Abstract: In the study presented, Design of Experiments (DOE) was combined with statistical analysis such as fractional factorial design and small central composite design Response Surface Methodology (RSM) to significantly increase the extracelullar recombinant CGTase yields in fermentation flasks. The new medium obtained by the statistical analysis for the significant medium components comprised of 12 g L-1 NZ Amine A, 24 g L-1 yeast extract, 9.44 g L-1 KH2PO4, 4.4 g L-1 K2HPO4, 4.58 mL L-1 glycerol, 7 mg L-1 sucrose and 3 mg L-1 CuCl2. Yields were improved about 68% from 9.54 to 16.07 kU mL-1 in flasks when using the optimized cultivation medium. The results suggest that the overexpression level of recombinant CGTase excreted into the culture medium using the recombinant Escherichia coli could be improved through medium optimization.
INTRODUCTION
Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is a unique extracellular enzyme that can degrade starch to form non-reducing cyclic malto-oligosaccharides called cyclodextrin (CDs) (Tonkova, 1998). CDs have a peculiar torus-shaped structure with hydrophobic interior cavity and hydrophilic exterior surface and therefore can form inclusion complexes with a variety of hydrophilic guest compounds (Szetjli, 1998). As a result, CDs can change physical and chemical properties of the encapsulated guest compounds.
Given the potential uses of CGTase and their high demand, requirement exists for the cloning and recombinant clones that will produce CGTase with high volumetric yield and the development of low cost industrial media formulation. Park et al. (1997) applied the fed-batch strategy for the fermentation of recombinant Escherichia coli to produce Bacillus macerans CGTase together with some modifications of the inducer used; a 31-fold increase in the CGTase activity was achieved. Besides, in order to enhance the production of recombinant CGTase, Kim et al. (2005) co-expressed the folding accessory proteins for the production of active CGTase of B. macerans in recombinant E. coli, using fed-batch fermentation and this resulted in 1200 U mL-1 CGTase activity. Expression of foreign protein in E. coli normally resulted with intracellular protein production. A few studies on the usage of recombinant E. coli for excretion of foreign protein into the culture medium were conducted by previous researchers. This include the overexpression of the phytase from E. coli as an extracellular production in bioreactor by using kil gene (Miksch et al., 2002), secretory expression of thermostable T1 lipase through bacteriocin release protein by Rahman et al. (2005) and secretion of heterologous CGTase of Bacillus sp. E1 from E. coli by Yong et al. (1996) with signal peptide. In this study, co-expression of the Bacteriocin Release Protein (BRP) with the expression of CGTase in recombinant E. coli enable the excretion of recombinant CGTase into the culture medium. However, none of the research regarding the medium optimization for the production of extracellular recombinant β-CGTase in E. coli was reported.
Designing a fermentation medium is of critical importance because medium composition will absolutely affect the product concentration, yield and volumetric productivity (Kennedy and Krouse, 1999). Medium optimization by classical methods, which only involves the change of one variable at a time, is definitely time-consuming. Besides, the interaction effects between the variables will be neglected. In this respect, the Response Surface Methodology (RSM), which is used to study the effects of several factors influencing the responses by varying them simultaneously and conducting a limited number of experiments, was the best choice for the medium optimization. This study applied the response surface methodology, the fractional factorial design and small central composite design, for the medium optimization with the objective to maximize the production of the extracellular recombinant CGTase by excretory E. coli overexpression system.
MATERIALS AND METHODS
Strains and plasmid: CGTase gene (cgt) from Bacillus sp. G1 was isolated and cloned in E. coli JM109 (F’, traD36, proAB, lacIqZΔM15) for the DNA manipulation and propagation. The E. coli K12 N3406 (Thr leu thi lacY tonA supE) that carried the pJL3-BRP gene (MoBiTec) was used as the real host for the overexpression of cgt, in which the cgt was placed under the control of the xylA promoter in pWH 1520 (MoBiTec) and was inducible by xylose. Meanwhile, the BRP gene was regulated by lac promoter and was induced by IPTG.
