ELSEVIER

Application of tris(hydroxymethyl)phosphine as a coupling agent for alcohol dehydrogenase immobilization

Fiona C. Cochrane, Helen H. Petach, and William Henderson
School o f Science and Technology, University o f Waikato, Hamilton, N e w Zealand

The use of tris(hydroxymethyl)phosphine as a coupling reagent for the immobilization of alcohol dehydrogenase onto chitosan films and for the attachment of the chitosan film to a glass support resulted in enzyme activities far above those obtained by adsorption of enzyme, and greater than those observed when using the more conventional glutaraldehyde-coupling protocol. The stability of the chitosan films was dramatically increased by covalent attachment to the glass using tris(hydroxymethyl)phosphine. Similar enzyme activities were found for coupling with tris(hydroxymethyl)phosphine and glutaraldehyde on aminopropyl silica and aminopropyl glass. The tris(hydroxymethyl)phosphine coupling extended the longevity of the alcohol dehydrogenase activity but did not alter the pH optima or Km of the enzyme.

Keywords: Tris(hydroxymethyl)phosphine; enzyme immobilization;alcohol dehydrogenase;aminopropylsilica; chitosan

Introduction
Numerous coupling agents are available for the covalent immobilization of enzymes onto supports. Coupling agents such as benzoquinone,1 carbodiimides, maleimide, and glutaraldehyde crosslink functional groups on the support (e.g., - N H 2, - C O 0 - , -SH) to similar functional groups on the surface of the enzyme? The most commonly used coupling reagent is glutaraldehyde, although its chemistry creates some inherent difficulties because of the continuing polymerization of the glutaraldehyde upon storage 3 and the reversibility of the Schiff base linkage. To overcome these difficulties of coupling with glutaraldehyde, we have recently reported a new coupling agent, P(CH2OH)3. 4 Hydroxymethylphosphines containing > P CH2-OH groups are well known to undergo Mannich-type condensation reactions 5,6 at room temperature with N - H group-containing compounds, giving aminomethyl phosphines, >P-CH2-N<; we recently showed that hydroxymeAddress reprint requests to Dr. Henderson at the School of Science and Technology, University of Waikato, Private Bag 3105, Hamilton, New Zealand Received 15 November1994; revised 5 June 1995; accepted30 June 1995

thylphosphines can be immobilized on chitosan and chitin supports. 7 The significant stability of the P-CH2-N linkage suggested that the inexpensive, water-soluble, and reasonably air-stable phosphine P(CH2OH)35,8 might be a useful coupling agent for the covalent immobilization of enzymes, with improved binding properties over existing coupling methods. The potential advantages of using P(CH2OH) 3 as a coupling agent include an increase in the number of immobilizing groups, together with an improved hydrolytic stability of the resulting P-CHz-N-enzyme linkages over, for example, the hydrolyzable C - - N - e n z y m e a n a l o g s formed from glutaraldehyde. Both coupling reagents ideally use nonessential NH 2 groups on the enzyme to minimize enzyme inactivation. Spacer arms between the enzyme and the support have been shown to maintain greater enzyme activity and stability than in the absence of spacer arms (e.g., glutamate dehydrogenase 9 and proteasesl bound to chitosan and glucose oxidase bound to nylon1). Furthermore, more flexible chain linkages such as polyether links have been suggested for the maintenance of higher levels of catalytic activity. 1~,12 The enzyme alcohol dehydrogenase (ADH) was chosen for an in-depth comparison between the new P(CH2OH) 3 coupling agent and glutaraldehyde, because of its impor-

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Papers
tance for optical biosensor applications and its sensitivity toward immobilization. Studies were carried out on the use o f the P ( C H 2 O H ) 3 c o u p l i n g agent and its effects on the properties of the e n z y m e , A D H . A derivative o f P(CH2OH)3 was also synthesized that incorporates a f l e x i b l e polyether chain linkage b e t w e e n two h y d r o x y m e t h y l p h o s p h i n e functional groups, to investigate the effect o f spacer arms on e n z y m e activity.

