Immobilized Microbial Cells - American Chemical Society

12 Evaluation of a Novel Microporous PVC-Silica Support for Immobilized Enzymes

Downloaded by UNIV OF ARIZONA on December 19, 2012 | http://pubs.acs.org Publication Date: August 16, 1979 | doi: 10.1021/bk-1979-0106.ch012

Its Use in a Flow-Through Reactor System for Production of Fructose BRUCE S. GOLDBERG, ALEXANDER G. HAUSSER, KEVIN R. GILMAN, and RICHARD Y. C H E N

Amerace Corporation, Microporous Products Division, Ace Road, Butler, NJ 07405 During the p a s t f i f t e e n years there has been a g r e a t d e a l of research i n the area o f immobilized enzymes. T h i s research e f f o r t has been focused i n three major areas. F i r s t i s the form of the enzyme t o be immobilized, whether the enzyme i s p u r i f i e d or i s contained w i t h i n whole c e l l s . The second has been i n the area o f i m m o b i l i z a t i o n techniques, whether by p h y s i c a l absorpt i o n , entrapment, o r chemical bonding. The t h i r d area o f research has been i n the development o f v a r i o u s types o f c a r r i e r s that can support the enzyme and be used i n v a r i o u s types o f reactors. The purpose o f t h i s paper i s t o d i s c u s s the t h i r d area, v i z . the enzyme support. Various c a r r i e r s t h a t have been used over the years f o r immobilizing enzymes can be c l a s s i f i e d i n t o three c a t e g o r i e s . The f i r s t i s hard p a r t i c u l a t e substances such as porous glass/ceramics and polymers. The second category i s polymers i n membranous form, such as r e c o n s t i t u t e d c o l l a g e n o r u l t r a f i l t r a t i o n membranes, where the enzyme i s trapped behind o r w i t h i n the membrane b a r r i e r . The t h i r d category i s c e l l u l o s e d e r i v e d m a t e r i a l s i n the form o f f i b e r s or beads. Almost a l l these m a t e r i a l s are used e i t h e r i n the form o f packed beds o r as membranes. In any case, the d i f f u s i o n a l r e s i s t a n c e s are major r e s t r i c t i o n s t o t h e i r use as e f f i c i e n t enzyme supports. We w i l l d i s c u s s and demonstrate a new type o f microporous c a r r i e r t h a t can be used very e f f i c i e n t l y as an immobilized enzyme support. Support

Characteristics

Amerace C o r p o r a t i o n has developed a novel microporous supp o r t which overcomes the disadvantages o f supports used p r e v i o u s l y . T h i s m a t e r i a l can be used i n a flow-through r e a c t o r i n which the s u b s t r a t e flow i s p e r p e n d i c u l a r t o the s u r f a c e o f the support; i e , the substrate passes through the support m a t e r i a l , r a t h e r than around i t . Reaction takes p l a c e as a r e s u l t o f the 0-8412-0508-6/79/47-106-173$05.00/0 © 1979 American Chemical Society

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


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substrate's d i r e c t l y c o n t a c t i n g the enzyme through bulk flow, r a t h e r than i t s having t o d i f f u s e i n t o the support before i t can contact the enzyme. B r i e f l y , the flow-through r e a c t o r has the advantages of e s s e n t i a l l y no e x t e r n a l or i n t e r n a l mass t r a n s f e r l i m i t a t i o n s , no substrate holdup, and no channeling; a l l of which c o n t r i b u t e t o a more e f f i c i e n t u t i l i z a t i o n o f immobilized enzyme. The s u b s t r a t e must c o n t a c t a l l r e a c t i v e s i t e s while p a s s i n g through the pores; t h e r e f o r e e l i m i n a t i n g d i s p e r s i o n or d i f f u s i o n r e l a t e d problems. One can perform s e q u e n t i a l r e a c t i o n s , s i n c e there i s no holdup of r e a c t a n t s and products from one r e a c t i o n step to the next. The support i s a microporous P V C - s i l i c a sheet having a poro s i t y i n the 70-80% range. The pore s i z e as determined by mercury i n t r u s i o n porosimetry i s i n the 0.2 μπι t o 2.0 ym range. The support i s extremely h y d r o p h i l i c , has a negative charge, and a surface area of 80 m /g. The m a t e r i a l i s non-compressible under normal c o n d i t i o n s , i s steam s t e r i l i z a b l e , and has a low dry d e n s i t y of 0.45 g/cm . The microporous support has r e c e i v e d FDA approval f o r d i r e c t food c o n t a c t . The t o r t u o s i t y of the pore s t r u c t u r e r e q u i r e s t h a t the s u b s t r a t e make i n t i m a t e c o n t a c t w i t h the a c t i v e enzyme as i t passes through the support m a t e r i a l . The a c t i v e s i t e s are a t t r i b u t e d t o the s i l i c a contained w i t h i n the porous matrix which allows the a d d i t i o n of o r g a n i c f u n c t i o n a l i t y . While t h i s paper deals with the chemistry and r e a c t i o n c h a r a c t e r i s t i c s of glucose isomerase, i t should be noted t h a t glucose oxidase, glucoamylase, aldose-1-epimerase, a-glucosidase, a l c o h o l dehydrogenase, p u l l u l a n a s e , and f u n g a l α-amylase have been s u c c e s s f u l l y immobilized onto t h i s support. 2