Expression of recombinant CGTase: Escherichia coli carrying the appropriate plasmids was grown in an initial medium consisting 12 g L-1 NZ Amine, 24 g L-1 yeast extract, 8 mL L-1 glycerol, 2.2 g L-1 KH2PO4 and 9.4 g L-1 K2HPO4, 100 μg mL-1 ampicillin and 34 μg mL-1 chloramphenicol, overnight at 37°C on a rotary shaker at 200 rpm as the seed culture. The experiment was carried out with 50 mL of culture shaken in 250 mL flasks at 37°C at 200 rpm. A 1 mL aliquot per 50 mL production medium served as its inoculum. When the cell optical density reached 0.3 at 600 nm, xylose at the final concentration of 1.0 mM and 50 μM IPTG was added and then the temperature was shifted to 20°C. Extracellular recombinant CGTase activity was measured after induction had started for 29 h (Lo et al., 2007).
CGTase enzyme assay: CGTase assay was conducted according to the Kaneko method (Kaneko et al., 1987). The reaction mixture containing 1 mL of 40 mg of soluble starch in 0.1 M potassium phosphate buffer (pH 6.0) and 0.1 mL of the crude enzyme from the culture after some appropriate dilution and the mixture was incubated in water bath at 60°C for 10 min. Reaction was stopped by adding 3.5 mL of 30 mM NaOH. Finally, 0.5 mL of 0.02% (w/v) phenolftalein in 5 mM Na2CO3 was added and mixed well. After leaving the mixture to stand for 15 min at room temperature, the reduction in colour intensity was measured with a spectrophotometer at 550 nm. A blank lacking the enzyme was tested simultaneously with each batch of samples. One unit of enzyme activity is defined as the amount of enzyme that forms 1 μmol of β-CD from soluble starch in 1 min.
Medium optimization by design of experiments: DOE analysis was performed using Design Expert Software (Stat-Ease Inc., Statistic made easy, Minneapolis, MN, USA, Version 6.0.4). A 215-10 fractional factorial design was applied to determine the statistically significant medium components with 3 center points repeated and incorporated 32 factorial points. Medium optimization was further expanded to a small central composite design with the results obtained from the screening process. The small central composite design consisted of 14 axial points, 5 center points and 22 factorial points. For all analysis, ANOVA test was considered to evaluate the significance for the experimental data using the p-value whereas p-value less than 0.05 indicates that the model terms are significant. Center-points for the factorial designs were applied to test the curvatures as well as the replication error for testing main and two-way interaction effects. Plots for the designs were constructed using the models generated and these plots were based on the best fitted model found by fitting the full quadratic models with the experimental data.
Batch fermentation: The cultivation of the recombinant E. coli was performed in the Biostat MD B. Braun stirred tank reactor consisted of barosilicate glass culture vessel with 2 L of total volume. Batch mode bioreactor was carried out for the recombinant CGTase expression. The optimized cultivation conditions (Lo et al., 2007) and new optimized medium were applied for the batch mode cultivation. Initial working volume was 75% of the total volume. The stirrer speed was maintained at 200 rpm and the oxygen was controlled around 20%. The overnight cultured inoculum was transferred to the working volume of medium and the cells were first maintained at 37°C. When the cell optical density reached 0.3 at 600 nm, xylose at the final concentration of 1.0 mM and 50 μM IPTG was added and then the temperature was shifted to 20°C. Extracellular recombinant CGTase activity was measured after induction had started for 36 h.
RESULTS AND DISCUSSION
Selection of medium components: As reported previously, the extracellular recombinant CGTase was successfully overexpressed in an excretory E. coli overexpression system; besides, flasks cultivation conditions were optimized in the early studies and the maximum yield for extracellular recombinant CGTase was 9.54 kU mL-1 (Lo et al., 2007). In a recent work, modified TB gave the highest expression level when the cultivation temperature was maintained at 37°C (Fig. 1). Previous researchers reported that addition of medium nutrients enhanced the expression level of the recombinant E. coli. Hence, the effect of different medium components was investigated and six categories of components were studied in the early one-at-a-time screening stage. The six categories involved are carbon sources (sugars and polyols), nitrogen sources, mineral salts, vitamins, trace elements and some protein folding agents. The original medium was enriched or replaced accordingly with the certain concentration as given by the references. The details and the yield of extracellular recombinant CGTase are shown in Table 1 and Fig. 2.
