Effect of agitation speed on the synthesis of Mucor miehei acid protease

Jose Escobar and Stanley M. Barnett

Department of Chemical Engineering, University of Rhode Island, Kingston, RI

Different experiments using M u c o r miehe i CBS 370.65 were carried out to study the effect of agitation speed on the production o f the mold acid protease. The experiments were conducted in shake flasks at a fixed substrate concentration o f 58 g l-1 of total carbohydrates and at shaker speeds from 80 to 380 rev min -t. Enzyme production was found to be directly proportional to the shaker speeds, with the highest concentration o f enzyme o f 1,400 Soxhlet Rennet units (SU) ml -t obtained at 380 rev min -1. The yield o f product to substrate at 380 rev min -t was determined to be 27,081.0 SU g- t substrate and the productivity of the process was 221 SU g-I h-l . Enzyme production was partially growth associated, and glucose supported both cell growth and enzyme production. Product formation and cell concentration were directly related to the rate of substrate consumption. The rate o f product formation decreased when product started to accumulate, suggesting that the process was affected by feedback repression.

Keywords: Rennet; acid protease; rate of substrate consumption; Mucor miehei

Introduction

Rennet is the extract obtained from the fourth stomach, or abomasum, of milk-fed calves. It contains mainly an acid protease called chymosin or rennin (E.C. 3.4.23.4). 1 In the last 10 to 20 years, there has been a shortage of true calf rennet on world markets, so it has been partially replaced by alternative milk coagulating enzymes of different origin. 1 The protease (E.C. 3.4.23.10) of one mold in particular, Mucor miehei, has generally been preferred as a substitute for true calf rennet because of its specificity in splitting similar pep- tide bonds in kappa-casein ,2 high ratios of milk-clotting activity to proteolytic activity, 1,3 similar calcium re- quirements, 4 and good cheese quality. 1,3,5

It has been reported that in a defined medium, a protein source must be present for the enzyme to be produced. 6 The use of casein (at least 0.08% w/v) in- duced higher enzyme yields. It was also noticed that the lack of glucose resulted in a dramatic decrease in enzyme production. However, no enzyme activity was

Address reprint requests to Dr. Barnett at the Department of Chemi- cal Engineering, University of Rhode Island, Kingston, RI 02881 Received 9 September 1992; revised 21 April 1993

observed when the glucose concentration was 160 g l - 1 or above. 6

Little work has been published about the variables that control the production of the acid protease of M. miehei. It is not known how cell concentration and substrate consumption affect enzyme production. The rate of substrate consumption and its relationship to the previous variables are fundamental to determine the kinetics of the fermentation. Kinetic equations are basic for bioreactor design and optimization. The rate at which the substrate is consumed is represented by r A. If the fermentation rate is limited by a single nutrient, a simple equation that relates r A to cell concentration Cc and substrate concentration C A is the Monod equation. 7 However, in many cases the Monod equation is not applicable, particularly at the end or at the beginning of a batch fermentation or when the system is mass transfer-limited, because in such a case rn will be de- pendent on mass transfer rates or degree of agitation. 7

Another important factor is the repression of prod- uct formation either by other metabolites or by the main product itself. Kinetically speaking, this behavior is found when the rate of product formation decreases when the product concentration increases. For this particular case, the Monod equation has to be modified to take into account the concentration of the repressor. 7

© 1993 Butterworth-Heinemann Enzyme Microb. Technol., 1993, vol. 15, December 1009

Papers

Lasure 6 reported that amino acids in concentrations above 1% repressed the production of the M. miehei protease; however, it was not clear if the phenomenon was due to repression or a failure of the induction mechanism.