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Enzyme Bonding

Procedure

The support p r e p a r a t i o n and i m m o b i l i z a t i o n procedures are as f o l l o w s . I n i t i a l l y , the sheet i s a c t i v a t e d by soaking f o r one hour i n a 1 M NaCl s o l u t i o n c o n t a i n i n g 1.5% polyethyleneimine (PEI) having a molecular weight of approximately 40,000. The support m a t e r i a l i s r i n s e d f r e e o f excess PEI and may be d r i e d f o r l a t e r use. The support m a t e r i a l may be cut to the proper s i z e and c o n f i g u r a t i o n before or a f t e r treatment with p o l y e t h y l eneimine. A l l work reported i n t h i s paper, unless otherwise s t a t e d , was done with 47 mm diameter d i s c s having a t h i c k n e s s of approximately 0.5 mm. The d i s c s were mounted i n r e a c t o r s com p r i s e d of 47 mm M i l l i p o r e Swinnex f i l t e r housings or were molded i n t o a stacked d i s c c o n f i g u r a t i o n , the concept of which i s p r e s e n t l y the s u b j e c t of a patent a p p l i c a t i o n . The r e a c t o r system i s assembled as i l l u s t r a t e d i n F i g u r e 1. The system i s f i r s t f i l l e d with d i s t i l l e d water and a 2.5% s o l u t i o n o f glutaraldehyde, at pH 9.5, i s pumped through the r e a c t o r a t 6 ml/min. f o r 1 h r . Excess glutaraldehyde i s removed from the r e a c t o r by f l u s h i n g with pH 7.5 b u f f e r s o l u t i o n c o n t a i n i n g 2 g/1 MgS04»7H 0 and 2 g/1 NaHC0 . B u f f e r i s pumped through the r e a c t o r u n t i l the e f f l u e n t 2