Synthesis of the bifunctional polyether coupling agent (HOCH2)2P(CH2CH20) n CH2CH2P(CH2OH)2 (1)

Synthesis of CI(CH2CH20)nCH2CIt2Ci. Poly(ethylene glycol) (MW 600) was converted to the polyether dichloride by reaction at ca. 50C with an excess of thionyl chloride, to which a few drops of pyridine catalyst had been added. The excess of thionyl chloride was removed under reduced pressure to yield the crude product. Polyethers have a tendency to retain acid, which was viewed to be highly undesirable, as it would react with the P(CH2OH) 3 in the subsequent step. Accordingly, the crude dichloride (ca. 15 g) was dissolved in isopropanol (ca. 60 ml) and treated with an excess of solid sodium hydrogen carbonate for several days. Filtration to remove salts, followed by the removal of solvent under reduced pressure, gave the crude polyether dichloride, which was used without further purification for the next step. The compound was analyzed by electrospray mass spectrometry. The compound when dissolved in water was neutral to litmus.

Materials and methods

Reagents
Tetrakis(hydroxymethyl)phosphonium chloride was obtained from Albright and Wilson Ltd. (U.K), as an 80% w/w aqueous solution. ADH (E.C. 1.1.1.1) from Saccharomyces cerevisieae, obtained from Sigma (St. Louis, MO), had a reported activity of 380 U mg -1 where 1 U is defined as the conversion of 1.0 ixmol of ethanol min -1 at pH 8.8, 25C. Chitosan was obtained as crude flakes from Sigma. Aminopropyltriethoxysilane and poly(ethylene glycol) average M W 600 were obtained from Aldrich. Silica gel (60-120 mesh) was obtained from BDH. Other chemicals were of analytical reagent grade, with the exception of thionyl chloride, which was of laboratory grade. Water was double-distilled and, for immobilization work, passed through a Milli-Q purification system prior to use. Buffered aqueous solutions were 30 mM tetrasodium pyrophosphate buffer at pH 8.8.

Synthesis of the crude coupling agent (HOCI-I2)2P

(CH2CH20)nCH2CH2P(CH2OH)2 (1). A solution of P(CHaOH) ~ was prepared under a nitrogen atmosphere by the addition of KOH (1.885 g) in water (5 ml) to P(CH2OH)4+C1 - (8.00 g of an 80% w/w aqueous solution) in water (30 ml). To this solution was added, with stirring under nitrogen, a solution of the polyether dichloride from above (10.7 g) in water (40 ml), followed by a catalytic amount (ca. 0.3 g) of sodium iodide. 31p-{1H} NMR spectroscopy showed the presence of only P(CH2OH) 3 with trace amounts of O=P(CH2OH) 3 and P(CH2OH)4 +, confirming that all acid had been removed from the polyether dichloride by the hydrogen carbonate treatment. The reaction mixture was refluxed under a nitrogen atmosphere for 5.5 h, at which point 31P-{1H} NMR showed the reaction to be two-thirds complete. An additional 12 h reflux effected completion of the reaction as evidenced by 31P-{1H} NMR, which showed a mixture of RP(CH2OH)3 + (28.9 ppm, ca. 63%), R2P(CH2OH)2 + (31.0 ppm, ca. 11%), and P(CH2OH)4 + (26.4 ppm, ca. 26%), where R is a polyether chain. [The presence of P(CH2OH)4 + was confirmed by a spiking experiment in which a small quantity of authentic P(CH2OH)4+C1 - was added to the NMR sample]. The reaction mixture was made up to 250 ml with water in a volumetric flask, to give a stock solution 0.134 N in phosphorus. The coupling agent was stored in this phosphonium salt form, which is air-stable. Immediately prior to use, the phosphoninm salt was converted to the free hydroxymethylphosphine by addition of an excess of triethylamine to the aqueous solution, analogous to the conversion of P(CH2OH)4 + to P(CH2OH)3

Instrumentation

31p-{1H} nuclear

magnetic resonance (NMR) spectra were recorded on a Jeol JNM-FX90 spectrometer at 36.23 MHz, with chemical shifts relative to external 85% H3PO 4 (0.0 ppm). Electrospray mass spectra were obtained using a VG Platform II mass spectrometer using a 1:1 v/v acetonitrile-water mobile phase. The compounds were dissolved in the mobile phase to give a solution typically of the approximate concentration 0.1 IxM. The diluted solution was injected into the spectrometer via a Rheodyne injector fitted with a 10 I*1 sample loop. A Thermo Separation Products SpectraSysetm P1000 LC pump delivered the solution to the mass spectrometer source at a flow rate of 0.01 ml min -~, and nitrogen was employed both as a drying and nebulizing gas. A cone voltage of 20 V was found to be satisfactory for all samples.

