Optimized Production of Active α-Glucosidase by Recombinant Escherichia coli. Evaluation of...

Optimized Production of Active r-Glucosidase by RecombinantEscherichia coli. Evaluation of Processes Using in Vivo Reactivationfrom Inclusion Bodies

Ha Le Thanh and Frank Hoffmann*

Department of Biochemistry/Biotechnology, Institute for Biotechnology, Martin-Luther-UniversityHalle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany

During fast production in recombinant Escherichia coli, the enzyme R-glucosidase fromSaccharomyces cerevisiae accumulates partially as inclusion bodies. The inclusionbodies are reactivated inside the cell upon temperature downshift. This in vivoreactivation was most efficient on complex medium with inclusion body production at42 °C and reactivation at 30 °C, yielding volumetric activities 85% higher than thoseof extended isothermal production at low temperature. On defined medium, however,in vivo reactivation was slow. In fed-batch cultivations, feeding controls the specificgrowth rate independent of the temperature. Here, high growth rates fostered inclusionbody formation even at low temperature. Intermediate growth rates permittedaccumulation of active R-glucosidase without affecting the total amount of R-glucosi-dase. Low growth rates yielded similar activities and additionally prevented inclusionbody formation. Moreover, high growth rates during production forestalled subsequentin vivo reactivation. Accumulation of activity after temperature reduction was possiblewith intermediate growth rates. The best time for temperature shift was concomitantto induction. Thus, in fed-batch culture, isothermal production at 30 °C and with aset growth rate of 0.12 h-1 controlled by feeding was most efficient for production ofactive R-glucosidase. Compared to production under optimal conditions on complexmedium, the specific and volumetric activities obtained were 3 and 45 times higher,respectively.

IntroductionThe specific growth rate is an important parameter to

optimize the production of active protein with recombi-nant Escherichia coli. Fast growth during production (1-3) or even before induction (4-6) accelerates recombinantprotein synthesis. With some systems, however, highgrowth rates deteriorate product accumulation (7-9 andadditional references in ref 10). Possible reasons includelower plasmid content (7, 11, 12) and a higher tendencyto form protein isoforms (13). Moreover, physiologicalchallenges may lead to induction of stress responses (14).Interestingly, optimum feeding profiles can include de-creasing feeding rates (15), and production can even beoptimum in the absence of further growth in quiescentcells (16). On the other hand, cells cultured at low ratemay become more fragile and lyse upon induction; thisdecreases the number of colony forming units (17, 18).Thus, the optimum growth rates depend on numerousproperties of the system under study, and the productactivity can show a sharp maximum with respect togrowth rate (1, 9, 10, 12, 19, 20).

Protein aggregation results in the formation of inclu-sion bodies. How growth rate influences the distributioninto active and aggregated product in bioreactor cultiva-tions has rarely been accounted for (20, 21). Growth-limiting feeding can improve folding compared to batch

cultures (21), but very low growth rates seem to impairfolding and intensify aggregation (20). In shake flasks,however, slow growth under unfavorable physical condi-tions can prevent inclusion body formation (e.g., 22), buton the other hand fast growth on complex medium canreduce aggregation compared to production on definedmedium (21). Another prime factor that promotes ag-gregation is high temperature (23). Temperature affectsthe stability of partially structured folding intermediates,favoring off-pathway intermolecular interactions (24).Additionally, temperature influences growth rate in batchor shake flask cultures.

Inclusion bodies reduce product degradation and fa-cilitate separation of the product from cellular proteins;hence they are welcome if in vitro refolding is possible(25). Inclusion bodies can also be disaggregated insidethe cell upon arrest of protein synthesis and reductionof the temperature (26). Proteins released from ag-gregates can be prone to reaggregation or degradation(27-30), but they also have been found to reach theiractive conformation (26, 31). Although proposed forapplication for some time (32), processes using in vivoreactivation have not been optimized so far, and espe-cially no reports about processes in reactor scale areavailable.

The enzyme R-glucosidase from Saccharomyces cerevi-siae principally is able to fold properly inside recombinantE. coli under conditions that prevent growth but other-wise accumulates as inclusion bodies (22). To reach arefolding yield of 40% in vitro, addition of small heat-

* To whom correspondence should be addressed. Tel:+49-345-552-4934. Fax: +49-345-552-7013. E-mail: [email protected].

