Medium improvement by orthogonal array designs for cholesterol oxidase production by Rhodococcus equi No. 23

Pivcess BiochemistO' Vol. 32, No. 8, pp. 697-7113, 1997 © 1997 Elsevier Science Ltd All rights reserved. Printed in Grcat Britain 0032-9592/97 $17.00 + 0.00

t LSEVIER PII:

S0032-9592~97)00031-9

Medium improvement by orthogonal array designs for cholesterol oxidase production by Rhodococcus equi No. 23 Ming-Tsung Lee, ~' Wen-Chang Chen b and Cheng-Chun

C h o u ~'*

"Graduate Institute of Food Science and Technology, National Taiwan University, 59, Lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan bDepartment of Bioengineering, Tatung Institute of Technology, Taipei, Taiwan (Received 27 November 1996; revised version received 25 February 1997; accepted 8 March 1997)

Abstract

Medium improvement for the production of cholesterol oxidase (CO, EC 1.1.3.6) by Rhodococcus equi No. 23 was investigated using an orthogonal array design in two steps. Results revealed that yeast extract, Tween 80 and zinc sulphate had positive effects on CO production, but magnesium sulphate had an inhibitory effect. In addition, interaction between cholesterol and sodium chloride also had a significant effect on enzyme production. The improved medium consisted of 2.0g/litre cholesterol, 8"0g/litre yeast extract, 1"0 g/litre NH4CI, 1'0 g/litre NaCI, 0-50 g/litre KH_,PO4, 0.25 g/litre Na2HPO4, 0-10 g/litre L-valine, 0' 15 g/litre L-tyrosine, 0"15g/litre MgSO47H20, 0-01 g/litre ZnSO4"7H20, 0-10g/litre FeSO4'7H20 and 4"0ml/litre Tween 80. CO production at 60 h (about 0-24 units/ml) was about four-fold greater than with the control medium. © 1997 Elsevier Science Ltd

Kevwords: orthogonal array designs, cholesterol oxidase, medium improvement, Rhodococcus equi No. 23.

coccus equi [1, 7]. Amongst these, R. equi No. 23 isolated by Watanabe and Adachi [7] from butter was reported to be a potential industrial strain for CO production because of its high yield [1, 8-10]. The optimal design of culture media is a relevant aspect to be considered in the development of fermentation processes but there are few studies concerning medium improvement for CO production by R. equi strain [1, 10]. The attainment of optimal conditions for multivariable fermentation processes is often tedious but it is possible to undertake a rational study by using adequate experimental, statistical designs to reduce the number of experiments [1 1,12]. The conventional 'variation of one factor at a time' approach of optimization is not only time-consuming but often incapable of reaching the true optimum due especially to interactions among factors. On the other hand, response surface methodology (RSM) has been reported to be a much more efficient technique for the optimization of medium composition. The method becomes impractical when a large number of components in the medium have to be considered since too many combinations

Introduction

Microbial cholesterol oxidase (CO, EC 1.1.3.6) catayses the conversion of cholesterol to 4-cholesten~-one. Interest in this enzyme is due to its increasing ~tilization for the determination of cholesterol in food md blood serum and the production of the precursor ~'or chemical synthesis of steroid hormones [1]. In addiion, CO can also be applied to the oxidation of dietary :holesterol which is partially related to cardiovascular lisease, and hence to improve human health [2]. Since the first report by Turfitt [3] on enzymic tegradation of cholesterol to 4-cholesten-3-one in Oroactinomyces erythropolis, the same enzymic reaction 1as been described in many microorganisms, including trthrobacter simplex [4], Corynebacterium cholesteroicum [5], Pseudomonas testosteroni [6] and Rhodo'To whom correspondence should be addressed. Present address: Graduate Institute of Food Science and Technology, National Taiwan University, 59, Lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan. 697

698

M.-T. Lee et al.