Production of P(CH2OH) 3
The P(CH2OH) 3 solutions were prepared by reaction of the phosphonium salt by treatment with 1 tool equivalent of base. The appropriate amount of P(CH2OHL+C1 - was dissolved in 5 ml of 100% ethanol and deoxygenated. One molar equivalent of KOH was then dissolved in 5 ml of buffer and added dropwise under nitrogen to the stirred phosphonium salt solution; the resulting solution was diluted with 40 ml of deoxygenated buffer. The resuiting P(CH2OH) 3 solution, which also contained the by-product formaldehyde, was then filtered under vacuum to separate the KC1 salt. Formaldehyde-free P(CH2OH)3 was prepared by reacting P(CH2OH)4C1 with excess triethylamine as described by Henderson et al.7 and Ellis et al. s Excess triethylamine was removed under vacuum. Hemiformal adducts were converted to P(CH2OH) 3 by stirring the mixture at 90C for 16 h under vacuum, with a nitrogen bleed to facilitate formaldehyde removal. The final product was crystallized by cooling to -20C for 12 h, yielding a transparent crystalline product. Purity was confirmed by 31p_{ 1H } NMR spectroscopy?

Production of ADH-chitosan films

Chitosan films were cast onto quartz plates (8 x 20 mm) either directly or following silanization. Silanization of the plates was carried out by refluxing quartz plates in 9% (v/v) aminopropyltriethoxysilane in dry toluene for 4 h followed by thorough rinsing with toluene, methanol, and water. The silanized plates were reacted with P(CHaOH) 3 (2.5 mg ml -~ in water) for 40 min at 20C, and unreacted P(CH2OH) 3 was removed by rinsing successively with deionized water. Chitosan (75 mg) dissolved in 5 ml 5% acetic acid was spread (130 txl) across a quartz plate and dried at 60C for 24 h. The films were neutralized with 30 mM tetrasodium pyrophosphate buffer and washed several times with distilled water at room temperature prior to enzyme coupling.

374

Enzyme Microb. Technol., 1996, vol. 18, April

Immobilization of alcohol dehydrogenase: F. C. Cochrane et al.

Alcohol dehydrogenase was covalently bound to chitosan films by reacting chitosan with either (a) 0.50 mg ml -~ glutaraldehyde in 30 mM tetrasodium pyrophosphate buffer, (b) 2.50 mg ml -~ P(CH2OH)3 (formaldehyde-free preparation) in buffer, or (c) 0.54 mg m1-1 of the polyether coupling reagent (1) in buffer for 90 min at 25C, followed by thorough washing with buffer. Activated chitosan films were contacted with ADH solutions (1.0 mg ml -l) in 30 mM tetrasodium pyrophosphate buffer for 2 h at 25C followed by 24 h at 4C. The films were rinsed with buffer, 1 M NaC1 and, finally, buffer until no ADH activity could be detected in the washings. The washed films were stored in buffer for 24 h prior to their use to be certain that nt~ further enzyme was dialyzed from the film. a deoxygenated environment to maintain the activity toward amine coupling. Chitosan is a copolymer of N-acetyl-D-glucosamine and D-glucosamine, and thus contains amine functional groups that are available for grafting to aminopropyl glass using P(CH2OH) 3 (Figure 1). The integrity of the chitosan films was found to increase dramatically from hours to months when the chitosan films were covalently attached to aminopropyl glass using P(CH2OH)3 as a coupling agent instead of forming films by merely depositing chitosan onto nonfunctionalized glass. All chitosan films used in this study were formed by covalent attachment using P ( C H ; O H ) 3. Figure 1 gives a schematic representation of the chemical linkages involved in the grafting of a chitosan film onto aminopropyl glass or silica, and the immobilization o f the e n z y m e A D H onto this c h i t o s a n support, both using P(CH2OH)3. The ability of P(CHzOH) ~ to couple A D H to a support is illustrated in Figure 2. Enzyme activities were measured after binding A D H to supports using P(CHzOH) 3, glutaraldehyde, or adsorption in the absence o f a coupling agent. The concentrations of the coupling reagents provided for the same number of functional groups to be available for the coupling reaction. For the three supports studied here, the P(CHzOH)3-coupled enzyme retained activity in excess of

