Immobilization of Rhodococcus AJ270 and Use of Entrapped Biocatalyst for the Production of Acrylic Acid
Rhodococcus AJ270 is adsorbed by Dowex 1 at 15.4 mg dry weight per g resin with maximum amidase specific activity observed at lower loadings. Bacteria form a monolayer on the resin surface, and adsorption is complete within 2 min AJ270 can be entrapped in
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Summary. Rhodococcus AJ270 is adsorbed by Dowex I at 15.4 mg dry weight per g resin with maximum amidase specific activity observed at lower loadings. Bacteria form a monolayer on the resin surface, and adsorption is complete within 2 min. AJ270 can be entrapped in agar and agarose gels (optimum loading: 20mg dry weight bacteria per cnr' gel). Adsorption and entrapment improve amidase thermal stability 3-4 fold, and entrapment shifts the pH optimum from 8 to 7. Adsorbed and free bacteria show similar values for K m and Vrnax, but entrapped bacteria have higher K m values. Compared with bacteria adsorbed to Dowex, the activity per crrr' of matrix of agar-entrapped AJ270 is eight-fold higher. In stirred-tank reactors, exposure to acrylic acid reduces the amidase activity of the biocatalyst in the hydrolysis of acrylamide. In column reactors, entrapped AJ270 suffers little reduction in amidase activity against 0.25 M acrylamide over 22 h continuous operation. Keywords. Acrylamide ; Amidase; Acrylate; Biocatalysis; Rhodococcus.
Introduction
When grown on acetamide, Rhodococcus AJ270 expresses high specific activities of nitrile hydratase and amidase [1], enzymes with broad specificity against a wide range of nitriles and amides [2,3]. Washed suspensions of AJ270 have been used for the enantioselective hydrolysis of (R,S)-2-phenylbutyronitrile [2] and of racemic ibuprofen amide, in the latter case yielding the pharmacologically active non-steroidal anti-inflammatory agent S-( +)-ibuprofen in 94% e.e. [1]. This versatile biocatalyst also catalyses the regioselective hydrolysis of certain dinitriles [2,4]. These applications used the rhodococcal biocatalyst in a 'one-shot' batch mode, whereas immobilization of the bacteria would allow multiple re-use of the catalyst, possibly in continuous reactors, and would certainly facilitate product recovery. This paper describes methods for the adsorption and entrapment of AJ270, reports changes in the properties of the amidase activity resulting from immobilization, and investigates the use of entrapped bacteria for the hydrolysis of acrylamide. This latter biotransformation is of commercial significance for the
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Corresponding author Present address: Helena Biosciences, Sunderland Business Park, Sunderland SR5 3XB, UK
H. Griengl (ed.), Biocatalysis © Springer-Verlag Wien 2000
130
J. Colby et al.
Table 1. Adsorption of AJ270 to anionic exchange resins . Bacteria (4.2 mg dry weight) were immobilized to the anionic exchangers (3 g wet weight), and the % of bacteria adsorbed determined as described in the Experimental section. For the data in the second column, the bacteria and resins were washed with buffer containing 0.01 M EDTA prior to immobilization % of bacteria adsorbed
Resin Amberlite IRA 904 Amberlite IRA 400 Amberlite IRA 402 Amberlite IRA 440c Amberlite IRA 900 Dowex 1X8-400 Dowex 2X8-400
-EDTA
+EDTA
6 21 2 6 4
22 24 7 7 14 94 50
96 60
production of acrylates, commodity chemicals produced globally on a large scale for the manufacture of a wide variety of p
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