!"#$%&'"$() +,-",&&.", /('&0 / ('&0 12 3&&
&!##4 5&.0"#, 6276
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Biochemical Engineering James M. Lee Washington State University
eBook Version 2.32
ii © 2009 by James M. Lee, Department of Chemical Engineering, Washington State University, Pullman, WA 99164-2710. This book was originally published by Prentice-Hall Inc. in 1992. All rights reserved. No part of this book may be reproduced, in any form or by any means, without permission in writing from the author.
iii
Contents Preface Chapter 1 Introduction Importance of bioprocessing and scale-up in new biotechnology. Chapter 2 Enzyme Kinetics Simple enzyme kinetics, enzyme reactor, inhibition, other influences, and experiments Chapter 3 Immobilized Enzyme Immobilization techniques and effect of mass transfer resistance Chapter 4 Industrial Applications of Enzymes Carbohydrates, starch conversion, cellulose conversion, and experiments Chapter 5 Cell Cultivations Microbial, animal, and plant cell cultivations, cell growth measurement, cell immobilization, and experiments Chapter 6 Cell Kinetics and Fermenter Design Growth cycle, cell kinetics, batch or plug-flow stirred-tank fermenter, continuous stirred-tank fermenter (CSTF), multiple fermenters in series, CSTF with cell recycling, alternative fermenters, and structured kinetic models Chapter 7 Genetic Engineering DNA and RNA, cloning of genes, stability of recombinant cells, and genetic engineering of plant cells Chapter 8 Sterilization Sterilization methods, thermal death kinetics, design criterion, batch and continuous sterilization, and air sterilization Chapter 9 Agitation and Aeration Basic mass-transfer concepts, correlations form mass-transfer coefficient, measurement of interfacial area, correlations for interfacial area, gas hold-up, power consumption, oxygen absorption rate, scale-up, and shear sensitive mixing Chapter 10 Downstream Processing Solid-liquid separation, cell rupture, recovery, and purification
iv
Preface to the eBook Edition
While I was revising the book for the second edition, I decided to test this concept of college textbook as ebook. If it works out, more professors will be encouraged to try this concept and people will be benefited with variety of textbooks available as ebook inexpensively compared to the published books. There are many other advantages for publishing textbooks as ebook including easy revisions and corrections, wide exposure of the text as a reference to general public, etc. James M. Lee
v
Preface to the First Edition