Production of ADH-aminopropyl silica and glass

Aminopropyl silica was prepared via a minor modification of the method described by Khatib and Parish. 13 Silica gel (60-120 mesh) was activated by refluxing in concentrated hydrochloric acid for 4 h, washed thoroughly with water to remove all traces of acid, and then dried in an oven at 320C. Aminopropyl silylation was achieved by refluxing activated silica gel (10 g) with aminopropyltriethoxysilane (4 ml) in dry toluene (dried by distillation from sodium metal under a nitrogen atmosphere) for 4 h. The silica was filtered off; washed extensively with dry toluene, methanol, water, and methanol; and then dried in an oven at 100C. Aminopropyl glass was prepared by reaction with aminopropyltriethoxysilane as described for the activation of quartz for the binding of chitosan films. ADH was covalently bound to the silica and glass by the same procedure as that used earlier for the chitosan films.

Quantitative determination of ADH activity

Alcohol dehydrogenase activity was assayed by a modification of the method of Bergmeyer et al.14 Assays were initiated by the addition of the immobilized enzyme or an aliquot of the free enzyme to a solution of 0.074 M ethanol, 0.13 mM NAD +, and buffer at pH 8.8, at 20C. Mixing was carried out for the chitosan films and glass supports by purging the reaction cuvette with a constant stream of nitrogen, while an orbital shaker was used for mixing for the silica support. The absorbance at 340 nm was measured at l-rain intervals after briefly stopping the flow of nitrogen. K,, values were determined from activities measured while only one substrate was present in saturating concentration. The activities of the free and immobilized ADH were determined in the pH range of 6.0-9.2 as described above, except that 30 mM NazHPO4/NaH2PO4 was used as the buffer from pH 6.08.0. Activity assays are reported as the average of five replicates.
N--

p--

H2c /
.OH Q grafted chitosan film ~" / ~ \OH
,CH2

gO

\ / N Jn

./
;Ha)a

Results and discussion

The production of the coupling agent P(CH2OH) 3, from P(CH2OH)4 +, is carried out in a deoxygenated environment to prevent the oxidation o f the phosphine that would render the hydroxyl groups inactive toward amine coupling. The degree o f oxidation o f the P(CH2OH) 3 can be readily monitored by 31p_ {1H } N M R spectroscopy, which distinguishes the p h o s p h i n e ( - 2 4 . 0 p p m ) and the p h o s p h i n e o x i d e (HOCH2)3P=O (49.0 ppm). Freshly prepared P(CHzOH) 3 solution stored under nitrogen was stable for several months, but was only useable for 1 day once exposed to the atmosphere. Thus, P(CH2OH)3 should be carefully stored in

o/,;\o
///////

3i

silica or glass

Figure 1 Schematic representation of the P(CH2OH)3-mediated immobilization of alcohol dehydrogenase onto an SiO2-grafted chitosan film Enzyme Microb. Technol., 1996, vol. 18, April 375

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500
ACTIVITY

(a)
ACTIVITY
( m U / c m 2)

1.5

(b)

(mU/g)

400 300 200 1000Aminopropyl Silica

1-

,00J t
. \

Free

........ ....... + P(CH2OH)3 ......... ~ ........ - - e- + Glutaraldehyde

750.5"-1

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Chitosan
Film

nChit0san Aminopropyl
Film

I1)

