measured by LC-MS/MS analytics and compared to the standard solution incubated at 48C without adding TE-buffer,methanol and chloroform.

For investigation of the cofactor stability in the presence of substrates of the biotransformation different concentra-tions of MAA and 2-propanol were added to a 0.05mol L à1potassium phosphate buffer solution,pH 6.0,containing 1m mol L à1of NAD,NADP,NADH,and NADPH as given in the legend of Figure 4.After incubation for 5h at 308C the cofactor mixtures were analyzed by LC-MS/MS analysis and compared to the standard solution incubated for 5h at 308C without any additives.

Kinetic Characterization of Lb ADH

The kinetic characterization of isolated Lb ADH was

performed in a 1mL quartz cuvette and measured spectro-photometrically at 308C (Photometer Shimadzu UV160,Shimadzu,Duisburg,Germany).The activity assays con-tained the investigated concentration of substrates and cofactors in 50mmol L à1potassium phosphate buffer,pH 6.0and 2mmol L à1MgSO 2.From the change of absorbance

at 340nm,activities were calculated by using a molar

extinction coef?cient of 6.22cm 2m mol à1for NADH and NADPH.In the kinetic measurement the concentration of one reactant was varied while all other concentrations were kept constant.All experiments were carried out as technical replicates;the data show the average of two measurements.Experimental details from kinetic investigation are pre-sented as supporting information.

Mathematical Model

The kinetic properties of Lb ADH were described using a

two-substrate Michaelis–Menten equation with competitive substrate inhibition or competitive product inhibition.Lb ADH kinetics were examined for the reduction of MAA,oxidation of (R )-methyl 3-hydroxybutyrate (MHB),oxida-tion of 2-propanol and reduction of acetone with regarding either NAD(H)or NADP(H)as cofactor.The kinetic

constants were estimated by nonlinear regression of initial rate measurements using Scientist 1for windows,version 3.0(MicroMath Research,St.Louis,MO,USA).The kinetic equations of the mathematical model for simulation of intracellular cofactor concentrations are as follows:r 1?v max ;1c MAA c

NADH k MAA m 11tc MHB

k MHB pi 1

tc MAA

ek NADH m 1

tc NADH T(1)

r 2?v max ;2c MHB c NAD

k MHB m 21tc

MAA MAA pi 2

tc MHB

ek NAD m 2tc NAD T

(2)

r 3?v max ;3c ace c

NADH k ace m 31tc 2àprop k 2àprop pi 3

tc ace ek NADH m 3

tc NADH T(3)

(4)

(5)

(6)

(7)

(8)

254

Biotechnology and Bioengineering,Vol.104,No.2,October 1,2009

石英吸收池

分光光度计

摩尔的

系数

活度竞争性的估算

动力学方程1-8用来描述lbADH