Carbon sources such as lactose, sucrose, fructose, maltose, glucose, mannitol and sorbitol were added to the original cultivation medium during the experiment. Except for fructose, maltose, glucose, mannitol and sorbitol, the rest of the carbon sources gave satisfactory enzyme activity results (10.15-13.65 kU mL-1 enzyme) compared to the control (9.79 kU mL-1 enzyme) given by the original cultivation medium (Table 1 and Fig. 2). The lowest yield activity was obtained with glucose (1.21 kU mL-1 enzyme), which contradicted with the defined medium well established by Riesenberg et al. (1990). As for nitrogen sources, NH4Cl, (NH4) 2SO4, NH4NO3, casamino acid and tryptophan were screened manually. According to Table 1, enzyme activities of 4.10, 4.36, 8.39 and 9.35 kU mL-1 enzyme were obtained with NH4Cl, NH4NO3, casamino acid and tryptophan, respectively, which is lower than the control enzyme activity (9.79 kU mL-1). The best performance was seen for (NH4)2SO4 (10.06 kU mL-1 enzyme).
In order to study the effects of the mineral salts and vitamins on the expression
level of the extracellular recombinant CGTase, the original medium was enriched
with salt components such as NaCl (LB), CaCl2, MgSO4·7H2O,
Na2HPO4·7H2O, NaH2PO4
orthophosphate NaOH and sodium acetate.
| Fig. 1: | Comparison for the expression level of the extracellular recombinant CGTase in media selection phase after the post induction for 24 h, before the cultivation conditions optimization. Modified TB obviously gave better performance than the others. (YT: YT medium, TB: Terrific broth, NZ: NZ medium, LB: Luria-Bertani medium) |
| Fig. 2: | Effects of substrate components toward the expression level of the extracellular recombinant CGTase (Types of substrate were detailed in Table 1) |
However, only CaCl2 gave positive effect towards the expression
level of extracellular recombinant CGTase (9.98 kU mL-1 enzyme),
due to the stabilizing effect of CaCl2 on the CGTase (Han
and Tao, 1999). Besides, vitamin B1, proline, trisodium citrate and citric
acid together with glucose were also added and only vitamin B1 enhanced the
expression level to 10.11 kU mL-1 enzymes. Finally, trace elements
such as CoCl2·6H2O, EDTA, FeCl3, FeCl2,
MnCl2·4H2O, ZnSO4·7H2O,
FeSO4·7H2O, CuCl2, CuSO4,
CuN2O3, H3BO3 and some protein folding
agents such as urea and Triton X-100 were added to the original medium. Better
performance was seen for CoCl2·6H2O, EDTA, ZnSO4·7H2O,
FeSO4·7H2O, CuCl2 and CuSO4,
which showed enzyme activities of 10.72, 10.80, 11.26, 11.03, 11.74 and 13.82
kU mL-1, respectively (Table 1).
| Table 1: | Categories and the concentrations of the medium components involved |
| *The selected medium components for the fractional factorial medium screening stage | |
All the protein folding agents reduced the expression level of the recombinant enzymes. Finally, the 10 components that gave the highest performance of enzyme activity were included in the fractional factorial design, together with the components from the original medium, as shown in Table 1.
Fractional factorial design for screening process: In order to formulate a better medium for the growth of recombinant E. coli harbouring plasmid pWH 1520-cgt and hence increase the expression and production of the extracellular recombinant CGTase, effects of medium components were further investigated through the fractional factorial design to study the interaction effects between the components remained. Besides, the first order model generated by the fractional factorial design was able to identify the significant components for the medium optimization small central composite design.
A total of 15 medium components were chosen after the one-at-a-time screening
method in which the initial medium components included are detailed in Table
1, together with the concentration studied. The statistical model predicting
the production of extracellular recombinant CGTase with the medium components
is given in the first order model, Eq. 1:
| (1) |
where, Y is the extracellular recombinant CGTase activity (kU mL-1) and the model was used to predict the enzyme activity.
The fitness of the regression Eq. 1 was confirmed by the determination coefficient R2 where R2 implies that the samples variation of 95.83% for the extracellular recombinant CGTase was attributed to the medium components investigated. The corresponding Analysis of Variance (ANOVA) is presented in Table 2, where only the significant components are considered where their p-values (Probability>F) are less than 0.05. Components X1 (NZ Amine A), X2 (yeast extract), X3 (K2HPO4), X4 (KH2PO4), X5 (glycerol), X8 (sucrose) and X15 (CuCl2) show p-value less than 0.05 and were considered to be significant while the rest having p-value more than 0.05 and hence were considered insignificant for the enhancement of expression level for the extracellular recombinant CGTase. Furthermore, the significance of the curvature with p-value less than 0.0001 anticipated an optimum concentration for each significant component within the range studied. In order to achieve a more accurate concentration for the components, an optimization design is required.