Glass

25[] [] [] + P(OH2OH) 3 Adsorbed

0
+ Glutaraldehyde 0 20 40 60 80

Time (days)
2 Activity of the alcohol dehydrogenase after immobilization onto aminopropyl silica, chitosan film, or a m i n o p r o p y l glass using three different coupling methods: adsorption, coupling with P(CH2OH)3, or glutaraldehyde. Comparisons between the immobilized materials are based on both (a) w e i g h t and (b) an area basis. Activity was measured in 30 mM tetrasodium pyrophosphate buffer at pH 8.8 and 20C. Standard deviations for the activity measurements were 10% of the value in all cases e x c e p t for the chitosan support, for which standard deviations were <20% of the total activity
Figure F i g u r e 3 Longevity of the covalently-bound alcohol dehydrogenase on chitosan films compared with free enzyme in solution. All samples were stored at 4C between assays. For the bound enzyme, the same chitosan film was used for each measurement

that observed for adsorbed enzyme, and thus appeared to facilitate enzyme binding. Furthermore, on the chitosan support, ADH activity, following coupling with P(CH2OH) 3, was significantly greater than that observed for coupling with glutaraldehyde. The longevity of the enzyme activity is retained for longer periods of time when the P(CH2OH) 3 was used as the coupling agent rather than glutaraldehyde (Figure 3). This finding is in agreement with the notion that the P(CH2OH)3 coupling reaction should yield an irreversible and robust linkage between the enzyme and the amino-derivatized support based on the chemistry of the Mannich condensation. Other properties of the ADH were compared for the enzyme immobilized by coupling either with glutaraldehyde or P(CH2OH)3 to determine whether the P(CH2OH)3 affected other characteristics (e.g., pH and K m values) of the immobilized enzyme. The pH optimum of the chitosan-immobilized ADH shifted to more acidic pH relative to the free enzyme (Figu r e 4 ) i r r e s p e c t i v e of w h e t h e r g l u t a r a l d e h y d e or P(CHzOH) 3 was used as the coupling agent. In contrast, the pH optimum remained unchanged when ADH was immobilized onto aminopropylsilica. Studies of urease bound to chitosan found that innnobiiization had no effect on ti~c pH optimum. 16 The shift of the pH optimum of the large ADH enzyme upon immobilizing onto chitosan may be a result of the strongly hydrophilic support 15 being able to interact with the enzyme and shield its contact with the surrounding solution. Furthermore, positively charged supports arc known to displace pH-activity curves to more acidic values. ~6 Immobilization affects the pH characteristics of the ADH, but the identity of the coupling agent seems to have little effect.
376 E n z y m e M i c r o b . T e c h n o l . , 1996, v o l . 18, A p r i l

Michealis constants, K m, were determined for both NAD + and ethanol, the substrates for the ADH reaction, for free enzyme and that of the immobilized ADH on chitosan films. Results are shown with the 95% confidence limits in Table 1. No significant difference was observed between K m values obtained for the two methods of coupling ADH onto chitosan films. This result thus provides further evidence that P(CH~OH)3 is coupling enzyme to the support without dramatically changing the enzyme structure. The 95% con-

100-

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i

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40

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- =
e-

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i

8:5

9,5

pH
Figure 4 pH optima for atcohot dehydrogertas~ cuvsier]tiy bound to chitosan films or free in solution, in the pH range 8.0-9.2. All assays were carried out in 30 mM tetrasodium pyrephosphate buffer adjusted to the appropriate pH with aqueous NaOH or HCI, For the bound enzyme, the assay f o l l o w e d a 15min presaturation with the buffer solution

Immobilization of alcohol dehydrogenase: F. C. Cochrane et al.

Table I Kr, values for ADH (yeast) in pH 8.8 tetrasodium pyrophosphate buffer at 20C Km (NAD ) (IJM) 95% Confidence interval (IJM)a 197.0-279.8 81.6-641.2 59.3-166.2 Km (ethanol) (raM) 9.16 16.39 18.03 95% Confidence interval (mM) a 9.12-9.21 13.72-24.93 14.67-29.84