1053Biotechnol. Prog. 2005, 21, 1053−1061

10.1021/bp050045+ CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical EngineersPublished on Web 06/15/2005

shock proteins or detergent is necessary (33, 34). Henceimproving the production of active R-glucosidase directlyin the cell is desirable.

In this study, cultivation parameters for production ofactive R-glucosidase are optimized. The applicability ofin vivo reactivation of inclusion bodies as a productionprocess is evaluated in comparison to direct productionof active protein in shake flask and bioreactor scale.

Material and MethodsStrain and Plasmids. Escherichia coli MC4100

araD139 ∆(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1ptsF25 rbsR was transformed with the plasmids pKK177-3/GLUCP1 encoding yeast R-glucosidase under the con-trol of the tac promoter (22) and pUBS520 encoding theminor argU tRNA and carrying the lacI repressor gene(35), which improves production of R-glucosidase (36).

Shake Flask Experiments. Shake flask cultureswere incubated on 200 mL of Luria-Bertani (LB) medium,supplemented with appropriate antibiotics (at final con-centrations of 100 µg mL-1 ampicilline and 50 µg mL-1

chloramphenicol), in 1000 mL shake flasks on a rotaryshaker at 140 rpm with a radius of 2.5 cm and 37 °C toan OD600 of 0.5, induced with 1 mM IPTG and transferredto different temperatures as indicated in the resultssection. For in vivo reactivation, the temperature shiftwas delayed as indicated in Results. When indicated,tetracycline was added to a final concentration of 25 µgmL-1 to arrest protein synthesis before the temperatureshift.

Culture Conditions. Cultivations in the bioreactorBIOSTAT C (B. Braun Biotech, Melsungen) with aworking volume of 10 L were performed on glucosemineral salt medium (37) supplemented with appropriateantibiotics as above, with an initial volume of 8 L.Physical parameters were controlled at 37 °C, above pH6.8, and 40% saturation of dissolved oxygen. The initialglucose concentration was 30 g L-1 for batch cultivationand usually 5 g L-1 for fed-batch cultivation. Precultureswere grown overnight on defined medium with 20 g L-1

glucose and were used to inoculate the bioreactor at 5%(v/v). With fed-batch cultivation, a glucose feeding startedautomatically upon depletion of glucose, detected by asudden decrease of stirrer speed due to reduced oxygenuptake. The glucose feeding rate increased exponentiallywith time to enable growth with constant rate µset, if µ0) µset (eq 1). As R-glucosidase production was incompat-ible with a constant growth rate above 0.12 h-1, higherinitial growth rates were achieved by using higher µ0values. The actual growth rate during the feeding phasedeclined from µ0 to µset. The feeding rate is defined by

where F is the feeding rate in g h-1; Xf, Vf, and tf are celldensity, culture volume, and cultivation time at feedingstart in g L-1, g L-1, and h, respectively; SF is the glucoseconcentration in the feeding solution in g g-1; YX/S is thebiomass yield coefficient in g g-1; t is the cultivation time;and µ0 and µset are the initial and set growth rate,respectively, in h-1.

The expression of R-glucosidase was induced by addi-tion of 0.4 mM IPTG 1 h after the feeding start.Temperature was subsequently reduced as described inthe results section.

Analytical Methods. The optical density of the cul-ture, OD600, was determined at a wavelength of 600 nm

in samples appropriately diluted with 0.9% (w/v) NaCl.For cell dry weight determination (CDW), 2 mL of culturesuspension was transferred into a preweighed Eppendorftube and was centrifuged for 3 min at 13 000 rpm and 4°C. The pellet was washed once with 0.9% (w/v) NaCland was dried at 60 °C under ambient pressure until theweight was constant (at least 24 h). CDW was determinedin quadruplicate.

For SDS-PAGE analysis, cell pellets resuspended inphosphate buffer pH 7 were incubated on ice with 33 mgL-1 lysozyme for 30 min and disrupted by sonication for20 s. Soluble and insoluble fractions were separated bycentrifugation for 20 min at 13 000 rpm. The insolublefractions were washed with phosphate buffer twice.SDS-PAGE analysis was performed according to themethod of Laemmli (38). Levels of R-glucosidase wereestimated by densitometry of Coomassie-stained gels.