Small central composite design for optimization process: The seven selected
significant medium components, NZ Amine A (X1), yeast extract (X2),
K2HPO4 (X3), KH2PO4 (X4),
glycerol (X5), sucrose (X8) and CuCl2 (X15),
were, further optimized using a small central composite design, with the new
range of concentration determined (Table 1).
| Table 2: | ANOVA for the fractional factorial screening design |
| df: Degree of Freedom | |
The software Design Expert (Stat-Ease Inc., Minneapolis, USA, Version 6.0.4)
was used for the data analysis and quadratic model building (Eq.
2) and the theoretical values of CGTase activity could be obtained with
the model fitting techniques:
| (2) |
where, Y represents the predicted CGTase activity (kU mL-1). The statistical model (Eq. 2) adequacy was analyzed by p-value and the correlation coefficient R is tabulated in Table 3. The p-value of the lack of fit is 0.8625 which is not significant indicating the experimental data achieved a reliable response. Adequate precision that measures the signal to noise ratio obtained is 15.803, which is greater than 4 and is desirable to indicate the responses achieved are reliable.
Maximization of the regression model (Eq. 2) was carried
out using iterative procedure (Rao et al., 2000;
Rosenbrock, 1960) to obtain the optimum concentrations
of each independent variable. The 3D response surface plots of the variables
presented in Fig. 3-6 provide a method to
visualize the relationship between the response and experiment levels of each
variable. The p-value of the regression model (Eq. 2) obtained
in Table 3 (0.0023) indicates that the global maximum in the
recombinant protein yield was confined within the experimental range. From Table
3 and based on the p-value, K2HPO4 (X3)
has the greatest impact towards the expression level (p-value is 0.0010) while
NZ Amine A (X1) has the least impact towards the expression level
with p-value obtained is 0.4763, the largest p-value obtained among the medium
components studied.
| Table 3: | ANOVA of the small composite optimization design and interaction effect of the medium components |
| R2 = 99.18%, R = 99.59%. Adequacy precision = 15.803 | |
| Fig. 3: | Response surface plot of extracellular recombinant CGTase activity: Effect of NZ Amine A (X1) and yeast extract (X2) while the other variables were fixed at their predicted concentration (Table 4) |
| Fig. 4: | Response surface plot of extracellular recombinant CGTase activity: Effect of NZ Amine A (X1) and K2HPO4 (X3) while the other variables were fixed at their predicted concentration |
The p-value in Table 3 shows that yeast extract (X2)
influences the expression level more than NZ Amine A (X1); while
the nitrogen sources, the yeast extract and NZ Amine A give more impact on the
expression level more than the carbon sources such as glycerol and sucrose.
| Fig. 5: | Response surface plot of extracellular recombinant CGTase activity: Effect of yeast extract (X2) and glycerol (X5) while the other variables were fixed at their predicted concentration |
| Fig. 6: | Response surface plot of extracellular recombinant CGTase Activity: Effect of K2HPO4 (X3) and Sucrose (X8) while the other variables were fixed at their predicted concentration |
The interaction effect of X1X3 (NZ Amine A and K2HPO4) gave the greatest influence towards the production of extracellular recombinant CGTase with the p-value of 0.0178 when compared to all the interaction effects observed. The observation of the p-value is reasonable in this study as the recombinant E. coli requires a lot of nitrogen sources to synthesize the recombinant CGTase and potassium (K+) is required by all organisms especially for those involved in protein synthesis (Madigan et al., 2003).
From Eq. 2, the negative fitted constant of X1 (NZ Amine A) suggests that the concentration should be fixed at a low level (12 g L-1) as well as the variable X2 (yeast extract) and variable X3 (K2HPO4), the concentrations for each variable were fixed at 24 g L-1 (Fig. 3) and 9.44 g L-1 (Fig. 4), respectively. Based on Fig. 5, when the concentration of yeast extract was fixed at 24 g L-1, in order to achieve the highest enzyme activity, a concentration of 4.6 mL L-1 glycerol was selected. When variable X3 (K2HPO4) was set at 9.4 g L-1, Fig. 6 predicts that the highest enzyme activity would be achieved by fixing the sucrose (X8) at 70 mg L-1. The positive fitted constant of variable X4 (KH2PO4) showed in Eq. 2 confines that the concentration for KH2PO4 should be chosen at the high level (4.4 g L-1), as well as for the CuCl2 (X15), the high level concentration of 3 mg L-1 was selected finally. There are 2 new added medium components to the original medium after the medium optimization was carried out.