This book is written for an introductory course in biochemical engineering normally taught as a senior or graduate-level elective in chemical engineering. It is also intended to be used as a self-study book for practicing chemical engineers or for biological scientists who have a limited background in the bioprocessing aspects of new biotechnology. Several characteristics lacking in currently available books in the area, therefore, which I have intended to improve in this textbook are: (1) solved example problems, (2) use of the traditional chemical engineering approaches in nomenclature and mathematical analysis so that students who are taking other chemical engineering courses concurrently with this course will not be confused, (3) brief descriptions of the basics of microbiology and biochemistry as an introduction to the chapter where they are needed, and (4) inclusion of laboratory experiments to help engineers with basic microbiology or biochemistry experiments. Following a brief introduction of biochemical engineering in general, the book is divided into three main sections. The first is enzyme-mediated bioprocessing, which is covered in three chapters. Enzyme kinetics is explained along with batch and continuous bioreactor design in Chapter 2. This is one of two major chapters that need to be studied carefully. Enzyme immobilization techniques and the effect of mass-transfer resistance are introduced in Chapter 3 to illustrate how a typical mass-transfer analysis, familiar to chemical engineering students, can be applied to enzyme reactions. Basic biochemistry of carbohydrates is reviewed and two examples of industrial enzyme processes involving starch and cellulose are introduced in Chapter 4. Instructors can add more current examples of industrial enzyme processes or ask students to do a course project on the topic. The second section of the book deals with whole-cell mediated bioprocessing. Since most chemical engineering students do not have backgrounds in cell culture techniques, Chapter 5 introduces basic microbiology and cell culture techniques for both animal and plant cells. Animal and plant cells are included because of their growing importance for the production of pharmaceuticals. It is intended to cover only what is necessary to understand the terminology and procedures introduced in the following chapters. Readers are encouraged to study further on the topic by reading any college-level