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has maintained a pH o f 7.5 f o r 15 minutes. When the e f f l u e n t pH has been s t a b i l i z e d a t 7.5 the enzyme s o l u t i o n i s r e c y c l e d through the r e a c t o r f o r 1 h r . Excess enzyme i s recovered by f l u s h i n g the r e a c t o r with the above b u f f e r and c o l l e c t i n g the e f f l u e n t . T h i s e n t i r e procedure i s c a r r i e d out a t room temperature. In the above sequence, the PEI i s chemi-adsorbed onto the surface o f the s i l i c a p a r t i c l e s i n the PVC matrix and confers organic f u n c t i o n a l i t y t o i t . The glutaraldehyde c r o s s l i n k s the PEI rendering i t t o t a l l y i n s o l u b l e and a l s o a c t s as a l e a s h f o r the enzyme pendent group with which i t i s subsequently r e a c t e d . The enzyme, which i n t h i s case i s glucose isomerase, was obtained from Novo L a b o r a t o r i e s and i s d e r i v e d from B a c i l l u s coagulans NRL-5666. The s o l u b l e enzyme was i s o l a t e d from a d r i e d c e l l p r e p a r a t i o n , as s u p p l i e d by Novo L a b o r a t o r i e s , by suspension i n b u f f e r and c e n t r i f u g a t i o n t o remove c e l l u l a r m a t e r i a l and other i n s o l u b l e i m p u r i t i e s . The r e s u l t i n g s o l u t i o n was f r a c t i o n ated by ammonium s u l f a t e p r e c i p i t a t i o n r e t a i n i n g the 55-70% f r a c t i o n s followed by d i a l y s i s t o o b t a i n a p u r i f i e d enzyme s u i t able f o r i m m o b i l i z a t i o n onto the microporous support. The p u r i t y o f the above prepared enzyme i s approximately 7,000 IGIU/g. One IGIU i s d e f i n e d as the amount o f enzyme r e q u i r e d t o convert one micromole o f glucose t o f r u c t o s e i n one minute a t 60°C i n a s o l u t i o n c o n t a i n i n g 40% w/w glucose, 2 g/1 M g S 0 » 7 H 0 , 1 g/1 NaHC0 having a pH o f 7.5 (measured a t room temperature). The a c t i v i t y o f s o l u b l e enzyme s o l u t i o n s were measured on a Yellow Springs b i o l o g i c a l oxygen analyzer (Model 53) which measures the enzyme c a t a l y z e d o x i d a t i o n o f glucose t o g l u c o n i c a c i d and by l i q u i d chromatography of g l u c o s e - s o l u b l e enzyme r e a c t i o n products. Depending on the p u r i t y and the h i s t o r y o f the dry enzyme p r e p a r a t i o n , recovery y i e l d s o f 90-100% expressed a c t i v i t y o f the immobilized enzyme have been achieved. The a c t i v i t y o f the s o l uble enzyme p r e p a r a t i o n a f t e r d i a l y s i s i s approximately 400 IGIU/ ml and a 2X q u a n t i t y o f enzyme i s u t i l i z e d f o r immobilization with roughly 50% o f the o f f e r e d s o l u b l e enzyme being recovered a f t e r i m m o b i l i z a t i o n and 45-50% of the o f f e r e d s o l u b l e enzyme r e s u l t i n g i n expressed a c t i v i t y on the r e a c t o r when operated under the above c o n d i t i o n s . Depending upon the a c t i v i t y o f the p u r i f i e d enzyme, the a c t i v i t y o f the c a r r i e r a f t e r i m m o b i l i z a t i o n i s t y p i c a l l y 600 IGIU/g. 4

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Analysis of D i f f u s i o n a l Limitations As mentioned e a r l i e r , one o f the important features o f t h i s immobilized enzyme support i s the f a c t t h a t i t permits design o f a flow-through r e a c t o r where the s u b s t r a t e must pass through a l l the pores. As a r e s u l t , the d i f f u s i o n l i m i t a t i o n s o f the support are reduced t o a minimal l e v e l . An experiment was performed t o determine the minimum v e l o c i t y below which d i f f u s i o n a l e f f e c t s


