S. Alan Barker

Application of living immobilized cells to the acceleration of the continuous conversions of ethanol (wort) to acetic acid (vinegar)—Hydrous titanium(IV) oxide-immobilized Acetobacter species

Abstract Various strains of Acetobacter species have been immobilized on hydrous titanium(IV) oxide or hydrous titanium(IV) chelated cellulose and used in the continuous conversion of a dilute aqueous alcoholic solution (in the form of‘charging wort’) into acetic acid (in the form of vinegar) in tower fermenter-type reactors. A strain of Acetobacter species producing extracellular polysaccharide aggregated in the presence of hydrous titanium(IV) oxide thereby enabling higher medium flow rates and an increased acetic acid output to be achieved. A strain of Acetobacter species not producing polysaccharide showed no effect with hydrous titanium(IV) oxide but did produce more acetic acid when a titanium(IV)-cellulose chelate was added to the fermentation, although aggregation was not observed. Mechanisms, which appear to conform to established results, are proposed for the aggregation of both strains of bacteria. Apparently, these water-insoluble titanium compounds can interact with the bacterial cells, increasing their density and thus making them more resistant to ‘wash out’ by increasing the rate at which they sediment in the fermenter. This enables a greater cell mass per unit volume to be achieved which in turn leads to an increase in conversion rate in the reactor .

Factors affecting microbial growth and polysaccharide production during the fermentation of Xanthomonas campestris cultures

Fermentations of Xanthomonas campestris have been carried out on laboratory and pilot plant scales using various organic nitrogen sources in order to test their effectiveness in polysaccharide (xanthan) production. It was discovered that high nitrogen concentrations give highest yields of crude product and result in a need for only short fermentation times to achieve maximum product formation. These products, however, have inferior solution rheology to those produced from low-nitrogen media due partly to their high concentrations of co-precipitated microbial cells and partly to differences in tertiary molecular structure.

The adsorption ofd-glucose and glucans by magnetic cellulosic, and other magnetic forms of hydrous titanium(IV) oxide

Abstract A more-effective means of activating cellulose than hitherto reported has been devised for the abstraction of carbohydrates from solution. Cellulose coated with hydrous titanium(IV) oxide readily adsorbs branched polysaccharides such as glycogen ( e.g. a coated cellulose containing 4.5% of titanium will adsorb 0.52 mg of glycogen/mg of titanium) and has a low affinity for monosaccharides. The hydrous oxide, as a dried powder, had a lower maximum adsorption (0.08 mg of glycogen and 0.02 mg of d -glucose/mg of titanium) showing the beneficial effect of its coating on cellulose. Conversely, freshly-prepared hydrous titanium(IV) oxide, which had been neither washed nor dried had a greater maximum adsorption of glycogen (4.46 mg/mg of titanium whilst the generation of the hydrous oxide in solutions of glycogen further increased the maximum adsorption of glycogen (8.40 mg/mg of titanium). Magnetic iron oxide coated with hydrous titanium(IV) oxide retains all the adsorption properties of the freshly-prepared hydrous titanium(IV) oxide and improves the handling and sedimentation properties of the material, including its response to magnets.

The selective adsorption of polysaccharides on polyaromatic surfaces

Abstract A new matrix, polymeric 1,3-diaminobenzene-coated Celite, which selectively adsorbs monomeric and polymeric carbohydrates, has been prepared. This matrix adsorbs glycogen and other branched polysaccharides, as opposed to neutral or charged monosaccharides. Other solid supports were coated with polymeric 1,3-diaminobenzene; some of these (coated Sephadex G10 and coated Bioglas 1,000) had better physical form for column packing. Coated Celite was considered to be the best support in view of its greater stability. The effects of ionic concentration, pH, temperature, and the concentration of carbohydrate solution on the adsorption of glycogen on to coated Celite were studied, and methods to prevent adsorption and remove adsorbed carbohydrate were investigated. A comparison is made with the adsorption of heterocyclic compounds by cross-linked dextran gels.