vi microbiology textbook as needs arise. Chapter 6 deals with cell kinetics and fermenter design. This chapter is another one of the two major chapters that needs to be studied carefully. The kinetic analysis is primarily based on unstructured, distributed models. However, a more rigorous structured model is covered at the end of the chapter. In Chapter 7, genetic engineering is briefly explained by using the simplest terms possible and genetic stability problems are addressed as one of the most important engineering aspects of genetically modified cells. The final section deals with engineering aspects of bioprocessing. Sterilization techniques (one of the upstream processes) are presented in Chapter 8. It is treated as a separate chapter because of its importance in bioprocessing. The maintenance of complete sterility at the beginning and during the fermentation operation is vitally important for successful bioprocessing. Chapter 9 deals with agitation and aeration as one of the most important factors to consider in designing a fermenter. The last chapter is a brief review of downstream processing. I thank Inn-Soo, my wife, and Young Jean, my daughter, for their support and encouragement while I was writing this book. This book would not exist today without many hours of review and editing of the manuscript by Brian S. Hooker, my former graduate student who is now on the faculty of Tri-State University, Jon Wolf, my previous research associate who is now working for Boeing, and Patrick Bryant, my present graduate student. I also thank Rod Fisher at the University of Washington for using incomplete versions of this book as a text and for giving me many valuable suggestions. I extend my appreciation to Gary F. Bennett at the University of Toledo for providing example problems to be used in this book. I thank the students in the biochemical engineering class at Washington State University during the past several years for using a draft manuscript of this book as their textbook and also for correcting mistakes in the manuscript. I also thank my colleagues at Washington State University, William J. Thomson, James N. Petersen, and Bernard J. Van Wie, for their support and encouragement. James M. Lee
Chapter 2. Enzyme Kinetics !"#"
%&'()*+,'-)&
!.#
!"!"
/-0123 4&5603 7-&3'-,8
!.9
!":"
4;<2+<'-)& )= 7-&3'-, ><(<03'3(8
!.#?
!"9"
4&5603 @3<,')( A-'B /-0123 7-&3'-,8
!.!:
!"C"
%&B-D-'-)& )= 4&5603 @3<,'-)&8
!.!?
!"?"
E'B3( %&=2+3&,38 )& 4&5603 F,'-;-'6
!.!G
!"H"
4I13(-03&'J 4&5603 7-&3'-,8
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2.1. Introduction 4&56038 <(3 D-)2)N-,<2 ,<'<268'8 'B<' <(3 1()'3-& 0)23,+238 -& &<'+(3" OB36 <(3 1()*+,3* D6 2-;-&N ,3228 P<&-0<2Q 12<&'Q <&* 0-,())(N<&-80R <&* <(3
2-6
Enzyme Kinetics
rmax
r p
K M C S
Figure 2.2 OB3 3==3,' )= 8+D8'(<'3 ,)&,3&'(<'-)& )& 'B3 -&-'-<2 (3<,'-)& (<'3"