have to be taken into account to optimize the medium composition [ 11 ]. Orthogonal arrays are highly fractionated factorial designs that allow the testing of multiple independent process variables within a single experiment. One of the most important properties of the designs is the orthogonality - - that is, the ability to separate the individual effects of several variables in an experiment. The designs can deal with varying factors with either two or more levels [12]. They have been successfully applied to the improvement of culture media for the production of primary and secondary metabolites in fermentation processes [13, 14]. The aim of this study was to evaluate the quantitative effects of 16 medium variables including carbon and nitrogen sources, mineral salts, amino acids and surfactant, individually and in combination, on CO production by R. equi No. 23 by employing orthogonal array designs (OAD) and statistical analyses. Materials and methods

Microorganism R. equi No. 23 (CCRC 13634), obtained from the Culture Collection and Research Center (CCRC), Hsinchu, Taiwan, was used in this study and was maintained in nutrient agar (Gibco Laboratories, Madison, USA) at 4°C and subcultured every 4 weeks.

Culture conditions The control medium for CO production was that of Arima et al. [15], consisting of 1.0g/litre cholesterol, 5.0 g/litre yeast extract, 1.0 g/litre NH4NO3, 0.25 g/litre K~_HPO4, 0"25 g/litre MgSO4"7H,O, and 0.001 g/litre FeSOa'7H20. The pH of the media was adjusted to 7.0 with 0.1 N HC1. Using an OAD to assess the effect of culture medium on CO production, culture media were prepared according to Table 2 or Table 4; 1 ml aliquots of an active seed culture were added to 100 ml of sterile culture medium in 250-ml Erlenmeyer flasks and incubated at 37°C in a rotary shaker (150revmin ~) for 72 h.

CO analysis For the determination of cholesterol oxidasc, samples of culture were centrifuged at 56(10g (5°C) for 15 rain and the supernatant fluid was used as the enzyme source. The assay of CO activity was based on the conversion of cholesterol to 4-cholesten-3-one according to the method described by Richmond [16]. One unit of CO is defined as the amount of enzyme required to produce 1 mmole of 4-cholesten-3-one per minute under the assay conditions. Cell density was

determined by measuring the culture absorbance at 600 nm using a spectrophotometer.

Statistical screening method: the orthogonal array design (OAD) Experimental design Eighteen basic orthogonal arrays were tabulated. The notation of each orthogonal array is expressed by its number of rows and columns, as well as the number of levels in each column. The L,,(2 ~~) orthogonal arrays, for example, have 16 rows and 15 two-level columns [17]. The OADs allow the investigation of up to N - 1 or less variables, if one or more two-factor interactions are assigned, with N experiments. In practice, all the experiments arc carried out according to a design matrix, which is based on the number of variables to be studied. Each row represents each trial (culture medium) and each column represents a different variable (medium component). Once the independent variables have been sclected, they are tcsted at two levels, a high (coded as 2) and a low (coded as 1) level, which in this study means two different nutrient concentrations. A number of variables are designated as 'dummy variables', because no change is made to them, but they are used to estimate the experimental error. The matrix of an Lz(,(2 ~5) orthogonal array was used in the row of separate experiments employed in medium development for CO production from R. equi No. 23 in batch shake cultures. All the experiments were perh)rmed in shake flasks according to the matrix, and for each experiment the response (i.e. CO activity) was recorded.

Data analysis" Statistical analyses using the procedure of a general linear model (GLM procedure) in the SAS package [18] were made to identify those medium variables and/or two-factor interaction that had a significant effect on CO production. The analysis of variance (ANOVA) for the experimental designs was obtained, and the significant level of each medium variable was determined by using Fisher's F-test and accepted only to the 95% level. Moreover, the average values of the experiments, Yx,~, for those significant medium variables, at low (level 11 and high (level 2) levels can bc calculated as follows:

Yx~il =

,Y_,R at (levcl i)

(i = 1 o r 2)

8

where R is the measured response for each test. If '/~~2j is larger than y ~ l , an increase in the concentra-