Further facile immobilization of enzymes on hydrous metal oxides and use of their immobilization reversibility phenomena for the recovery of peptide antibiotics

Abstract Practical aspects of the facile immobilization of enzymes on hydrous metal oxides, a well-established means of enzyme-movement restriction, are described. Various enzymes (e.g. glucoamylase, peroxidase, dextranase) have been immobilized by chelation of several hydrous metal oxides, those of titanium(IV) and zirconium(IV) proving to be the most satisfactory for practical purposes. Localization of the gel into a granular form could be achieved successfully with good enzyme-immobilization characteristics by using ion-exchange resin as an internal matrix. The immobilization process was highly efficient for the relative proportions of hydrous oxide to enzyme used, with usually >90% of the available protein being insolubilized. Retention of enzyme activity was generally very good and was stable to reuse and to conventional buffer conditions. Activities of the immobilized enzymes were partially stable to lyophilization or drying of the hydrous oxide gels. Modification of the hydrous metal oxide surface by drying or treatment with phosphate or carbonate led to a decrease in complexing ability. The effect of carbonate can be circumvented by lowering the pH of the solution to around 5 and removing any carbon dioxide formed, by aeration. Such treatment allowed compounds to chelate to hydrous zirconium oxide(IV) in the presence of carbonate and therefore the hydrous oxide could be applied successfully to the concentration of peptide antibiotics from the fermentation medium in which they are being produced, including production at low concentrations.

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.

Effect of oxyanions on the d-glucose isomerase catalysed equilibrium: 2. Effect of germanate on the equilibrium of d-glucose and d-fructose with immobilized d-glucose isomerase

Abstract The addition of germanate anions to high d -glucose feed syrups, which are passed through an immobilized d -glucose isomerase [xylose isomerase, d -xylose ketol-isomerase, EC 5.3.1.5] column, displaces a ca. 50/50 d -glucose/ d -fructose mixture (produced in the absence of germanate) in favour of d -fructose. A maximum conversion of 94% from a d -glucose feed (40% w/v) is obtained with no detrimental effect on the enzyme. This is related to the germanate: sugar ratio. Optimization of the d -fructose yield from d -glucose germanate substrate has been carried out. The effects due to temperature, pH and concentration were taken into consideration. Confirmation of the quantitative identification of the d -fructose was obtained by isotope dilution analysis. The theory behind the displacement is also discussed, and shows close agreement with practical results.

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.

Insoluble complexes of amino-acids, peptides, and enzymes with metal hydroxides

Investigation of a number of gelatinous metal hydroxides has established that several (e.g. those formed from TiIV, ZrIV, FeIII, VIII, and SnII) are capable of forming with enzymes insoluble complexes which are enzymically active. From the practical viewpoint TiIV and ZrIV proved the most satisfactory. Comparatively high retentions of enzyme specific activity may be achieved. Complexes of these metal hydroxides may also be formed with peptides and amino-acids. All these complexes are considered to be formed by the re-occupation of ligand sites on the metal ions by the incoming molecules. The metal hydroxide–amino-acid complexes can also subsequently bind an enzyme molecule, to give a product the pH–activity profile of which may differ from that of the soluble form of the enzyme. Thus by this technique it may be possible to alter the pH optimum of an enzyme. The peptide ligand can be displaced from the complex by phosphate or fluoride ions, or by any other such entity capable of forming a more stable complex with the metal ion involved.

Preparation of a water-insoluble trans-2,3-cyclic carbonate derivative of macroporous cellulose and its use as a matrix for enzyme immobilisation

The degree of substitution of macroporous cellulose withtrans-2,3-cyclic carbonate groups has been controlled by moderating the reaction with water. Exercise of this control enabled the preparation of a matrix with physical and chemical properties which facilitated the covalent binding of chymotrypsin A in such a way that the activity of the insoluble enzyme was appreciable towards a high molecular weight substrate (casein) as well as towards a low molecular weight substrate (tyrosine ethyl ester). Under the optimum conditions of preparation the bound protein had a relative activity towards casein of 26% and towards the ester of 65% of the activity of the free enzyme.