!" OB3 (3<,'-)& (<'3 *)38 &)' *313&* )& 'B3 8+D8'(<'3 ,)&,3&'(<'-)& AB3& 'B3 8+D8'(<'3 ,)&,3&'(<'-)& -8 B-NBQ 8-&,3 'B3 (3<,'-)& (<'3 ,B<&N38 N(<*+<226 =()0 =-(8' )(*3( ') 53() )(*3( <8 'B3 8+D8'(<'3 ,)&,3&'(<'-)& -8 -&,(3<83*" :" OB3 0
/ 4
4/
P!"CR
4/
: >4
P!"?R
k
Enzyme Kinetics
2-7
W()A&X8 U-&3'-, -&=3(3&,3 )= 'B3 3I-8'3&,3 )= 'B3 3&5603.8+D8'(<'3 ,)0123I A<8 0<*3 2)&N D3=)(3 'B3 ,B30-,<2 &<'+(3 )= 3&56038 A<8 U&)A&Q 9M 63<(8 D3=)(3 'B3 813,'()1B)')03'(-, *3'3,'-)& )= 8+,B ,)0123I38" E&3 )= 'B3 )(-N-&<2 'B3)(-38 ') <,,)+&' =)( 'B3 =)(0<'-)& )= 'B3 3&5603.8+D8'(<'3 ,)0123I -8 'B3 Y2),U <&* U36Z 'B3)(6" OB3 0<-& ,)&,31' )= 'B-8 B61)'B38-8 -8 'B<' 'B3(3 -8 < ')1)N(<1B-,<2Q 8'(+,'+(<2 ,)01<'-D-2-'6 D3'A33& <& 3&5603 <&* < 8+D8'(<'3 AB-,B )1'-0<226 =<;)(8 'B3 (3,)N&-'-)& )= 'B3 8+D8'(<'3 <8 8B)A& -& [-N+(3 !":"
+
Figure 2.3 \),U <&* U36 'B3)(6 =)( 'B3 3&5603.8+D8'(<'3 ,)0123I"
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ichaelis-Menten approach P_-,B<32-8 <&* _3&'3&Q #G#:RJ %' -8 <88+03* 'B<' 'B3 1()*+,'.(323<8-&N 8'31Q 4S" P!"?RQ -8 0+,B 82)A3( 'B<& 'B3 (3;3(8-D23 (3<,'-)&Q 4S" P!"CRQ <&* 'B3 82)A 8'31 *3'3(0-&38 'B3 (<'3Q AB-23 'B3 )'B3( -8 <' 3S+-2-D(-+0" OB-8 -8 <& <88+01'-)& AB-,B -8 )='3&
!
OB-8 -8 < (3<8)&
(<,'-,<226Q -' -8 <28) )+( D38' -&'3(38'8 ') +83 <8 2-''23 3&56038 <8 1)88-D23 D3,<+83 )= 'B3-( ,)8'8"
2-8
Enzyme Kinetics :
3012)63* -& B3'3()N3&3)+8 ,<'<26'-, (3<,'-)&8 -& ,B30-,<2 U-&3'-,8" 4;3& 'B)+NB 'B3 3&5603 -8 8)2+D23 -& A<'3(Q 'B3 3&5603 0)23,+238 B<;3 2<(N3 <&* ,)012-,<'3* 'B(33.*-03&8-)&<2 8'(+,'+(38" OB3(3=)(3Q 3&56038 ,<& D3 <&<2)N)+8 ') 8)2-* ,<'<268'8 -& ,B30-,<2 (3<,'-)&8" [+('B3(0)(3Q 'B3 =-(8' 8'31 =)( <& 3&5603 (3<,'-)& <28) -&;)2;38 'B3 =)(0<'-)& )= <& 3&5603.8+D8'(<'3 ,)0123IQ AB-,B -8 D<83* )& < ;3(6 A3
!" Briggs-Haldane approach PW(-NN8 <&* V<2*<&3Q #G!CRJ OB3 ,B<&N3 )= 'B3 -&'3(03*-<'3 ,)&,3&'(<'-)& A-'B (3813,' ') '-03 -8 <88+03* ') D3 &3N2-N-D23Q 'B<' -8Q d PC ES R /dt ] M" OB-8 -8 <28) U&)A& <8 'B3 pseudo steady-state P)( quasi-steady-stateR <88+01'-)& -& ,B30-,<2 U-&3'-,8 <&* -8 )='3& +83* -& *3;32)1-&N (<'3 3I1(388-)&8 -& B)0)N3&3)+8 ,<'<26'-, (3<,'-)&8" :" Numerical solution: /)2+'-)& )= 'B3 8-0+2'<&3)+8 *-==3(3&'-<2 3S+<'-)&8 *3;32)13* =()0 4S8" P!"CR <&* P!"?R A-'B)+' 8-012-=-,<'-)&"
2.2.1. Michaelis-Menten Approach %= 'B3 82)A3( (3<,'-)&Q 4S" P!"?RQ *3'3(0-&38 'B3 );3(<22 (<'3 )= (3<,'-)&Q 'B3 (<'3 )= 1()*+,' =)(0<'-)& <&* 8+D8'(<'3 ,)&8+01'-)& -8 1()1)('-)&<2 ') 'B3 9 ,)&,3&'(<'-)& )= 'B3 3&5603.8+D8'(<'3 ,)0123I <8J
:
[)( B3'3()N3&3)+8 ,<'<26'-, (3<,'-)&8Q 'B3 =-(8' 8'31 -8 'B3 <*8)(1'-)& )= (3<,'<&'8 )& 'B3 8+(=<,3 )= < ,<'<268' <&* 'B3 83,)&* 8'31 -8 'B3 ,B30-,<2 (3<,'-)& D3'A33& 'B3 (3<,'<&'8 ') 1()*+,3 1()*+,'8" /-&,3 'B3 =-(8' 8'31 -&;)2;38 )&26 A3