Medium improvement by orthogonal array designs

According to the design rules of the matrix of an L,,(2 ~5) orthogonal array, the entries of column 3 were obtained by multiplying the entries of columns 1 and 2 [12, 17, 21]. Therefore, column 3, in this study, was assigned as A × C (i.e. is the interaction between cholesterol and NaCI) when columns 1 and 2 wcrc assigned as main effects of A (cholesterol) and C (NaCI), respectively. C O activity was determined for each experimental design (Table 2), and the mean of squares for each treatment and error, which allow the determination of the significant level of each constituent, using Fisher's F-test, wcrc calculated. The A N O V A for the experimental results obtained by O A D 1 is given in Table 3. The main effects of yeast extract (B), MgSO4"7H20 (E) and Twccn 80 (G) had significant effects, and the interaction between cholesterol (A) and NaCI (C) also showed significant effects on the response. The average values of the experiments, for those significant medium variables (B, E, G, and A × C), are also shown in Table 3. Results obtained from the

lion of the variable X would be beneficial t o the enzyme production. Results

and

699

discussion

Preliminary experiments indicated that cholesterol and ,east extract were, respectively, the best carbon and uitrogcn sources for C O production by R. equi No. 23. Moreover, it was also found that addition of Twecn 80 ,~r NH4CI increased the enzyme production. Therefore, h e s e factors should bc taken into account in I c v c l o p i n g the fermentation process for CO producion by the test microorganism. Quantitative improvement of the medium was pcro r m e d with the L,,,(2 '5) ortfiogonal array design in wo steps: the first was used to determine which components have a significant effect on C O production by ?. equi No. 23, and the second to adjust their conccnrations and to test the influence of another ingredient.

,)rthogonal an'ay design no. I (OAD 1) X.n L,,(2 '5) orthogonal array experimental design was )erformed and the concentration of each constituent at evels 1 and 2 is given in Table 1. Results obtained r o m the screening of medium c o m p o n e n t s showed hat the addition of NaCI would p r o m o t e C O producion [19]. It was supposed that sodium ions might be mcessary for transporting substances in and out h r o u g h the cell m e m b r a n e and maintaining the )smotic pressure of the cells [2(I]. Therefore, the interiction effect between cholesterol and NaCI was lssigned and O A D 1 consists of 16 runs that allowed he study of eight parameters and one two-factor intertction (Table 2).

Table I. Experimental field for OAD 1 Constituent

Symbol

Concentration (g/litre) Level I

Level 2

I "00 4"00 0' 10

2"00 5.00 1'00

D

I '00

E

0'25

FeSO4-7H20

F

0.10

2"00 0"50 0.50

Tween 80 KHePO4 NaeHPO4

G H I

1.00 0-25 0-25

Cholesterol Yeast extract NaCI N H4CI MgSO4"7H20

A B ("

2-00 0"50 0-50

Fable 2. Medium improvement by OAD 1 for CO production by R. equi No. 23 Run

I

2 3 4 5 6 7 8 9 10 ll 12 13 14 15 16

A

("

A ×C

B

E

F

G

D

H

1

e

c

c

c

c

1

2

3

4

5

6

7

8

9

1()

11

12

13

14

15

1 1 1 1

1 1 1 I

1 1 2 2 "~

1 1 2 2 9

I 2 1 2 1

1 _'~ I

1 .'~ 1

1 2 2

1 2 2

1 .'~ 2

I _'~ 2

"~ 2 2 2

1 1 2 2 1

1 "~ 1

"~ 2 2 2

1 I 2 2 1

1 2 2

1 2 2

2 1 1

2 1 1

2 I 2

1 I I 1

2 2 2 2

1 1 2 2

2 ~ 1 1

I I 2 2

2 2 I I

I 2 1 2

2 "~ 2

1

1

2

2

I

1

1

2

2

1

I

2

1

1

2

I "~ 1

~ 1 2 2 1 2 I 2 I _'~ 1

Symbols are the same as those in Table I; e is the error effect. 'Coded level I, high level; coded level 2. low level.