As a summary, the predicted optimum concentration for the medium components is NZ Amine A (X1) 12 g L-1; yeast extract (X2) 24 g L-1; K2HPO4 (X3) 9.44 g L-1; KH2PO4 (X4) 4.4 g L-1; glycerol (X5) 4.6 mL L-1; sucrose (X8) 70 mg L-1; CuCl2 (X15) 3 mg L-1, while the predicted maximum yield for the extracellular recombinant CGTase was 14.78 kU mL-1. For further verification, the new medium formulation was implemented in the cultivation and an optimal yield of extracellular recombinant CGTase activity at 16.07 kU mL-1 was attained. Before the new medium optimization was applied for the production of extracellular recombinant CGTase, an activity of 9.54 kU mL-1 was achieved with the optimized cultivation condition (Lo et al., 2007) and 1.68-fold increment in the activity yield was observed with the new formulated medium. Table 4 show the new medium optimization and the extracellular recombinant CGTase activity obtained. NZ Amine A and yeast extract remained the same concentration as the original medium, while the concentration for K2HPO4 and glycerol was quite similar with the original medium (Table 4). However, the concentration for KH2PO4 increases from 2.2 to 4.4 g L-1. In this case, the potassium phosphate is considered to supply nutrient K+ and to remain as a buffer as well.
There have been a number of studies conducted on formulation of medium components
for both cultivation of recombinant clone and wild type, or selection of the
best medium for some overexpression studies. Khushoo et
al. (2004) compared some media such as LB, 2xYT and TB, which are well
known for the expression of recombinant protein and finally found that TB resulted
in 4-fold higher extracellular L-asparaginease II in recombinant E. coli.
In this case, modified TB was chosen as the initial medium for the cultivation.
Modified TB consisting of phosphate buffer for better production of extracellular
recombinant CGTase compared to TB. K2HPO4 and KH2PO4,
the main components in the phosphate buffer, which help to maintain the
pH of the culture along the cultivation period, were added in the modified TB
used. After a long cultivation period, the pH of the fermentation medium normally
changes to acidic due to the production of acetic acid caused by the glucose
metabolism (March et al., 2002; Park et al.,
1997). Due to that condition, K2HPO4 (X3) and
KH2PO4 (X4) remained significant in this case.
Based on Fig. 6, which reveals that only minor changes of
pH occurred during the cultivation period indicates that the advantages of using
phosphate buffer solution consisting of K2HPO4 and KH2PO4
towards the production of extracellular recombinant CGTase. This suggests that
phosphate buffer is capable of neutralizing the acids generated by E. coli
in the course of metabolizing lactose as a partial carbon source (Ma
et al., 2006). A similar concept was also applied by Xu
et al. (2000) when the phosphate salts, K2HPO4
and KH2PO4, were added into the medium for the production
of human Epidermal Growth Factor (hEGF) in the recombinant E. coli K12.
Maximal hEGF expression was detected at the initial pH of about 6.8 (Xu
et al., 2000).
| Table 4: | Summary of the optimized medium |
Trace elements that are essential to microbial nutrition were investigated in this study. However, CaCl2 and MgSO4 were excluded due to the interference of these elements with the autolytic process caused by the Bacteriocin Release Protein (BRP) (Dekker et al., 1999) and thus will inhibit the production of extracellular recombinant CGTase where BRP mainly helps to excrete the expressed CGTase into the culture medium. Among the trace elements studied, only CuCl2 remained as the important component that will influence the production of recombinant CGTase. In order to maintain the integrity of periplasmic and outer membrane components especially for the proper folding of Bacteriocin Release Protein (BRP), RpoE of E. coli, which is a sigma factor of the extracytoplasmic function protein family, is activated by Cu(II) ion (Egler et al., 2005). In this study, which involved the co-expression of BRP to obtain the extracellular recombinant CGTase, CuCl2 included in the new medium is important.