9
%' 83308 'B<' 'B3 (<'3 )= 8+D8'(<'3 ,)&8+01'-)& 8B)+2* D3 3I1(3883* <8
dC S dt
k#CS CE k! CES
OB3&Q -' N-;38 < ,)&'(<*-,')(6 (38+2' 'B<' 'B3 8+D8'(<'3 ,)&,3&'(<'-)& 8'<68 ,)&8'<&' P*C S/ *' ] MR D3,<+83 'B3 =-(8' (3;3(8-D23 (3<,'-)&Q 4S" P!"CRQ -8 <88+03* ') D3 -& 3S+-2-D(-+0 Pk # C S C E . k ! C E /` ] MR" /-&,3 'B3 (<'3 )= (3<,'-)& -8 *3'3(0-&3* D6 'B3 83,)&* 82)A3( (3<,'-)&Q 4S" P!"?RQ 'B3 1(3,3*-&N 3I1(388-)& -8 A()&N" %&8'3<*Q 'B3 (<'3 )= 8+D8'(<'3 ,)&8+01'-)& 0+8' <28) D3 A(-''3& D6 'B3 83,)&* (3<,'-)& <8
Enzyme Kinetics r
dC P
dCS
dt
dt
k:C ES
2-9 P!"HR
a&2388 )'B3(A-83 813,-=-3*Q 'B3 ,)&,3&'(<'-)& -8 3I1(3883* <8 0)2<( +&-'Q : 8+,B <8 U0)2b0 )( 0)2b\" OB3 ,)&,3&'(<'-)& )= 'B3 3&5603.8+D8'(<'3 ,)0123I C ES -& 4S" P!"HRQ ,<& D3 (32<'3* ') 'B3 8+D8'(<'3 ,)&,3&'(<'-)& C S <&* 'B3 =(33.3&5603 ,)&,3&'(<'-)& C E =()0 'B3 <88+01'-)& 'B<' 'B3 =-(8' (3;3(8-D23 (3<,'-)& 4S" P!"CR -8 -& 3S+-2-D(-+0" OB3&Q 'B3 =)(A<(* (3<,'-)& -8 3S+<2 ') 'B3 (3;3(83 (3<,'-)& 8) 'B<' ES P!"KR k#CS CE k!C W6 8+D8'-'+'-&N 4S" P!"KR -&') 4S" P!"HRQ 'B3 (<'3 )= (3<,'-)& ,<& D3 3I1(3883* <8 < =+&,'-)& )= C S <&* C E Q )= AB-,B C E ,<&&)' D3 3<8-26 *3'3(0-&3*" %= A3 <88+03 'B<' 'B3 ')'<2 3&5603 ,)&'3&'8 <(3 ,)&83(;3*Q 'B3 =(33.3&5603 ,)&,3&'(<'-)& C E ,<& D3 (32<'3* ') 'B3 -&-'-<2 3&5603 ,)&,3&'(<'-)& C E M C EM
E S CE C
P!"GR
/)Q &)A A3 B<;3 'B(33 3S+<'-)&8 =()0 AB-,B A3 ,<& 32-0-&<'3 C E <&* C ES ') 3I1(388 'B3 (<'3 3I1(388-)& <8 'B3 =+&,'-)& )= 8+D8'(<'3 ,)&,3&'(<'-)& <&* 'B3 -&-'-<2 3&5603 ,)&,3&'(<'-)&" W6 8+D8'-'+'-&N 4S" P!"KR -&') 4S" P!"GR =)( C E <&* (3<((<&N-&N =)( C ES Q A3 )D'<-& C ES
C EM C S k ! k #
P!"#MR
C S
/+D8'-'+'-)& )= 4S" P!"#MR -&') 4S" P!"HR (38+2'8 -& 'B3 =-&<2 (<'3 3S+<'-)& r
dC P dt
dCS dt
k:CE M CS k ! C S k #
r0
P!"##R
AB-,B -8 U&)A& <8 _-,B<32-8._3&'3& 3S+<'-)& <&* -8 -*3&'-,<2 ') 'B3 301-(-,<2 3I1(388-)& 4S" P!"9R" K M -& 4S" P!"##R -8 U&)A& <8 'B3 _-,B<32-8 ,)&8'<&'" %& 'B3 _-,B<32-8._3&'3& <11()<,BQ K M -8 3S+<2 ') 'B3 *-88),-<'-)& ,)&8'<&' K # )( 'B3 (3,-1(),<2 )= 3S+-2-D(-+0 ,)&8'<&' K eq <8
dC S dt
k:C ES
[)( 'B3 W(-NN8.V<2*<&3 <11()<,BQ 'B3 (<'3 3I1(388-)& =)( 8+D8'(<'3 ,<& D3 3I1(3883* D6 'B3 =-(8' (3;3(8-D23 (3<,'-)& <8 3I12<-&3* -& 'B3 &3I' 83,'-)&"
2-10
Enzyme Kinetics K M
k! k#
K #
CS C E C ES
# K 3S
P!"#!R
OB3 +&-' )= K M -8 'B3 8<03 <8 C S" cB3& K M -8 3S+<2 ') C SQ r -8 3S+<2 ') )&3 B<2= )= r 0
3&5603 <,'-;-'6 mg )= 1()'3-&
mmol )= 1()*+,' (323<83* mL s mg )= 1()'3-&
F8 'B3 3&5603 -8 1+(-=-3*Q 'B3 813,-=-, <,'-;-'6 A-22 (3<,B < 0
2-14
Enzyme Kinetics dC P dt dC ES
dt
ES k :C
k#CS CE
k !CES k:C ES
P!"#9R
P!":MR
dC S