R-Glucosidase activity was measured as describedpreviously (39). The resulting volumetric activities areexpressed as units of R-glucosidase per milliliter ofculture. For the calculation of specific activities, theprotein concentration in the soluble cell fraction wasdetermined according to Bradford (40) with bovine serumalbumin as standard. Specific activities are expressed asunits of R-glucosidase per milligram of total solubleprotein (U mg-1).

Results

Production of R-Glucosidase in Shake Flask Cul-tures on Complex Medium. R-Glucosidase was pro-duced in shake flask cultures with recombinant E. colion complex medium. Higher temperatures acceleratedgrowth and product accumulation and also resulted inenhanced final R-glucosidase concentrations in the rangeof 24-37 °C (data not shown). Above this range, thegrowth rate and the initially high production rate rapidlydeclined 2 h after induction, giving at 42 °C final totalproduct levels 40% lower than at 37 °C. The R-glucosidaseactivity and concentration of soluble R-glucosidase, how-ever, were highest at 24 °C (22; Figure 1 and data notshown); nearly no increases above preinduction activitieswere found at 37 °C or above. The beneficial effect of lowtemperature has been attributed to the ensuing slowgrowth, as production at extreme pH values also in-creases the specific R-glucosidase activity (22; Figure 1,open symbols). The gain of specific activity was compro-mised, however, by loss in biomass formation, leaving thevolumetric activities unchanged (Figure 1B). With opti-mum production conditions, a specific activity of 3 U mg-1

or, starting with a culture of OD600 0.5, a volumetricactivity of 0.7 U mL-1 was obtained after 4 h. Thus, toachieve useful volumetric quantities, production condi-tions that support further growth are desirable.

At 37 °C, slower growth at a low pH value could hardlyimprove the specific activity (Figure 1A), thus the directimpairment by higher temperature is the predominantimpact on R-glucosidase folding. Other means are neededto improve the productivity of the process.

In Vivo Reactivation of r-Glucosidase InclusionBodies. R-Glucosidase inclusion bodies were producedin shake flask cultures on complex medium at 37 °C.After 2 h, about 50 mg g-1 of the recombinant proteinaccumulated, but less than 10% was found in the solublecell fraction (data not shown). Subsequently, the ag-gregated protein was reactivated inside the living cell.For this in vivo reactivation, tetracycline was added toarrest protein synthesis, and the culture was transferredto 24 °C. This initiated disaggregation of inclusion bodies

F )XfVf

SF‚

µ0

YX/S‚ exp[µset(t - tf)] (1)

1054 Biotechnol. Prog., 2005, Vol. 21, No. 4

and in vivo reactivation of R-glucosidase. While thespecific activity increased 4 times within 3 h after arrestof protein synthesis and reached 2 U mg-1, the volumetricyield was compromised by the lack of further culturegrowth (Figure 2).

To initiate reactivation by stopping selectively thesynthesis of the recombinant protein, the inducer IPTGwas removed 2 h after induction by washing centrifugedcells with medium twice under aseptic condition at 4 °C.No changes in the R-glucosidase concentration wasobserved during the treatment, excluding degradation ofinclusion bodies (data not shown). The cells were resus-pended in fresh medium and cultivated further at 24 °C.The culture growth continued afterward. The increaseof the specific activity, however, leveled off soon, as theproduct is diluted by cell growth (Figure 2). Thus, it ispossible to initiate reactivation by removal of the inducer,and the volumetric activity was improved slightly.

Alternatively, reactivation was initiated by a merereduction of the cultivation temperature. This way, notonly was continuous growth achieved, but also thespecific activity increased faster than after arrest ofprotein synthesis (Figure 2A), probably due to a contribu-tion by de novo synthesis of active R-glucosidase. Thevolumetric activity obtained was 2.4 times higher thanwith reactivation mediated by arrest of protein synthesis.Thus, a mere temperature shift was the most efficientway to trigger in vivo reactivation of R-glucosidaseinclusion bodies.

Temperature Profiles for in Vivo Reactivation.The effect of the temperature profile on the reactivationprocess was examined. Higher specific R-glucosidaseactivities were obtained during reactivation at 24 °C thanat 30 °C, but as growth was slower by about the samefactor, the reactivation temperature did not change theprofile of the volumetric activity (Figure 3), in accordancewith the impact of temperature on R-glucosidase activityduring isothermal production (Figure 1). A higher tem-

perature of 42 °C during production imposed a moresevere stress on the cells, as is seen by the slow growth,but on the other hand improved the subsequent reactiva-tion after temperature reduction: 2-fold higher specificactivities were obtained after production at 42 °C thanafter production at 37 °C (Figure 3). Using this temper-ature profile, the volumetric R-glucosidase activity ob-tained after 2 h production plus 3 h of reactivation was85% higher than that obtained by continuous productionfor 5 h under optimum conditions (Figure 3). Thus,combined de novo synthesis of active R-glucosidase plusin vivo reactivation by temperature downshift are moreefficient then the isolated processes.