In accordance with the kinetic relationship between growth and product formation, fermentations are clas- sified as types I, II, and III. 8 These three groups are also known respectively as growth-associated, mixed growth-associated, and non-growth-associated, 9 de- pending on whether the product is directly derived from the substrate used for primary energy metabolism (type I) or produced in a secondary pathway (type II). 8 If product formation and primary metabolism occur at completely separate times, a fermentation of type III will occur. 8 When the specific rates for growth (/~), substrate consumption, and product formation (Qp) fol- low similar or parallel patterns, the fermentation is classified as type I. 8 Growth-associated fermentations are easier to implement in a continuous fashion because they can usually be run in a single reactor. 8 Specific rates are defined as the derivatives versus time of cell, substrate, and product concentration divided by cell concentration. 8,9

The objective of our study was to evaluate the effect of agitation rates on the production of Mucor miehei acid protease. To do so, kinetic parameters such as substrate consumption, rate of substrate consumption, and cell and enzyme concentrations were evaluated against different shaker speeds. The relationship be- tween product and growth was analyzed to determine whether the enzyme is growth-associated or non- growth-associated. The possibility of the enzyme ' s be- ing affected by feedback repression was also studied.

Materials and methods

Microorganisms

The microorganism Mucor miehei CBS 370.65 was used for this research. The cultures were maintained on potato dex- trose agar slants at 4°C. Every 2 months the cultures were transferred to new slants to keep them viable.

Mycelia from the slants were inoculated on potato dex- trose agar in 2-1 Fernbach flasks. The mold was incubated at 37°C. After 3 to 4 days, spore suspensions were prepared by scraping the surface and rinsing the mycelium with 75 ml of sterile distilled water. The spore suspensions were assayed with a hemacytometer and found to be in the range of 1 × 106 to8 × 106 sporesml 1.

Culture medium

A suitable medium to determine cell growth with high enzyme yields was developed for this research. The medium composi- tion was as follows: glucose monohydrate, 1.80%; soluble starch, 1.80%; malt extract, 3.10%; Bactopeptone 0.8%; ca- sein, 0.80%; KH2PO 4, 0.20%; the remaining percentage was distilled water. All chemicals employed were reagent grade.

Experiments with flasks and fermenter

Cell growth, enzyme production, glucose, carbohydrate con- centration, and pH were analyzed with respect to time. Shake

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Figure 1 M. miehei acid protease production. Shake flasks at 380 rev min -1. (+) Cell weight; ([E]) total carbohydrates; (x) en- zyme concentration; ( I ) pH

flasks were inoculated with the spore suspension volume necessary to obtain concentrations of I x l05 to 2 × 105 spores ml J.10.11

The shake flask fermentation was carried out for 8 days at 37°C. All experiments were done in duplicate. The results stated in this paper are the average of the two values. At least 16 flasks were used for each experiment, and two flasks were withdrawn and assayed daily. The cultures were shaken in 250-ml flasks containing 50 ml media from 80 to 380 rev min-1 using a rotatory-incubator shaker (Lab-line). Growth was determined daily by dry cell weight per volume (g 1 ~). The mycelium was separated and dried at 80°C overnight and then weighed.

Assays

Total carbohydrates

Hydrolyzed starch and glucose accounted for almost all the carbohydrates present in the media. In consequence, total carbohydrates were assumed to be equal to the final glucose concentration (plus a correction factor), after the total hydro- lysis of the sample.~2 Glucose was determined using a YSI Model 23A Glucose Analyzer (Yellow Spring Industries).

Enzyme assay

The enzyme assay was based on the procedure described by Ernstrom 13 and Rand and Ernstrom. 14 An equivalence was established between the rennet units from the former method and Soxhlet rennet units (SU). 2 All results were expressed in Soxhlet rennet units per milliliter (SU ml- 1) unless otherwise specified.

Results and discussion

Initial experiments

Figure 1 is a typical example of the general fermenta- tion curves for the production of M. miehei enzyme. Cell growth increased rapidly in the early stages. En- zyme synthesis started in the first 24 h when nutrient consumption was high. It was found that glucose was

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Synthesis of M. m i e h e i acid protease: J. Escobar and S, M. Barnett

Table 1 Effect of agitation rate on (a) biomass and (b) enzyme Table 2 Effect of agitation rate on (a) substrate consumption production and (b) rate of substrate consumption ( - r a)

(a) Biomass (g 1-1) Agitation rate (rev min -1)

(a) Substrate consumption (g 1-1)