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become e v i d e n t . A l l subsequent experiments were run a t v e l o c i t i e s above the minimum v a l u e . F i g u r e 1 i l l u s t r a t e s the experimental setup used to d e t e r mine t h i s c r i t i c a l v e l o c i t y . A 47 mm diameter r e a c t o r , having an expressed a c t i v i t y of 145 IGIU/g was p l a c e d i n a 60°C temperature c o n t r o l l e d bath. Substrate was r e c i r c u l a t e d through t h i s r e a c t o r at v a r i o u s v e l o c i t i e s and the time r e q u i r e d f o r the 108 ml of r e a c t o r and r e s e r v o i r volume t o reach 5% conversion a t each s u p e r f i c i a l v e l o c i t y was recorded and the expressed a c t i v i t y c a l c u l a t e d . F i g u r e 2 i l l u s t r a t e s a p l o t of the r e s u l t i n g data. T y p i c a l l y , above a s u p e r f i c i a l v e l o c i t y o f 0.1 cm/min the expressed a c t i v i t y remained constant a t 145 IGIU/g. Kinetic Evaluation To i l l u s t r a t e the e f f i c i e n c y of enzyme u t i l i z a t i o n with t h i s microporous enzyme support, 30 g of support m a t e r i a l was immob i l i z e d with 22,000 IGIU of p u r i f i e d enzyme. The r e s u l t i n g r e a c t o r had an expressed a c t i v i t y of 726 IGIU/g a t 60°C. A 40% w/w s o l u t i o n of 99% pure dextrose c o n t a i n i n g 2 g/1 MgS04-7H 0 and 1 g/1 of NaHC03 a t pH 7.5 was converted to a 45% f r u c t o s e product i n 6.5 min and an e q u i l i b r i u m product of approximately 50% f r u c tose i n l e s s than 12 min residence time. The flow r a t e r e q u i r e d to o b t a i n a 45% converted product was 6 ml/min. A second experiment was performed t o demonstrate the s u p e r i o r u t i l i z a t i o n of enzyme by a microporous sheet flowthrough r e a c t o r versus a packed column c o n t a i n i n g c o n t r o l l e d pore g l a s s . C o n t r o l l e d pore g l a s s p a r t i c l e s 40-80 mesh were obtained from E l e c t r o Nucleonics C o r p o r a t i o n and c h e m i c a l l y m o d i f i e d by standard methods 0^ t o i n t r o d u c e c o v a l e n t l y bound, a l i p h a t i c amino f u n c t i o n a l i t y on the e x t e r n a l and i n t e r n a l s u r f a c e s . Two grams of the amino-CPG were degassed and suspended i n 100 ml of 10% pH 8 glutaraldehyde s o l u t i o n f o r 1 h r . The CPG p a r t i c l e s were e x t e n s i v e l y washed t o remove excess glutaraldehyde using pH 7.5 Hepes b u f f e r c o n t a i n i n g 2 g/1 MgS04-7H 0. Ten ml of enzyme s o l u t i o n c o n t a i n i n g 0.43 u n i t s / m l a t pH 7.5 was added to the p a r t i c l e s and allowed t o r e a c t f o r 1 hr with g e n t l e a g i t a t i o n . Excess enzyme was r i n s e d from the CPG p a r t i c l e s and based upon l o s s of a c t i v i t y from the enzyme s o l u t i o n , the l o a d i n g was 0.66 u n i t s / m l (CPG has a bulk d e n s i t y of 0.36 g/ml). The 1.2 ml volume of the p a r t i c l e s was loaded i n t o a s m a l l 0.6 cm diameter column and used i n the subsequent experiment. 2

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Four 26 mm diameter MPS d i s c s were t r e a t e d with PEI, mounted i n a standard 26 mm M i l l i p o r e Swinnex f i l t e r housing and s e t up as p r e v i o u s l y d e s c r i b e d . A 10% pH 8 s o l u t i o n of glutaraldehyde s o l u t i o n was pumped through the r e a c t o r f o r 1 h r . The r e a c t o r was r i n s e d with 200 ml of pH 7.5 Hepes b u f f e r c o n t a i n i n g 2 g/1 MgS04»7H20 f o r 1 h r . Enzyme s o l u t i o n , 30 ml having an a c t i v i t y of 0.43 u n i t s / m l , was r e c i r c u l a t e d through the r e a c t o r f o r 1 hr a f t e r which time excess enzyme was f l u s h e d from the r e a c t o r u s i n g


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\ REACTOR \

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CONSTANT TEMPERATURE

Figure 1.

Flow diagram of reactor system for diffusion studies

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Superficial Velocity (cm/mirO

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Figure 2. Effect of superficial velocity on enzyme utilization efficiency. Reactor size: diameter—47 mm, thickness—0.45 mm, weight—0.43 g. Conditions: pH— 7.5, temperature—60°C. Substrate: dextrose—45% w/v, MgSO^ · 7H 0—2 g/L, NaHCOs—1 g/L. 2