Impact of Medium Composition on in Vivo Re-activation. In vivo reactivation was examined on definedglucose mineral salt medium, which is preferred for thedevelopment of a process on bioreactor scale. While theinclusion bodies can be resolved after arrest of proteinsynthesis upon overnight incubation both on defined andcomplex medium, the disaggregation was significantlyretarded on defined medium (Figure 4). Consequently,the specific activity, which amounted to 0.25 U mg-1 after2 h production with both media, increased to 3 U mg-1

within 6 h of reactivation on complex medium butreached only 1.5 U mg-1 during this time span on definedmedium.

Figure 1. Influence of production temperature on R-glucosidaseactivity. Shake flask cultures of E. coli MC4100:pKK177-3/GLUCP1:pUBS520 were incubated in Luria-Bertani (LB) me-dium at 37 °C at pH 7 (solid symbols) or pH 5 (open symbols) toan OD600 of 0.5, then induced with 1 mM IPTG and shifted tovarious temperatures ([, 24 °C; 9, 30 °C; 2, 37 °C; b, 42 °C).Time is given relative to the time of induction. (A) SpecificR-glucosidase activity, in units per milligram of total solubleprotein, (B) volumetric R-glucosidase activity, in units permilliliter of culture.

Figure 2. Method to initiate in vivo reactivation of inclusionbodies. Shake flask cultures were incubated at 37 °C in LBmedium to an OD600 of 0.5, and then R-glucosidase productionwas induced by addition of IPTG to a final concentration of 1mM. Two hours later, the reactivation of R-glucosidase frominclusion bodies was initiated by a transfer of the culture to 24°C directly (b) or with prior arrest of protein synthesis with 25µg mL-1 tetracycline (2) or with prior washing of the culturetwice with fresh medium to remove IPTG (9). (A) SpecificR-glucosidase activity, (B) volumetric R-glucosidase activity, (C)optical density OD600 of the culture. Incubation time is givenrelative to time of temperature shift.

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Production of r-Glucosidase on Defined Mediumin Fed-Batch Cultivation. R-Glucosidase productionwas examined under controlled conditions in the biore-actor. In fed-batch cultivations, growth rates can beadjusted independent of the temperature by growth-limiting glucose feeding. Biomass was provided duringan initial batch phase at 37 °C; maximum growth rateand biomass yield coefficient were µmax ) 0.73 ( 0.02 h-1

and YX/S ) 0.50 ( 0.05 g g-1, respectively. Upon depletionof initially supplied glucose, the feeding started. Thespecific growth rate was feed-forward controlled to dif-ferent values. One hour after start of feeding, R-glucosi-dase synthesis was induced by addition of IPTG, and thecultivation temperature was reduced to 30 °C or keptconstant at 37 °C (Figure 5).

Specific growth rates µset higher than 0.12 h-1 cannotbe constantly maintained for longer times because of theinterference of R-glucosidase production with glucoseuptake (44), resulting in accumulation of glucose in themedium. To evaluate nevertheless the impact of highergrowth rates on R-glucosidase production, an initialglucose feeding rate corresponding to µ0 ) 0.2 h-1 wascombined with an exponential increase with µset ) 0.12h-1 as detailed in Material and Methods. This way,glucose was limiting throughout the production period,and no glucose accumulated up to 10 h after induction(data not shown).

In all cultivations, the R-glucosidase concentrationincreased linearly with a constant rate of about 10 mg

g-1 and 7 h after induction reached a level of 70 mg g-1.Neither growth rate nor temperature changed the ac-cumulation rate significantly (Figure 5D). Both param-eters, however, determined the distribution of the productin soluble and insoluble cell fraction.