Response (units/ml)

0.108 (1"11 t) 0.144

"~

2

2

1

I

1

1

0"151

I

-v 1

_~ I

I 2

I 2

2 I

~ 1

2 I I _'~ 1 2 2 1 _'~ I

2 1 2 1 2 I 1 2 1 2

2 1 1 ~'~ 2 I 1 2 2 1

2 1 2 1 1 2 2 1 I 2

I 2 1 ,~~' 2 I 2 1 1 2

I 2 2 I I 2 I 2 ,.~ 1

0"074 0"09S 0"06 I

0"075 0" 105 0"039 0-103 0"070

(1"121 0"08,8 0"205 0"251

M.-T. Lee et al.

700 Table 3. Results of ANOVA for CO production by OAD 1 Source

Degree of freedom

Sum of square

Mean of square

F-value

0 '00144 0"00593 0-00114 0.00005 0-00370 0"00083 0'00652 0.00124 0"00109 0'01975 0.00048

2.98 12"31 * 2"36 0"11 7-68* 1.71 13'52* 1457 2.27 40"98*

1 5 - 10 = 5 1 6 - 1 = 15

0 '00144 0-00593 11.00114 11.011005 11-00370 0.1101183 /I.00652 0.00124 0.00109 0.01975 0-00241 0'114405

A B

C D E F G H I

A×C Error Total

*Significant at 5% level. Fisher's F-test, F = 6"61 with 1 and 5 degrees of freedom. Average values of CO activity of significant factors: A × C (cholesterol and NaCI) YA~I)¢'(I) = 0'522/4 = 0"131 YA~ i)t,(2) = 0'3118/4 = 0'1177

YA~2~'~I) = 11"317/4 = 0'1179 YA~2)C~2~= 0'665/4 = 0'166 B (yeast extract) Yn~l) = 0'752/8 = 0.094

YB~2)= 1-11611/8= 11.133

E (MgSO4"7H20) YEll) = 1"1128/8= 0' 129 YE~2)= 0.784/8 = 0.098 G (Tween 80) Y~;~t) = I).745/8 = 0.093

Yc;~2~= 1.067/8 = 0-133

experiments of various combinations of two-factor interaction (A × C) revealed that the average value of CO activity is highest with Y:,~21cc21 followed by YA~)¢'¢I~, YA(2)('(I) and YACn)¢'¢2). Therefore, the initial concentrations of cholesterol (A) and NaCI (C) were fixed at their high levels, 2.0 and 1.0g/litre, respectively, in the next improvement step. YI~(21 was found to be larger than YB(I), YE(2) smaller than Yf~¢~ and Y(;(2) larger than Yc;¢~. It may be concluded that yeast extract and Tween 80 had a positive effect on CO production and their initial concentrations were increased in the next improvement step. In contrast, MgSO4 exhibited a negative effect on enzyme production, therefore its concentration was decreased. The main effects of NHaCI (D), FeSO4"7H20 (F), KH2PO4 (H) and Na2HPO4 (I) were found to be of no significance on CO production, as shown in Table 3, and their concentrations were not modified. The addition of Tween 80 into the culture media is useful to dissolve cholesterol, which is not watersoluble but is an excellent inducer of CO [1]. Moreover, it was supposed that the surfactant might change the permeability of the cell membrane and enhance the secretion of intracellular products into the culture medium [20]. This result also agrees with reports of Liu et al. [4] and Kreit et al. [22] for CO production by A. simplex and Rhodococcus sp. G K 1, respectively. On the other hand, Watanabe et al. [1] reported that Tween 80 could also be degraded by R. equi No. 23. Wu [10] also reported that addition of Tween 80 to the

culture medium of R. equi No. 23 resulted in the reduction of CO production and the pH of broth, probably due to the release of oleic acid from Tween 80. Higher CO production also occurred at an initial pH of 7.0 [19]. The medium for CO production by Rhodococcus sp. G K 1 [22], R. equi No. 23 [1, 7-9] and A. simplex [4] also contained phosphate. Therefore, in this study, a mixture of KH2PO4 and Na2HPO4 was used as the buffering reagent to maintain the pH value near the optimum for CO production, although the main effects of these two factors were not found to be significant (Table 3).