ADH (free) ADH on chitosan + P(CH2OH)ab ADH on chitosan + glutaraldehyde

230.3 127.4 80.0

"The confidence intervals for the K,, values were calculated from the linear regression of the Lineweaver-Burk plot (n = 28) bP(CH2OH)a was used as the coupling agent for the immobilization CGlutaraldehyde was used as the coupling agent for the immobilization

fidence intervals illustrate the greater uncertainty in the estimation of Km for the immobilized ADH. This greater uncertainty arises partly because of the differences between chitosan plates. Similar ADH activities were obtained for P(CH2OH)3 prepared in both the presence and absence of the sideproduct formaldehyde. The small amount of formaldehyde produced during synthesis does not seem to preferentially bind to the available NH 2- groups and render the surface less active for protein immobilization. Thus, the simpler preparation of P(CH2OH) 3 from P(CH2OH)4 + and OH- (in which formaldehyde is produced as a side product, but not removed) should be sufficient for most enzyme immobilizations. Enzymes are generally found to be more active in solution than when bound to surfaces, and thus, a flexible linkage between the enzyme and the support may more closely approximate the solution environment and yield higher enzyme activities. Accordingly, we wished to investigate the effectiveness of a hydroxymethylphosphine coupling agent incorporating such a long linkage. P(CH2OH) 3 was derivatized with a polyethylene glycol chain, Scheme 1, to form the coupling agent (1), enabling a direct comparison with the short underivatized coupling agent P(CH2OH) 3. The polyethylene glycol, polyether dichloride, and crude polyether bis(phosphonium) salt were investigated by electrospray mass spectroscopy, a technique which is very well suited for the study of polyetbers and phosphonium salts. The spectrum for the polyethylene glycol showed the expected oligomer distribution, centered on the m/z 600 region, with a separation of 44 mass U between peaks due to variable numbers o f - C H z C H 2 0 - monomer units. Individual peaks were assigned to M + H20 + H +, where M is the polyethylene glycol; for example, the peak at rrdz 608.3 corresponded to the species HO(CH2CHzO)lzCH2CHaOH + H20 + H +. The spectrum for the polyether dichloride was similar, excepting that the fine structure of each peak was more complex because of the presence of 35C1 and 37C1 i~otopcs, ihe spectrum tor the crude bis(phosphonium salt) was more complex, but appeared to consist of two superimposed oligomer distribution patterns. The first, centered on m/z 400 can be ascribed to the series of desired oligom e r i c b i s ( p h o s p h o n i u m ) salts. For example~ for
[ ( H O C H 2 ) 3 P ( C I t 2 C H 2 0 ) 1 2 C H 2 C I - t z P t C H 2 0 H ) 3 ] z* (.M =

starting material. P(CH2OH)4 + was confirmed as the peak at m/z 155. Complications arise from the oligomeric distributions characteristic of polyether materials of this type, together with the complicating phosphorus chemistry, which in addition to the monoalkylated products afford some dialkylated product plus P(CH2OH)4 . Nevertheless, we wished to ascertain whether enzyme immobilized via a long-chain polyether coupling agent of this type effects any dramatic increase in enzyme activity. When the coupling agent (1) was used to activate chitosan films prior to enzyme binding, ADH activities were l.2 mU cm -2, similar to results from using the P(CH2OH) 3 coupling agent of 1.3 mU cm -2, based on using the same molar concentration of functional groups in the coupling reagents. Therefore, results on chitosan films do not indicate an advantage for the use of polyether spacer arms on chitosan.
H O ~ O H 1. SOCI2

2. NaHCO3
CI,~,,,ww,,,,~CI

P(CH2OH)3. Nal catalyst

(HOCH2)ap+~,,c,,w,,~,,,v~P(C H20 H)3+

CI" CI"

excess NEt3

(HOC H2)2P,,ww,,,,,-o~P(CH2OH)2

(1)

804), this would give a peak in the electrospray mass spectrum at m/z 402. The second oligomer distribution, centered around m/z ca. 630 could be ascribed to monophosphonium salts, plus any chloropo!yetber byproducts and unreactcd

= polyether chain Scheme 1 Synthesis of the difunctional polyether coupling ag~;lt (I), Side ploduct~ are not shown