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the above b u f f e r s o l u t i o n . The l o a d i n g on the r e a c t o r was c a l c u l a t e d to be 0.7 u n i t s / m l based upon l o s s of a c t i v i t y from the immobilizing s o l u t i o n . The volume o f t h i s r e a c t o r was 1.0 ml. The two r e a c t o r s were evaluated by measuring the degree of conversion of a 7.2% w/v f r u c t o s e s u b s t r a t e (ph 7.0) a t s e v e r a l flow r a t e s when operated i n a s i n g l e pass mode. The r e s u l t i n g data i s shown i n F i g u r e 3 which shows the per cent conversion obtained versus residence time. I t can be noted t h a t i t takes 5.5 min to o b t a i n a 19.1% conversion product i n the porous g l a s s packed column, whereas the r e a c t o r c o n t a i n i n g the MPS immobilized enzyme achieves a conversion of 34% i n the same residence time. The MPS i s 78% more e f f i c i e n t i n u t i l i z i n g an e q u i v a l e n t a c t i v i t y of enzyme. pH

Study

The experimental setup f o r the e v a l u a t i o n of the e f f e c t o f pH and temperature on the expressed a c t i v i t y o f the MPS immobi l i z e d enzyme i s shown i n F i g u r e 4. The r e a c t o r was 47 mm i n diameter and contained 2 g of support m a t e r i a l having an ex pressed a c t i v i t y o f approximately 650 IGIU/g. An approximate 10% converted product was obtained a t 60°C and pH 7.5 on a s u b s t r a t e c o n t a i n i n g 40% w/w dextrose, 2 g/1 MgS04«7H 0, 1 g/1 NaHC03, and 1 g/1 NaHS03. The flow r a t e through the r e a c t o r was maintained at 5 ml/min throughout the experiment. F i g u r e 5 shows the e f f e c t of pH a t v a r y i n g temperatures on the expressed a c t i v i t y of glucose isomerase i s o l a t e d from B a c i l l u s coagulans and immobilized on MPS. I t may be noted t h a t the optimum pH (measured a t room temperature) i s not a f f e c t e d by v a r y i n g the temperature and t h a t the per cent change of a c t i v i t y as a f u n c t i o n of pH remains r e l a t i v e l y constant w i t h changing temperatures. F i g u r e 6 i s an Arrhenius p l o t o f the pH 7.5 data i n F i g u r e 5. Using the formula In Κ = In A - Ε /RT an a c t i v a t i o n energy o f 19.6 Κ cal/mole i s obtained. 2

Half-Life

Study

Using the i n f o r m a t i o n from the pH-temperature experiment we then b u i l t a l a r g e s c a l e flow-through r e a c t o r and operated i t a t 60°C and pH 7.5 t o o b t a i n a h a l f - l i f e on the immobilized enzyme. T h i s r e a c t o r was 47 mm i n diameter and c o n s i s t e d of 80 stacked sheets of support m a t e r i a l having a combined weight of 30 g. F i g u r e 4 i l l u s t r a t e s the experimental setup which c o n s i s t e d of a r e s e r v o i r maintained a t room temperature c o n t a i n i n g the s u b s t r a t e which contained 40% w/w dextrose, 2 g/1 MgS0 -7H 0, 1 g/1 NaHC03, and 1 g/1 NaHS03 having a pH o f 7.5 (measured a t room tempera ture) . T h i s s u b s t r a t e was pumped by a v a r i a b l e speed pump through a p r e f i l t e r c o n t a i n i n g an 8 ym paper f i l t e r and an Amerace 0.45 ym f i l t e r and then through the r e a c t o r which was p l a c e d i n a 60°C constant temperature water bath. The s u b s t r a t e 4

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3 4 5 Residence Time (min.)

S

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Figure 3. Percent conversion vs. residence time. Substrate: fructose—7.2% to/ν (4M), pH—7.5, temperature—25°C. ( Ο ) MPS flow-through reactor, ( X ) controlled pore-glass reactor.

SPEED PUMP Figure 4.

GAUGES

Flow diagram of reactor system for constant conversion of dextrose



Figure 5. Rehtive activity vs. pH as a function of temperature. Substrate: dextrose—40% w/w, MgSO · 7H 0—2 g/L, NaHCO —l g/L, NaHSO —l g/L. h

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Figure 6. Arrhenius plot for Bacillus coagulans in flow-through reactor. Substrate: dextrose—40% w/w, MgSO^ · 7H 0—2 g/L, NaHCO —l g/h, NaHSO —1 g/L, pH—7.5. 2