Orthogonal array design no. 2 (OAD 2) The number of unmodified variables in O A D 1 allows the study of at least one additional ingredient in the next improvement step. In preliminary experiments, it was found that two amino acids, L-valine and L-tyrosine, could promote CO production. In addition to calcium chloride (CaCI2"2H20), manganese sulphate (MnSO4-3H20), zinc sulphate (ZnSO4"7H20) and thiamine had also been used as medium components for enzyme production by other Rhodococcus spp. [22]. Therefore, a second L,,(2 I~) experimental design ( O A D 2) was used, and the concentration of each constituent is given in Table 4. O A D 2 allows the study of nine parameters in 16 runs (Table 5): three parameters (B, E and G) with significant effect, which were

Medium improvement by orthogonal array designs

o b s e r v e d in O A D l ( T a b l e 3), a n d the six n e w v a r i a b l e s L-valine, c-tyrosine, CaCI2-2H20, MnSO4.3H20, ZnSO4"7H20 and thiamine. C O activity was d e t e r m i n e d for each e x p e r i m e n t a l design ( T a b l e 5), a n d the A N O V A for the experim e n t a l results o b t a i n e d by O A D 2 is given in T a b l e 6. O n l y Z n S O 4 " 7 H 2 0 (I) h a d a significant effect o n e n z y m e p r o d u c t i o n . T h e a v e r a g e v a l u e s o f the experim e n t s with zinc s u l p h a t e , Y , t ) a n d YJ(2), are s h o w n in T a b l e 6, a n d it is e v i d e n t that Y,21 is larger t h a n Y,~), s u g g e s t i n g that an i n c r e a s e of the c o n c e n t r a t i o n o f zinc s u l p h a t e m a y e n h a n c e C O p r o d u c t i o n . T h e effect of the c o n c e n t r a t i o n of zinc s u l p h a t e o n e n z y m e p r o d u c tion was e x a m i n e d f u r t h e r , a n d it was f o u n d that C O

q able 4. Experimental field for O A D 2 ( onstituent

Symbol

-Valine '~ east extract I -Tyrosine "t hiamine 1~IgSO4-7H20 ( aCI2"2H20 ~!ween 80 1~|nSOa'3H20 2 nSO4'7H20

Concentration (g/litre)

A B C D E F G H I

Level 1

Level 2

0-10 5.00 0.10 0.00 0-15 0.00 2.00 0-00 0"00

0.2(I 8.00 0-20 0.01 0'25 0.02 4.00 0.01 0"01

701

~Iable 5. Medium improvement by O A D 2 fl)r CO production by R. equi No. 23 I',un

1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

A*

C

e

B

E

F

G

D

H

I

e

e

e

e

e

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1"

1 1 1 I

1 1 1 1

1 1 2 2

1 I 2 2

1 1 2 2

1 1

1

1

1

2

2

2

2

1

1

1

2

2

2

2

2 2 2 2

2 2 2 2

1 1 2 2

1 1 2 2

2 2 1 1

I

2

2

2

2

1

1

1

2

2

I 1 1 I

2 2 2 2

I 1 2 2

2 2 1 1

1 1 2 2

2 2

1 1

1 2

I

2

1

2

1

2

2 2 2 2

I 1 1 1

1 I 2 2

2 2 1 1

2 2 1 1

1 2 2 1 1 2 2 1 2 1 1 2 2 1 1

1 2 2 1 2 I 1 2 1 2 2 I 2 1 1

1 2 2 I 2 l 1 2 2 I 1 2 1 2 2

2

2

I

2 2 2 2 1 1

2

2 2

2

1 1 1 1

2 2

2 2

1

1

2

2

1

1 2 2 1 1 2 2 I 1 2 2 1 1 2 2

1

I

2

1

1

2

1

2

2

1 2

Basal medium: cholesterol, 2"0g/litre; NHaCI, l'l)g/litre; FeSO4.7H20, 0"1 g/litre; NaC1, Na2HPO4, 0'25 g/litre. ~ymbols are the same as those in Table 4; e is the error effect. Coded level 1, high level; coded level 2, low level.