E n z y m e M i c r o b . T e c h n o l . , 1996, v o l . 18, A p r i l

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This lack of effect may result because chitosan provides a flexible hydrophilic environment which is not dramatically altered by coupling via a polyethylene glycol spacer. In conclusion, tris(hydroxymethyl)phosphine is a versatile new reagent in immobilization methodology. In this work we have demonstrated that ADH can be effectively immobilized onto transparent chitosan films, by using P(CH2OH) 3 as the coupling agent for binding both the chitosan film onto the glass and enzyme onto the film. This offers potential for the development of an active and stable preparation for use in optical biosensors.
5. 6. coupling reagent for the covalent immobilisation of enzymes. J. Chem. Soc. Chem. Commun. 1994, 2181-2182 Weil, E.D. Phosphorus-based flame retardants. In: Handbook of Organophosphorus Chemistry (Engel, R., ed.). Marcel Dekker, New York, 1992, chapter 14 Frank, A. W., Dalgle, D. J. and Vail, S. L. Chemistry of hydroxymethyl phosphorus compounds. Part II: Phosphonium salts. Text. Res. J. 1982, 678-693 Henderson, W., Olsen, G.M. and Bonnington, L. S. Immobilised phosphines incorporating the chiral biopolymers chitosan and chitin. J. Chem. Soc. Chem. Commun. 1994, 1863-1864 Ellis, J. W., Harrison, K. N., Hoye, P. A. T., Orpen, A. G., Pringle, P.G. and Smith, M.B. Water-soluble tris(hydroxymethyl)phosphine complexes with nickel, palladium, and platinum: Crystal structure of [Pd{P(CH2OH)3 }4] " CHaOH. Inorgan. Chem. 1992, 31, 3026-3033 Petach, H. H. and Driscoll, J. Transparent chitosan derivatives for the immobilization of glutamate dehydrogenase. Biotechnol. Bioeng. 1994, 44, 1018-1022 Hayashi, T. and lkada, Y. Protease immobilization onto porous chitosan beads. Z Appl. Polym. Sci, 1991, 42, 85-92 Bonnington, L. S., Petach, H. H. and Henderson, W. Polyphosphine oxides as supports for enzyme immobilisation. Eur. Polym. J. 1995, 31, 981-985 Molinari, H. and Montanari, F. Heterogeneous phase-transfer catalysts: High efficacy of catalysts bonded by a long chain to a polymer matrix. J. Chem. Soc. Chem. Commun. 639-641. Khatib, I. S. and Parish, R. V. Insoluble ligands and their applications: I. A comparison of silica immobilized ligands and functionalised polysiloxanes. J. Organomet. Chem. 1989, 369, 9-16 Bergmeyer, H.H., Gawehn, K. and Grassl, M. Enzymes as biochemical reagents. In: Methods of Enzymatic Analysis, 2nd ed. (Bergmeyer, H.U., ed.). Academic Press, New York, 1974, 428429 Kurita, K., Kamiya, M. and Nishimura, S.-I. Solubilization of a rigid polysaccharide: Controlled partial N-acetylation of chitosan to develop solubility. Carbohyd. Polym. 1991, 16, 83-92 Krajewska, B., Leszko, M. and Zaborska, W. Urease immobilized on chitosan membrane: Preparation and properties. J. Chem. Tech. Biotechnol. 1990, 48, 337-350

7.

8.

Acknowledgments
The authors gratefully acknowledge financial support from the Waikato Medical Research Foundation, and thank Albright and W i l s o n Ltd. for a generous supply of [P(CH2OH)4] C1. The New Zealand Lottery Grants Board is acknowledged for a grant-in-aid toward the purchase of a mass spectrometer.

9.

10. 11.

12.

References
13. 1. Beh, S. K., Moody, G.J. and Thomas, J. D. R. Studies on spacer molecules and coupling agents for immobilising glucose oxidase on nylon mesh for glucose oxidase electrodes. Analyst 1989, 114, 1421-1425 Everse, J. and Ginsburgh, C. L. Immobilized enzymes in biochemical analysis. Methods Biochem. Anal. 1981, 25, 135-198 Kirkegy, S., Jakobsen, P., and Moe, D. Glutaraldehyde--' 'pure and impure": A spectroscopic investigation of two commercial glutaraldehyde solutions and their reaction products with amino acids. Anal. Lett. 1987, 20, 303-315 Petach, H. H., Henderson, W. and Olsen, G. M. P(CH2OH)3: A new 14.

2. 3.

15.

16.

4.

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Enzyme Microb. Technol., 1996, vol. 18, April