of the improved procedure of enzyme coupling to macroporous cellulose particles gave a water-insoluble derivative of papain having further enhanced proteolytic activity. Other properties of the water-insoluble derivatives of papain and of similarly prepared water-insoluble conjugates of urease (EC 3.5.1.5) and cholinesterase (EC 3.1.1.8) with cellulose are also reported.