Agitation rate (rev min -1)

Time(h) 80 150 270 380 Time (h) 80 150 270 380

0 0 0 0 0 0 54.7 51.3 60.5 58.0 24 6.1 15.5 7.3 15.7 24 47.5 43.0 38.1 38.8 48 11.0 16.6 10.3 21.8 48 43.9 34.8 24.6 22.8 72 13.2 23.1 16.0 25.1 72 40.3 26.5 11.0 6.8 96 15.8 26.1 21.0 26.4 96 37.9 17.3 7.7 6.5

120 17.5 26.1 23.1 27.4 120 35.6 8.0 4.4 6.3 144 21,5 26.2 20.6 26.4 144 32.7 4.5 4.2 5.6 168 21.0 25.7 20.5 25.5 168 29.8 4.5 4.0 4.9

Maxima 21.5 26.2 23.1 27.4

(b) Enzyme production (SU m1-1)

Agitation rate (rev min -1)

Time (h) 80 150 270 380

(b) Rate of substrate consumption ( - q ) (g I-1 h- l )

Agitation rate (rev min -1)

Time (h) 80 150 270 380

0 0 0 0 0 24 6 70 278 523 0 0.312 0.400 1.072 1.126 48 37.0 271 606 806 24 0.224 0.350 0.775 0.792 72 80 368 915 977 48 0.159 0.310 0.526 0.514 96 126 387 1,121 1,240 72 0.116 0.270 0.323 0.293

120 145 406 740 1,401 96 0.096 0.250 0.168 0.129 144 159 424 774 1,165 120 0.098 0.240 0.059 0.021 168 211 553 451 182 144 0.123 0.140 0.002 0.030

Maxima 211 553 1,121 1,401 168 0.170 0.002 0.016 0.025

the most concentrated carbohydrate (50%) present in the substrate. All carbohydrates were rapidly metabo- lized from the beginning of the bioprocess. There was reduction in the enzyme activity after reaching a maxi- mum (for this particular experiment at 120 h). For all experiments, it was found that a decrease in enzyme

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20

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Figure 2 Dependence of enzyme and biomass concentration on shaker speeds (rev min-1), Profiles at a given time. (B) Enzyme at 96 h; (+ ) enzyme peak values; (x) cell at 96 h; (rq) cell peak values. (- - - ) cells

activity was associated with an increase in pH to values higher than 7.0 (see Figure 1). It is known that the enzyme of M. miehei CBS 370.65 is unstable at pH greater than 6.5,15 and it was also demonstrated that there is total loss of enzyme activity at pH 8.0 after 72 h at 38°C. 15 Based on the previous facts, it is important to maintain pH below 6.5 to minimize losses in the enzyme activity.

Table 1 compares M. miehei biomass production (g l-1) and enzyme concentration (SU ml-1) at different shaker speeds (rev rain- 1). Cell and enzyme peak con- centrations at 80 rev rain -~ were respectively 21.5 g 1-1 and 211 SU m1-1 versus 27.0 g 1-1 and 1400 SU m1-1 at 380 rev rain -1 after 150 h. The previous results indicate that agitation rates strongly influence protease synthesis and, at a lesser degree, cell growth. Figure 2 illustrates the previous point clearly by comparing rennet and cell production profiles at 96 h and peak values as a function of revolutions per minute. Product concentration was strongly affected by shaker speeds at the lower values. The cause of the previous observa- tion seems to be a mass transfer problem, and it may be related to inadequate aeration rates and/or transfer of nutrients to the microorganism.

Rates o f substrate consumption and yields

Substrate consumption and its time derivative (ra) were affected by the shaker revolutions (see Table 2). The

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300 . 1600

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-1 -2 0 168

Figure 3 Comparison of (*) rate of consumption (rA), (+) en- zyme concentration (Cp), and (11) biomass (Cc) versus substrate concentration (CA)

value of r A, also called the rate of substrate consump- tion, was strongly influenced by the rate of agitation and decreased drastically at lower shaker speed. In Figure 3, r A is plotted against enzyme concentration (Cp) and cell concentration (Co) versus substrate con- centration for the experiment at 380 rev min- 1. Prote- ase synthesis and cell concentration were directly re- lated to the rate of consumption, as is clearly seen on Figure 3. Similar curves were obtained at 270 rev min- 1 (not shown). The relationship between product forma- tion and rate for 380 and 270 rev min-1 fits a linear equation very well when substrate is still available (cor- relation R 2 = 0.984). These results emphasize the im- portance of having high rates of substrate consumption (rA) to achieve high protease yields.