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passing through the r e a c t o r was discharged i n t o a r e s e r v o i r and analyzed d a i l y . Pressure gauges were l o c a t e d b e f o r e and a f t e r the p r e f i l t e r to determine the pressure or contaminant b u i l d - u p on the p r e f i l t e r or r e a c t o r . As i s normal commercial p r a c t i c e , the conversion was maintained a t a constant 45% f r u c t o s e l e v e l by s u i t a b l y a d j u s t i n g the flow r a t e through the r e a c t o r . F i g u r e 7 shows the experimental a c t i v i t y decay curve f o r t h i s immobilized enzyme r e a c t o r system. I n i t i a l residence time to o b t a i n a 45% converted product from the s t a r t i n g dextrose substrate was i n the order of 10.6 min. The f i r s t and second h a l f - l i v e s ( t . and t . ) were d e f i n e d as the times a t which the flow r a t e s were 1/2 and 1/4 o f the i n i t i a l flow r a t e , respectively. In t h i s p a r t i c u l a r case, the i n i t i a l flow r a t e was 5.1 ml/min i n c r e a s i n g to about 5.6 ml/min over a 200 hr p e r i o d and then decreasing i n a f i r s t order manner. For t h i s p a r t i c u l a r enzyme a t 60°C on t h i s support t was 920 hrs w h i l e t - ^ 1600 h r s . During the h a l f - l i f e experiment there was no problem with pressure b u i l d u p e i t h e r on the p r e f i l t e r or on the r e a c t o r and the e f f l u e n t coming from the r e a c t o r was water white. T h i s b u f f e r e d system d i d not show any pH drop across the r e a c t o r . Other s t u d i e s performed on non-buffered s u b s t r a t e s with a l l other c o n d i t i o n s being the same showed pH drops i n the range of 0.5 to 0.8 pH u n i t s . An experiment was run comparing the h a l f - l i f e of the flowthrough r e a c t o r w i t h t h a t o f commercially a v a i l a b l e polymeric granules immobilized with the same enzyme s t r a i n u t i l i z i n g a packed bed r e a c t o r . The r e a c t o r s were operated the same as p r e v i o u s l y d e s c r i b e d . Table I summarizes the two systems. We found t h a t the p r o d u c t i v i t y of the flow-through r e a c t o r c a l c u l a t e d a t t . was i n the order of 4100 g of dry product/g of support compared to 1500 g of dry product/g of support f o r the bed r e a c t o r system. On an enzyme u t i l i z a t i o n b a s i s , the p r o d u c t i v i t y f o r the flow-through r e a c t o r was 8.56 g dry product per u n i t of expressed a c t i v i t y as compared t o 6.54 g dry product per u n i t of expressed a c t i v i t y on the bed r e a c t o r . The product coming through the flow-through r e a c t o r was water white. T y p i c a l l y , the product of the packed bed r e a c t o r m a t e r i a l i s h i g h l y c o l o r e d and has a strong odor f o r the f i r s t 100 h r s .


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Discussion The work r e p o r t e d here demonstrates t h a t the microporous p l a s t i c sheet (MPS ) i s a v i a b l e support f o r glucose isomerase. B a c i l l u s coagulans glucose isomerase immobilized on MPS has a g r e a t e r e f f i c i e n c y than the same enzyme immobilized on cont r o l l e d pore g l a s s . The c o n t r o l l e d pore g l a s s e x h i b i t s an apparent e f f i c i e n c y o f 0.56 r e l a t i v e t o MPS. Pore d i f f u s i o n l i m i t a t i o n s of the g l a s s beads and bed channeling cause t h i s inefficiency. The r a t i o of the p r o d u c t i v i t y of B a c i l l u s coagulans glucose


PVC-Silica

GOLDBERG ET A L .

Support

183


12.

1

Ο

200

400

600

Time (hrs.)

800

1000

1200

Figure 7. Half life study of glucose isomerase on Amerace flow-through reactor. Conditions: pH—7.5, temperature—60°C. Substrate: dextrose—40% w/w, MgSO · 7H 0—2 g/L, NaHSOs—1 g/L, NaHCO —l g/L. u

2

s


184


Table I


COMPARISON OF FLOW-THROUGH VERSUS PACKED BED REACTOR

Amerace MPS Reactor

Commercially Available Polymeric Beads

I n i t i a l Expressed A c t i v i t y , IGIU*/g

572

193

Half-Life

920

710

1600

938

t . , hours ±/ ζ

Quarter-Life

t

1

^ r 4

hours

2

1

R e l a t i v e Reactor Volume Productivity 1.

g HFCS (dry)/g Support a t t

2.

g HFCS (dry)/IGIU* a t t , Initial Activity

^

4064

1450

8.56

6.54

Product Q u a l i t y 1.