With the initially fast feeding, the specific growth ratedeclined from 0.2 to 0.12 h-1 as set by the feeding rate(Figure 5A). Coincidentally, R-glucosidase initially ac-cumulated exclusively in the insoluble cell fraction evenat 30 °C but after the decline of the growth rate wasproduced also in active form (Figure 5B, E, F). With aconstant growth rate of µset ) 0.12 h-1, the actual specificgrowth rates matched the set value (Figure 5A), andR-glucosidase was produced in active form from thebeginning. Initially fast growth hence prevented ac-cumulation of soluble R-glucosidase. With a slower feed-ing rate corresponding to µset ) 0.06 h-1, the profiles forspecific activities were identical as with µset ) 0.12 h-1

(Figure 5B). The formation of inclusion bodies, however,was nearly completely avoided, and the total amount ofR-glucosidase was 60% lower (Figure 5D, F).

Temperature influenced the sustainability of produc-tion in active form. Active R-glucosidase could be pro-duced at 37 °C, but the specific activity already reacheda low constant level 3 h after induction (Figure 5B). Witha cultivation temperature of 30 °C, in contrast, theaccumulation of soluble, active R-glucosidase continuedunperturbed during the whole cultivation (Figure 5B, E).Decreasing the temperature further to 24 °C did not, incontrast to the shake flask experiments, change thespecific activity, as the growth rate was controlled by thefeeding rate (Figure 5B). Thus, specific growth rate andtemperature exert a strong and interdependent influenceon accumulation of active R-glucosidase; the specificactivities were highest with a growth rate µset e 0.12 h-1

and a temperature at or below 30 °C. Hence the optimum

Figure 3. Optimization of temperature profiles for in vivoreactivation. Shake flask cultures were incubated at 37 °C inLB medium to an OD600 of 0.5, induced by IPTG, and incubatedfurther at 37 ([, 9) or 42 °C (b). Two hours later, the cultureswere transferred to 24 ([) or 30 °C (9, b) for reactivation ofR-glucosidase. The time of temperature shift is indicated by anarrow. For comparison, profiles are shown for isothermalproduction at 24 °C (]). (A) Specific R-glucosidase activity, (B)volumetric R-glucosidase activity, (C) optical density OD600.Incubation time is given relative to time of induction.

Figure 4. Impact of medium on in vivo reactivation. Shakeflask cultures were grown on LB medium (solid symbols) or onglucose-mineral salt medium (open symbols) at 37 °C to OD600of 0.5 and induced with 1 mM IPTG. Two hours after induction,cultures were transferred to 30 °C after arrest of proteinsynthesis by addition of tetracycline. Time is given relative totime of induction. (A) Specific R-glucosidase activity, (B) specificR-glucosidase concentration in the insoluble cell fraction deter-mined from Coomassie stained gels by densitometry.


volumetric activities were obtained at 30 °C with µset )0.12 h-1 (Figure 5C).

Bioreactor Processes for in Vivo Reactivation.For in vivo reactivation in the bioreactor, R-glucosidaseinclusion bodies were produced for 2 h in batch or fed-batch mode, under conditions that prevent accumulationof active R-glucosidase. In batch culture, cells grewinitially with a specific rate of µmax ) 0.56 h-1 during theproduction phase at 37 °C. The specific production rateqP declined from nearly 60 mg g-1 h-1 exponentially witha time constant of 0.8 h, and the product concentrationreached a stationary level of 30 mg g-1 within 2 h (Figure6A). Thus, even without addition of an antibiotic, produc-tion is not sustained during fast production on definedmedium.

In fed-batch culture, the growth rate declined from µ) 0.2 to 0.12 h-1 as set by the glucose feeding rate. Theproduction initially was 3 times slower than in the batchprocess, but the exponential decay time was 7 timeslonger, enabling sustained production throughout thecultivation (Figure 6A).

Two hours after induction the temperature was re-duced to 24 °C to initiate reactivation. At this time, theconcentrations of R-glucosidase happen to be the samein both processes. The batch culture had difficultiesadapting properly to the low temperature, growing with

only µ ) 0.05 h-1 after temperature reduction (Figure6C). At the same time, essentially no accumulation ofactive R-glucosidase took place (Figure 6B). Thus, inaddition to the slow reactivation seen on defined mediumwith tetracycline addition (Figure 4), also no de novosynthesis of R-glucosidase was observed.

With the fed-batch process, a growth rate of 0.09 h-1

was maintained and a specific R-glucosidase activity of1.8 U mg-1 accumulated within 2 h (Figure 6B).