Response (units/ml)

0-146 0"218 0'169 0'2(17 0"208 0-157 0.258 0.125 0.137 0.176 0.158 0.194 0.137 0.143 0.240 0.184

l'()g/litre; KHePO4, ().5g/litrc;

' "able 6. Results of A N O V A for CO production by O A D 2 ;,ource

Degree of freedom

Sum of square

Mean of square

F-value

0-00087 11-00284 0'00012 ()'WOO14 0'00146 0"00075 0'00062 0' 00061 0"01092 0.00080

1.1)9 3"54 0" 16 0-18 l "83 0.94 0'77 0"76 13.61 *

15--9=6 16-- 1 = 15

0"00087 0-110284 0-00012 0"00014 0'00146 0"00075 0-00062 0 "00061 0"01092 0"110478 O'02322

A B

C D E F G H I

Error Fotal

Significant at 5% level. Fisher's F-test, F = 6.61 with I and 5 degrees of freedom. \verage values of CO activity of the significant factor: I (ZnSO4"7H20) "ul~ = 1"219/8 = 11"152 Yl~2)= 1"638/8 = 0"205

702

M.-T. Lee et al.

production reached a maximum at 0.01 g/litre ZnSO4"7H20 and then levelled off (data not shown). Cell growth requires specific metal ions and some organic growth factors and in many cases the need for these substances arises from their roles as cofactors of metabolic and biosynthetic enzymes. Among the metal ions (Fe, Mg, Mn, Ca and Zn) tested, only Zn cation showed a positive effect on CO production, since it may bc used as an enzyme cofactor and also required for protein synthesis and ccll division [20]. Thc samc kinds of metal ions were also added to the medium of Krcit et al. [22] for CO production by Rhodococcus sp. GK I. Only Fe and Mg ions were used in the media of Aihara et al. [8] and Watanabe et al. [1, 7, 9] for R. equi No. 23, and Mg and K ions in that of Liu et al. [4] for A. simph'x.

0.30 0.25 0.20 0.15 >

0.10 e..) < © 0.05 L.) 0.00

i

5 4 3

CO production kinetics © The composition of the improved medium, obtained above from the two-step OADs for CO production is (g/litre): cholesterol, 2.11; yeast extract, 8.11; NaCI, 1-0; NH4CI, 1.0; KH2PO4, 0.50; Na,HPO4, 0.25; L-valine, (1.10; L-tyrosine, 0.15; MgSO4.7H =O, 0.15; ZnSO4.7H=O, 0.01; FeSO4"7H~O, 0.10; Tween 80, 4.0 (ml/litre). The comparative behaviour of CO production by R. equi No. 23 in Erlenmeyer flasks, using control and improved media, is plotted in Fig. 1. Maximum extracellular CO production (0.06 and 0.24unit/ml in control and improved media, respectively) was reached after about 6(I h of cultivation. In conclusion, OAD methodology can be used efficiently and succcssfully for the improvement of multivariable biological systems. In this study, the improved medium, obtained by OAD in two steps, showed a four-fold increase in CO production. A significant incrcasc in growth of R. equi No. 23 was also observed. References

1. Watanabe, K., Shimizu, H., Aihara, H., Nakamura, R., Suzuki, K. and Komagata, K., Isolation and identification of cholesterol degrading Rhodococcus strains from food of animal origin and their cholesterol oxidasc activities. Journal ~f General and Applied MicrobiohLey, 1986, 32, 137-147. 2. Kaunitz, H., Cholcstcrol and repair processes in arteriosclerosis. Lipids, 1978, 13, 373-374. 3. Turfitt, G. E., The microbiological degradation of steroids. 2. Oxidation of cholesterol by Proactinomyces spp.. Biochemical Journal, 1944, 38, 492-496. 4. Liu, W. H., Meng, M. H. and Huang, C. F., Production of cholesterol oxidase by streptomycinresistant and constitutive mutant of Arthtvbacter simplex. Journal o1" the Chinese Agricultural ~Twmistry SocieO,, 1987, 25, 23-30. 5. Shirokanc, Y., Nakamura, K. and Mizusawa, K., Purification and some properties of an cxtraccllular

2 1

0 0

12

24

36

48

60

72

Culture Time (h) Fig. 1. Comparative kinetics of CO production with control and improved media by R. equi No. 23. A, Control medium; ,., improved medium.