P!":#R k#CS C E k ! CES dt 4S8" P!"#9RQ P!":MRQ <&* P!":#R A-'B 4S" P!"GR ,<& D3 8)2;3* 8-0+2'<&3)+826 A-'B)+' 8-012-=-,<'-)&" /-&,3 'B3 <&<26'-,<2 8)2+'-)& )= 'B3 1(3,3*-&N 8-0+2'<&3)+8 *-==3(3&'-<2 3S+<'-)&8 <(3 &)' 1)88-D23Q A3 &33* ') 8)2;3 'B30 &+03(-,<226 D6 +8-&N ;<(-)+8 0<'B30<'-, 8)='A<(38 8+,B <8 _<'B2)260<'B /)='A<(3Q c-22-0<&'-,Q dOR" %' 8B)+2* D3 &)'3* 'B<' 'B-8 8)2+'-)& 1(),3*+(3 (3S+-(38 'B3 U&)A23*N3 )= 32303&'<(6 (<'3 ,)&8'<&'8Q k # , k ! , <&* k :. OB3 32303&'<(6 (<'3 ,)&8'<&'8 ,<& D3 03<8+(3* D6 'B3 3I13(-03&'<2 '3,B&-S+38 8+,B <8 1(3.8'3<*6.8'<'3 U-&3'-,8 <&* (32
Example 2.2 W6 3012)6-&N 'B3 ,)01+'3( 03'B)*Q 8B)A B)A 'B3 ,)&,3&'(<'-)&8 )= 8+D8'(<'3Q 1()*+,'Q <&* 3&5603.8+D8'(<'3 ,)0123I ,B<&N3 A-'B (3813,' ') '-03 -& < D<',B (3<,')( =)( 'B3 3&5603 (3<,'-)&8 *38,(-D3* D6 4S8" P!"CR <&* P!"?R" OB3 -&-'-<2 8+D8'(<'3 <&* 3&5603 ,)&,3&'(<'-)&8 <(3 M"# <&* M"M# 0)2b\Q (3813,'-;326" OB3 ;<2+38 )= 'B3 (3<,'-)& ,)&8'<&'8 <(3J k # ] 9M \b0)28Q k ! ] C # # 8 Q <&* k : ] M"C 8 "
Solution: O) *3'3(0-&3 B)A 'B3 ,)&,3&'(<'-)&8 )= 'B3 8+D8'(<'3Q 1()*+,'Q <&* 3&5603.8+D8'(<'3 ,)0123I <(3 ,B<&N-&N A-'B '-03Q A3 ,<& 8)2;3 4S8" P!"#9RQ
Enzyme Kinetics
2-15
Table 2.2 _<'B2
%Content of BrownFunc.m %Create this file and place in the default m file directory function dydt = BrownFunc(t,y) global k1 k2 k3 Ce0 % More comprehensible names for the variables Cs = y(1); Ces = y(2); Cp = y(3); % Dynamic equations dydt(1) = -k1*(Ce0-Ces)*Cs + k2*Ces; % dCs/dt dydt(2) = k1*(Ce0-Ces)*Cs - k2*Ces - k3*Ces; % dCes/dt dydt(3) = k3*Ces; % dCp/dt dydt = dydt'; %MATLAB v5 wants a column vector 0.1 0.09 0.08 ) 0.07 L / l o m0.06 (
Substrate Enzyme-Substrate Complex Product
n o i t 0.05 a r t n e 0.04 c n o C
0.03 0.02 0.01 0
0
20
40
60 80 Time (sec)
100
120
140
Figure 2.4 dB<&N3 )= 8+D8'(<'3Q 3&5603.8+D8'(<'3 ,)0123IQ <&* 1()*+,' ,)&,3&'(<'-)&8 =)( 4I<0123 !"!"
P!":MRQ <&* P!":#R A-'B 'B3 3&5603 ,)&83(;<'-)& 3S+<'-)& 4S" P!"GR D6 +8-&N )&3 )= 0<'B30<'-,<2 8)='A<(38" O
2-16
Enzyme Kinetics
2.3. Evaluation of Kinetic Parameters %& )(*3( ') 38'-0<'3 'B3 ;<2+38 )= 'B3 U-&3'-, 1<(<03'3(8Q A3 &33* ') 0
r # r
K M r0
# r0
r r0
C S r 0
K M # r0
K M
r C S
P!":!R
P!"::R
P!":9R
F& 3S+<'-)& )= 'B3 =)(0 )= 4S" P!":!R A<8 N-;3& D6 \<&N0+-( Pd<(D3((6Q #GH?R =)( 'B3 '(3<'03&' )= *<'< =()0 'B3 <*8)(1'-)& )= N<8 )& < 8)2-* 8+(=<,3" %= 'B3 _-,B<32-8._3&'3& 3S+<'-)& -8 <112-,
Enzyme Kinetics
2-17
20
15
C S r
K M
1
10
rmax 5
rmax 0
20
40 C S
60
80
Figure 2.5 OB3 \<&N0+-( 12)' P K M ] #MQ r 0
0.4 1
K M
r 0.2
rmax 1
rmax -0.05
0
0.05
0.10
0.15
1
C S
Fi ure 2.6 OB3 \-&3A3<;3(.W+(U 2)' K M ] #MQ r 0
OB3 \-&3A3<;3(.W+(U 12)' -8 0)(3 )='3& 3012)63* 'B<& 'B3 )'B3( 'A) 12)'8 D3,<+83 -' 8B)A8 'B3 (32<'-)&8B-1 D3'A33& 'B3 -&*313&*3&' ;<(-