Using the same feeding profile and a reactivationtemperature of 30 °C, the specific activity increased moreslowly than at 24 °C (Figure 7), but as a growth rate ofµ ) 0.12 h-1 was maintained, the volumetric activity wascomparable at both temperatures. With a constantgrowth rate µset ) 0.12 h-1 from the beginning, a specificR-glucosidase activity of more than 1 U mg-1 accumu-lated already during the production phase at 37 °C. Afterthe temperature shift to 30 °C, the slope of the specificactivity was 3 times as high as with the fast feeding atthe same temperature, giving after 3 h a yield of 5 U mg-1

(Figure 7). With µset ) 0.12 h-1 and a production tem-perature of 42 °C, growth stopped after induction andless than 20 mg g-1 R-glucosidase accumulated within 2h. After the temperature shift to 30 °C, R-glucosidaseactivity was detected only after a lag phase of 3 h, giving3.5 U mg-1 after 8 h of reactivation (data not shown).

Figure 5. Cumulative impact of temperature and growth rate controlled by limiting glucose feed on R-glucosidase accumulationand activity. E. coli MC4100:pKK177-3/GLUCP1:pUBS520 was cultivated in a 10-L bioreactor on glucose mineral salt medium at 37°C. After depletion of glucose, a glucose feeding was started with a rate increasing exponentially with time (eq 1 in Materials andMethods) to enable growth with a specific rate of µset ) 0.06 h-1 (down triangles) and 0.12 h-1 (up triangles). Alternatively, thefeeding was started with a rate corresponding to µ0 ) 0.2 h-1 and increased exponentially corresponding to µset ) 0.12 h-1 (eq 1,squares). One hour after feeding start, production of R-glucosidase was induced by addition of IPTG to a final concentration of 0.4mM and the cultivation temperature was kept at 37 °C (solid symbols) or reduced to 30 °C (open symbols) or 24 °C (open symbols,dotted lines). Time is given relative to the time of induction. (A) Cell density X from dry mass measurement, relative to the celldensity at induction, Xind (3.2-6.5 g L-1); (B) specific R-glucosidase activity; (C) volumetric R-glucosidase activity is given relative tothe cell density at induction Xind, to enable comparison of the performance in cultures with different Xind; and specific R-glucosidaseconcentrations per grams cell dry weight, estimated by densitometry of Coomassie-stained SDS-PAGE gels of (D) total cell protein,(E) soluble cell fraction. and (F) insoluble cell fraction, separated by centrifugation for 20 min at 13 000 rpm after cell disruption.


Thus, although defined medium is less appropriate forin vivo reactivation than complex medium, a reasonableamount of active R-glucosidase could be produced by thetemperature-downshift protocol in fed-batch mode withµset ) 0.12 h-1.

Optimum Time of Temperature Shift. To testwhether higher concentrations of inclusion bodies assubstrate for reactivation can enhance the reactivationyield, the best time point for the temperature shift wasdetermined. In fed-batch cultivation with a feeding ratecorresponding to µset ) 0.12 h-1, R-glucosidase wasproduced for 2, 4, or 7 h at 37 °C before the temperaturedownshift to 30 °C. This is compared to a cultivation withisothermal production, i.e., after the growth phase at 37°C, the temperature was reduced to 30 °C directly uponinduction (Figure 8). As before, the profiles of cell densityand R-glucosidase concentration approximately super-imposed (data not shown). Upon temperature shift to 30°C, the R-glucosidase activity increased with similarslopes independent of the time of temperature shift.Stationary values were reached 8-10 h after inductionin all cases, in agreement with the total concentration ofR-glucosidase (Figure 8). Hence the earlier the temper-ature was reduced, the higher the final levels of activeR-glucosidase obtained. With isothermal production at 30°C, the specific and volumetric activities reached 9 Umg-1 and 33 U mL-1, respectively.

Comparison of the Process Scheme. While directproduction of R-glucosidase in active form is possible in

shake flask cultivation on complex medium at lowtemperature, the specific activity is low and growth isretarded under these conditions, leading under optimumconditions to a volumetric R-glucosidase activity of 0.75U mL-1 (Table 1). In contrast, production of inclusionbodies at high temperature, which enables fast growth,combined with in vivo reactivation of inclusion bodies atlow temperature yielded higher activities: the specificactivity and OD600 were 40% and 70% higher than withisothermal production, resulting in duplication of thevolumetric activity (Table 1). In fed-batch cultivation ondefined medium, direct production at low feeding rateswas sustainable and efficient, giving specific activities 3times higher than those obtained with direct productionin shake flasks (Table 1). Moreover, the optical densitywas 20 times higher. This process was used only for proofof principle; for production purposes, the cell density at