The carbon source content in the medium will affect the cell density of E. coli and the new medium containing 2 carbon sources, which are glycerol and sucrose. This is different with the typical medium for recombinant E. coli that normally consisting glucose as the main carbon source. It has been shown by a few research groups (Wong et al., 2008; March et al., 2002; Liu et al., 2000; Lee, 1996) that, during the biochemical synthesis of proteins, production of nonessential metabolites can waste carbon and energy that might otherwise be directed towards the protein product, for example the formation of acetate and acetic acid during aerobic growth of E. coli on glucose. Two carbon sources (glucose and glycerol) were compared for cell growth and IGF-1 production of recombinant E. coli (Yoon et al., 1997), glycerol was found to be a better carbon source than glucose for IGF-1 expression in high-cell density culture. Besides, in comparison with different carbon sources in the medium, Liu et al. (2000) found that the specific levels of PGA (penicillin G Acylase) activity obtained in the glucose medium were the lowest, which were likely due to the catabolic repression. Lower rate of transport for glycerol into the cell, compared with the glucose, caused a reduction in the flux of carbon through glycolysis, which greatly reduces the acetate formation (Lee, 1996). Addition of polyols such as glycerol, mannitol and sorbitol improved the production of active recombinant CGTase expressed in E. coli (Kim et al., 1999). Nevertheless, sucrose (X8) instead of glucose was selected as one of the significant carbon sources for the new medium as glucose was screened out in the very first manually screening stage. There have been several attempts to introduce cheap carbon source especially sucrose substrates such as beet and cane molasses in order to obtain the protein produced by recombinant E. coli more economically (Lee and Chang, 1993). The use of sucrose or any other non-metabolizing sugars were found to prevent the aggregation of β-lactamase and thus improved the production of this recombinant enzyme in E. coli (Gowden and Georgiou, 1988). In this study, two carbon sources: glycerol (X5) and sucrose (X8) have significant influence towards the production of extracellular recombinant CGTase based on the screening results.
Bioreactor scale production of extracellular recombinant CGTase: In
the study, the production scheme could be scaled up and same expression level
could be obtained as the shake flask scale and the batch fermentation was performed
in 2 L bioreactor using the optimized medium. As shown in Fig.
7, the pH of the culture was maintained between 7.21-7.08 using the optimum
medium, while the pH of the culture dropped from 7.14-6.51 using the original
medium. The observation confirms that the new potassium phosphate buffer solution
maintained the culture pH along the fermentation hour better than the original
medium. Besides, with the optimized medium concentration, the cell density was
increased successfully in the shake flask scale (Fig. 7) to
an optical density reading 9.56 while by using the original medium (Lo
et al., 2007), the highest cell density observed was less than 8.
However, the cell density decreased when the cultivation was performed in the
bioreactor scale (Fig. 7), as well as the expression level
(Fig. 8). Based on Fig. 8, 18.11 kU mL-1
enzyme activity was observed during the shake flask scale at 36 h post induction
time. However, only 12.94 kU mL-1 enzyme activity was observed at
the bioreactor scale and as much as 28.5% of product yield reduction was obtained.
| Fig. 7: | Comparisons for the pH profile (between the new and original medium) and the cell density profile (between the shake flask scale and the bioreactor scale) along the fermentation time |
| Fig. 8: | Comparisons for the production yield (between the shake flask scale and the bioreactor scale) along the fermentation time (EA: Enzyme activity) |
This production yield appears to be largely reduced and the results are similar with the initial scaling-up study reported by Chen et al. (2007). Notably, the product formation rate was affected by the scale-up scheme for the expression level of the extracellular recombinant CGTase and the decrement might be due to the changes on scale-up process such as oxygen transfer rate, heat transfer surface-to-volume ratio, quality of mixing, shear and superficial air velocity (Humphrey, 1998).
CONCLUSION
Medium optimization was successfully carried out by using the Fractional Factorial Design (FFD) and small Central Composite Design (SCCD) which is a Response Surface Methodology (RSM). The new medium consisted of 12 g L-1 NZ Amine A, 24 g L-1 yeast extract, 9.44 g L-1 KH2PO4, 4.4 g L-1 K2HPO4, 4.58 mL L-1 glycerol, 70 mg L-1 sucrose and 3 mg L-1 CuCl2. A predicted 14.78 kU mL-1 extracellular recombinant CGTase was obtained. After an experimental verification, an activity of 16.07 kU mL-1 was attained. The production of extracellular recombinant CGTase increased nearly 1.68-fold when the new medium was utilized.