2-34
Enzyme Kinetics
2.7. Experiment: Enzyme Kinetics Objectives: OB3 )DT3,'-;38 )= 'B-8 3I13(-03&' <(3J #" O) N-;3 8'+*3&'8 <& 3I13(-3&,3 A-'B 3&5603 (3<,'-)&8 <&* <88<6 1(),3*+(38 !" O) *3'3(0-&3 'B3 _-,B<32-8._3&'3& U-&3'-, 1<(<03'3(8 D<83* )& -&-'-<2.(<'3 (3<,'-)&8 -& < 83(-38 )= D<',B (+&8 :" O) 8-0+2<'3 D<',B <&* ,)&'-&+)+8 (+&8 D<83* )& 'B3 U-&3'-, 1<(<03'3(8 )D'<-&3*
Materials: #" /13,'()1B)')03'3( !" #MNb\ N2+,)83 8'<&*<(* 8)2+'-)& :" g2+,)83 <88<6 U-' PL)" #?.ahQ /-N0< dB30-,<2 d)"Q /'" \)+-8Q _ER 9" d322)D-)83 C" d322)D-<83 3&5603 PL);)560 #KKQ L);) L)(*-8U W-)-&*+8'(-<28 %&,"Q i<&D+(6Q dOR )( )'B3( ,322+2<83 3&5603 ?" M"MC_ P0)2b\R 8)*-+0 <,3'<'3 D+==3( P1V CR H" ?MM 0\ N2<88 '3013(-&N D3<(03( %&8'(+03&' d)"Q dB-,
Calibration Curve for Glucose Assay: #" >(31<(3 N2+,)83 8)2+'-)&8 )= MQ M"CQ #"MQ :"MQ C"M <&* H"MNb\ D6 *-2+'-&N #MNb\ N2+,)83 8'<&*<(* 8)2+'-)&" !" a8-&N 'B383 8'<&*<(*8 <8 8<01238Q =)22)A 'B3 <88<6 1(),3*+(3 *38,(-D3* -& 'B3 D(),B+(3 1();-*3* D6 /-N0< dB30-,<2 d)" :" >2)' 'B3 (38+2'-&N
Enzyme Kinetics
2-35
Experiment Procedures: #" >(31<(3 < M"M!_ ,322)D-)83 8)2+'-)& D6 *-88)2;-&N :"9! N -& CMM 0\ )= M"MC_ L)+( #MM 0\ )= ,322)D-)83 8)2+'-)& A-'B < ,3('<-& ,)&,3&'(<'-)& P!MQ #MQ CQ !Q )( #0_R -&') 'B3 (3<,')(Q '+(& )& 'B3 8'-((3(Q <&* A<-' +&'-2 'B3 8)2+'-)& (3<,B38 CM d" %&-'-<'3 'B3 3&5603 (3<,'-)& D6 <**-&N # 0\ )= ,322)D-<83 8)2+'-)& ') 'B3 (3<,'-)& 0-I'+(3 <&* 8'<(' ') '-03" C" O
Data Analysis: #" d<2,+2<'3 'B3 -&-'-<2 (<'3 )= (3<,'-)& D<83* )& 'B3 C <&* #M 0-&+'3 *<'<" !" i3'3(0-&3 'B3 _-,B<32-8._3&'3& U-&3'-, 1<(<03'3(8 <8 *38,(-D3* -& 'B-8 ,B<1'3(" :" /-0+2<'3 'B3 ,B<&N3 )= 'B3 8+D8'(<'3 <&* 1()*+,' ,)&,3&'(<'-)&8 =)( D<',B <&* ,)&'-&+)+8 (3<,')(8 D<83* )& 'B3 U-&3'-, 1<(<03'3(8 )D'<-&3*" d)01<(3 )&3 D<',B (+& A-'B 'B3 8-0+2<'3* (38+2'8" [)( 'B-8 (+&Q '
2.8. Nomenclature FM
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Enzyme Kinetics
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2.9. Problems JKL
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Enzyme Kinetics
2-45
!"!# F& 3&5603 P,<'B318-&R B6*()26538 \.N2+'<062.\.'6()8-&3 ') ,<(D)D3&5)I6.\.N2+'<0-, <,-* <&* \.'6()8-&3" %' B<8 D33& =)+&* P[(<&'5 <&* /'31B3&8)&Q J. Biol. Chem.Q 169Q :CGQ #G9HR 'B<' 'B3 N2+'<0-, <,-* =)(03* -& 'B3 B6*()268-8Q -&B-D-'8 P,)013'-'-;326R 'B3 1()N(388 )= 'B3 (3<,'-)& D6 =)(0-&N < ,)0123I A-'B ,<'B318-&" OB3 ,)+(83 )= 'B3 (3<,'-)& -8 =)22)A3* D6 <**-&N '6()8-&3 *3,<(D)I62<83 AB-,B 3;)2;38 dE !. /+D8'(<'3 j0)2b0\
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2.10. References W<-236Q m" 4" <&* i" [" E22-8Q Biochemical Engineering Fundamentals" L3A n)(UQ LnJ _,g(