Figure 6. Evaluation of bioreactor cultivations for in vivoreactivation of R-glucosidase. Batch cultivation (open symbols;initial glucose concentration of S0 ) 30 g L-1) and fed-batchcultivation (solid symbols; S0 ) 5 g L-1; subsequent feeding withµ0 ) 0.2 h-1 and µset ) 0.12 h-1, cf. eq 1) were performed onglucose mineral salt medium in a 10-L bioreactor. Cultures weregrown at 37 °C and were induced with 0.4 mM IPTG 4 h afterinoculation or 1 h after feeding start, respectively. Two hoursafter induction, the temperature was shifted to 24 °C to initiatereactivation of inclusion bodies (indicated by dashed lines). (A)Specific concentration c of R-glucosidase determined from Coo-massie stained gels by densitometry (b, O) and specific productformation rate qP (9, 0) calculated as qP ) d(cX)/dt/X, (B) specificR-glucosidase activity, (C) cell density X relative to the celldensities at the time of induction, Xind (2 g L-1 for batch and4.3 g L-1 for fed-batch cultivation). Time is given relative to timeof induction.

Figure 7. Impact of culture growth rate on in vivo reactivationof R-glucosidase. Inclusion bodies of R-glucosidase were producedin fed-batch cultures in a 10-L bioreactor with an initial growthrate of µ0 ) 0.2 h-1 and an exponential increase of µset ) 0.12h-1 (2) and with µset ) 0.12 h-1 throughout the feeding phase(b). Reactivation was initiated 4 h after induction by temper-ature shift to 30 °C (indicated by dotted line). (A) SpecificR-glucosidase activity, (B) cell density X relative to the celldensities at the time of induction, Xind (5.2 and 6.6 g L-1,respectively). Time is given relative to the time of induction.

Figure 8. Optimum time of temperature shift to initiate in vivoreactivation. Fed-batch cultures with a feeding rate enablinggrowth with µset ) 0.12 h-1 were incubated at 37 °C in a 10-Lbioreactor and induced 1 h after feeding start with 0.4 mMIPTG. The temperature was reduced to 30 °C at different timesafter induction, i.e., 2 h (9), 4 h (2) or 7 h ([). This is comparedto direct production under optimum condition (i.e., temperaturereduction to 30 °C immediately before induction, O). SpecificR-glucosidase activity was determined as above. Time is givenrelative to the time of induction. Arrows indicate times oftemperature downshift.


induction could be easily increased further using higherglucose concentrations for the batch phase (e.g., 30 g L-1

instead of 5 g L-1 used here), and by longer feedingphases before induction (instead of 1 h as used here),further increasing the benefit from fed-batch cultivation.However, even with this short process, the volumetricactivity was increased 45 times (Table 1).

Discussion

Optimum Process Conditions for Production ofActive r-Glucosidase. Production of R-glucosidase dur-ing rapid growth challenges the cells with severe stress(41-44), which in batch culture reduced the growth rateand impaired adaptation upon temperature changes(Figure 6). In fed-batch cultivation with slower growthrate, in contrast, continuous growth and production forat least 7 h after induction were achieved (Figure 5A,B). Process conditions that result in moderate synthesisrates can generally enhance the sustainability of proteinproduction (17, 45, 46). Moreover, slower growth in-creased the specific activity of R-glucosidase. With a setgrowth rate of µset ) 0.12 h-1, the specific activityobtained was twice as high as that with an initial growthrate of µ ) 0.2 h-1 and 3 times higher than that of thebest protocol on complex medium. An even lower growthrate of 0.06 h-1 did not ameliorate the specific activityfurther but efficiently prevented formation of R-glucosi-dase inclusion bodies. Thus three states can be distin-guished, going from very slow to higher growth rates: (i)exclusive accumulation in active conformation, (ii) samespecific R-glucosidase activity plus formation of inclusionbodies, (iii) constant concentration of total R-glucosidasebut low activity as the strong production interferes withcell physiology and proper protein folding.