6. 7.

8.

9.

10.

11. 12. 13.

3/#hydroxysteroid oxidase produced by Corynebacterium choh'sterolicum. Journal of Fermentation Technology, 1977, 55, 337-346. Talalay, P. and Dobson, M. M., Purification and properties of a /]-hydroxysteroid dehydrogenase. Journal of Biological Chemisoy, 1953, 205, 823-837. Watanabc, K. and Adachi, S., Cholesteroldegrading enzyme: cholesterol-degrading bacteria isolated from foods in animal origin and production of cholesterol oxidasc. Japanese Journal o[" Dahy Food Science, 1985, 34, A195-202. Aihara, H., Watanabe, K. and Nakamura, R., Characterization of production of cholesterol oxidases in three Rhodococcus strains. Journal o[" Applied Bacteriology, 1986, 61,269-274. Watanabc, K., Aihara, H., Nakagawa, Y., Nakamura, R. and Sasaki, T., Properties of the purified cxtraccllular cholesterol oxidase from Rhodococcus equi No. 23. Journal o[" Agriczdtural and Food Chemisto', 1989, 37, I 178-1182. Wu, C. Y., Developing a h)w-cholcstcrol egg yolk product by enzyme system of Rhodococczzs equi No. 23. Master thesis, National Taiwan University, 1994. Kennedy, M. J.. Reader, S. L. and Davies, R. J., The kinetics of developing fermentation media. Process Biochemistry, 1994, 29, 529-534. Logothctis, N. and Wynn, H. P., Designing experiments. In Quality Througlz Design. Oxford University Press, New York, 1989, pp. 90-159. Silveira, R. G., Kakizono, T.. Takemoto, S., Nishio,

Medium improvenlent by orthogonal array designs

4.

5.

6.

7.

N. and Nagai, S., Medium optimization by an orthogonal array design for the growth of Methanosarcina barkeri. Journal of Fermentation Bioengineering, 1991, 72, 211-25. Cruz, P. M., Christen, P. and Farrcs, A., Medium optimization by a fractional factorial design for lipase production by Rhizopus delemar. Journal of Fermentation Bioengineering, 1993, 76, 94-97. Arima, K., Nagasawa, M., Bae, M. and Tamura, G., Microbial transformation of stcrols. Part I. Decomposition of cholesterol by microorganisms. Agricultural and Bioh)gical Chemisto,, 1969, 33, 1636-1643. Richmond, W., The development of an enzymatic technique for the assay of cholesterol in biological fluids. Scandinavian Journal of Clinical and Laborato~. Investigation, 1972, 29 (Suppl. 26), abstract 3.25. Dcvor, R. E., Chang, T. H. and Suthcrland, J. W.,

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Two-level fractional factorial designs. In Statistical Quality Design and Control. Macmillan, New York, 1992, pp. 674-719. SAS, Statistical Analysis System. SAS Institute, Ralcigh, NC, 1986. Lcc, M. T., Production of cholesterol oxidase by Rhodococcus equi No. 23. PhD thesis, National Taiwan Univcrsity, 1994. Talaro, K and Talaro, A., Foundation in Microhiology. Win. C. Brown Communication Inc., Dubuque, IN, 1993. Jeff-Wu, C. F., Mao, S. S. and Ma, F. S., SEL: a scarch mcthod based on orthogonal arrays. In Statistics: Textbook and Monographs, Vol. 1(19, cd. S. Ghosh. Marcel Dekker, New York, 1990, pp. 279-286. Kreit, J., Germain, P. and Lefcbvre, G., Extracellular cholesterol oxidase from Rhodococcus sp. cells. Journal (~["Biotechnolo,~', 1992, 24, 177-188.