I n i t i a l Color

water white

very dark

2.

I n i t i a l Odor

none

strong odor of p r o t e i n

* I n i t i a l expressed a c t i v i t y o f the r e a c t o r 24 hours s t a r t up

after


12.

GOLDBERG ET A L .

PVC-SîlîCa

Support

185


isomerase immobilized i n commercially a v a i l a b l e polymeric beads to t h a t enzyme immobilized on MPS i s 0.76. The lower p r o d u c t i v i t y o f the polymeric beads i s apparently due t o a combination o f pore d i f f u s i o n l i m i t a t i o n , other f a c t o r s i n h e r e n t i n the nature of the polymeric beads, and the method by which they are utilized. Since pore d i f f u s i o n i s l i m i t e d and channeling i s a t a minimum, MPS allows f o r more e f f i c i e n t u t i l i z a t i o n o f enzyme. Shorter r e a c t i o n times are p o s s i b l e with MPS due t o the use o f the flow-through r e a c t o r concept and the higher a c t i v i t i e s attainable. Summary From a r e a c t o r engineering-design standpoint the flowthrough r e a c t o r would r e q u i r e about one-half the s i z e o f a packed bed r e a c t o r . T h i s r a t i o can be even g r e a t e r depending on the s p e c i f i c a c t i v i t y o f the enzyme u t i l i z e d i n the i m m o b i l i z a t i o n . The higher the enzyme p u r i t y , the smaller the s i z e o f the flowthrough r e a c t o r . Other advantages o f the flow-through r e a c t o r are r a p i d and e f f i c i e n t change-over o f r e a c t o r s and the e l i m i n a t i o n o f c o m p r e s s i b i l i t y and other h y d r a u l i c problems u s u a l l y a s s o c i a t e d with packed bed r e a c t o r s . T h i s development i s the s u b j e c t o f a r e c e n t l y i s s u e d U. S. patent(2) and other pending patent a p p l i c a t i o n s .

ABSTRACT EVALUATION OF A NOVEL MICROPOROUS PVC-SILICA SUPPORT FOR IMMOBILIZED ENZYMES AND ITS USE IN A FLOW-THROUGH REACTOR SYSTEM FOR PRODUCTION OF FRUCTOSE A microporous polyvinyl chloride-silica filled plastic sheet was evaluated as an immobilized enzyme support using a flow-throughreactor concept. The substrate was pumped through the microporous plastic sheet (MPS ) containing immobilized glucose isomerase chemically bound to the internal pore structure. The physical properties of the plastic sheet and the advantages of its use as a flow-through reactor as compared to a packed bed reactor are presented. The immobilization chemistry is described along with a listing of various enzymes which have been successfully immobilized onto the support. A detailed study, including half-life, is reported on the production of fructose utilizing glucose isomerase isolated from Bacillus coagulans. Reaction kinetics including the effects of temperature and pH on the relative activity of the immobilized enzyme was studied and the activation energy calculated. A minimum velocity was determined above which the diffusional affects in the microporous sheet are


186


eliminated. Typical operating conditions for the immobilized flow-through reactor were 60°C, a pH of 7.5 (at 25°C), 40% w/w dextrose substrate containing 1 g/1 of NaHCO, 1 g/1 NaHSO, 2 g/1 MgSO •7H O. 3

4

3

2

Literature Cited


(1)

(2)

Weibel, M. K. et al, "Immobilized Enzymes: A Prototype Device for the Analysis of Glucose in Biological Fluids Employing Immobilized Glucose Oxidase," Anal. Biochem., 52 502 (1973) Goldberg, Bruce S. (assigned to Amerace Corporation) "Immobilized Proteins," U. S. Patent #4,102,746, July 25, 1978

RECEIVED

March 29, 1979.


Immobilized Microbial Cells - American Chemical Society

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