Temperature analogously influenced production inshake flask cultures, resulting in faster production butlower specific activities at higher temperatures. As thevery rapid production at 42 °C terminated within the first2 h after induction, highest R-glucosidase concentrationsin shake flask experiments were obtained at 37 °C.Additionally, high temperatures interfere with proteinfolding (23, 24). Consequently, the benefit from usinglower growth rates was at 37 °C much less pronouncedthan at 30 °C (Figures 1 and 5), and R-glucosidaseaccumulated mainly as inclusion bodies.

Processes for in Vivo Reactivation of InclusionBodies. Proteins deposited in inclusion bodies can bedisaggregated and reactivated upon arrest of proteinsynthesis (26). Disaggregation and reactivation of R-glu-cosidase upon arrest of protein synthesis were slow ondefined medium, especially after production during un-limited growth. For some proteins, complex medium canreduce formation of inclusion bodies during fast produc-tion (21). Here we show that also reversion of aggregationto yield active protein can be accelerated by complexmedium.

Nevertheless, the specific activity did increase aftertemperature reduction on defined medium in fed-batchcultivation with slow feeding. The slope of the activity

was independent of the time of temperature shift, withoutany advantage from higher concentrations of inclusionbody as substrate for disaggregation. Thus, the activitymay stem from de novo synthesis rather than from invivo reactivation. Moreover, the ability to accumulateR-glucosidase, either soluble or insoluble, deterioratesduring R-glucosidase production despite ongoing culturegrowth. Thus, the final concentration of soluble, activeR-glucosidase was higher the earlier the temperature wasreduced. Optimum R-glucosidase yields, both with respectto specific and volumetric activities, were obtained witha temperature shift from 37 °C, which was used duringthe growth phase, to 30 °C immediately upon induction.They were 3 and 45 times higher, respectively, than thoseobtained under optimal production conditions on complexmedium in shake flasks.

On complex medium, in contrast, in vivo reactivationwas efficient. The most potent trigger was a merereduction of the cultivation temperature. The tempera-ture during the production phase was an importantparameter, and the activity yields were highest afterproduction at 42 °C. Compared to prolonged isothermalproduction at 24 or 30 °C, the in vivo reactivation schemeprovided nearly 2-fold higher volumetric activities. Afterproduction at high temperature, proteolytic degradationof disaggregated R-glucosidase is reduced, and reactiva-tion benefits from higher levels of the disaggregatingchaperones DnaK and ClpB (47). Also the enhanced denovo synthesis that is observed after heat-shock (48, 49)might contribute to the higher yields of active R-glucosi-dase. The reactivation scheme might be especially ap-pealing when using expression system with constrainsin the choice of temperature for production, e.g., with thewidely employed T7-promoter that is essentially re-pressed at 30 °C (4) or with temperature-induciblepromoters. Further improvement seems possible byoverproduction of DnaK and ClpB, the disaggregatingchaperone that can limit the disaggregation rate (50). Invivo reactivation is thus an appropriate method toproduce active protein in small-scale cultures on complexmedium.

Conclusion

The most efficient protocol for R-glucosidase productiondepends on scale and medium used. A growth-limitingfeeding profile triplicates the specific R-glucosidase activ-ity compared to that of shake flask cultures and ad-ditionally improves the volumetric yield by high celldensities. Fed-batch cultivation at 30 °C and with µset )0.12 h-1 was found to be an efficient procedure to produceactive R-glucosidase. Further reduction of the set growthrate is not indicated, as it decreases the volumetricactivity, but might be useful if avoidance of inclusion bodyformation is an issue. Although defined medium is notsuitable for refolding of aggregated R-glucosidase insidethe cell, a scheme for in vivo reactivation of inclusionbodies for provision of active R-glucosidase has beenestablished on complex medium. Compared to isothermalproduction, processes with the appropriate temperature

Table 1. Comparison of Different Approaches for Production of Active r-Glucosidase

direct productiona

(complex medium)in vivo reactivationa

(complex medium)fed-batch cultivationb

(defined medium)

spec activity (U mg-1) 3 4.3 9final OD600 3 5.2 60vol activity (U mL-1) 0.75 1.48 33

a Production in shake flask cultivation for 5 h after induction, OD600 at the time of induction was 0.5. b Production in bioreactor for 18h after induction, OD600 at the time of induction was 10.


profile resulted in nearly 2 times higher volumetricproductivity.

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Note Added after ASAP Publication. There was anerror in Figure 5 in the version published ASAP June15, 2005. The corrected version was published ASAP July5, 2005.

Accepted for publication May 11, 2005.

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