Table 3 shows substrate conversion yields to prod- uct (YcP/CA) and to ceils (Ycc/cA), and productivity at different agitation rates. Calculations were performed only for positive growth and enzyme production. As can be seen, yields and productivities were similar above 270 rev rain- 1. At the two lowest agitation rates, the productivity of the process was reduced by the fact that substrate that otherwise would have been used for enzyme production was not being transformed into the protease, particularly at 150 rev min-1 (see also Table 1). At 80 rev min- 1, the microorganism was under such

T a b l e 3 C o n v e r s i o n y ie lds to p roduc t YCP/CA, tO cells YCC/CA, and p r o d u c t i v i t y at d i f f e ren t ag i ta t ion r a t e s

Agitation rate (rev min -1)

80 150 270 380

YCP/CA 8,450 11,960 21,200 27,100 (SU g-1 substrate)

Productivity 50 71 221 225 (SU g-1 h- l)

Ycc/ca 0.979 0.772 0.412 0.534 (g cell g-1 subs.)

I I I I I I 24 48 72 98 120 144

lqrne (hours)

Figure 4 Comparison of relative specific rates for product (Op) and cells (/z) (- - -) versus time. (11) Qp, (+ ) /x at 80 rev min -1. (*) Qp, (n) /x at 270 rev min -1. (x) Qp, (O)/x at 380 rev min -1

mass transfer limiting conditions that mainly cells were produced and protease formation was almost negligi- ble. Besides experimental errors, the previous observa- tion helps to partially explain such high yield of cells from substrate as those shown in Table 3 at 80 rev min- I. The dependence of the rate of consumption on shaker speed, particularly below 270 rev min- 1, makes the Monod equation inadequate to describe these ex- periments.

Other kinetic studies

Relative rates for product and cells were compared on Figure 4, by comparing Qp]Qpmax vs. /Z//Zma x. Protease production is mixed growth-dependent because growth and enzyme production do not always follow similar patterns particularly for Qp at 380 rev min -l. Figure 1 illustrates this point clearly: after cell growth stopped at around 72 h, enzyme production continued for 50 more hours, increasing from 1,000 to 1,400 Ru ml- A similar case may be stated at 80 rev min-~ where enzyme production started almost 24 h after growth began. Hence growth and product formation were only partly linked. These results suggest that Mucor miehei enzyme production takes place in a secondary pathway which is separate from primary metabolism.

From Figure 5, when comparing the rate of product formation (dCp/dt) versus enzyme concentration (Cp), it can be observed how the rate decreases while the enzyme increases. This behavior is typical of feedback repression. This result may suggest that increases in the concentration of the product repress the enzyme synthesis as the product accumulates. A decrease in the substrate available for consumption might explain this behavior too; hence, further studies need to be done to determine if repression affects this fermen- tation.

Conclusions

For the rennet-like enzyme produced by Mucor miehei, it was found that enzyme stability is important to main- tain the fermentation process below pH 6.5. Enzyme

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20

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Figure 5 Comparison of enzyme concentration (Cp) and its de- rivatives (dCn/dt). 0, (Cp); and *, (dCp/dt) at 270 RPM. x, (Cp); and +, (dCp/dt) at 380 RPM

synthesis of M u c o r miehe i CBS 370.65 was found to be dependent on cell and substrate concentrat ion, but particularly on the rate of substrate consumption. Ade- quate mass transfer rates were critical to achieve faster enzyme yields. The relationship between product con- centrat ion and rA could be described adequately by a first-order equation for most of the fermentation pro- cess. However , more studies should be done to deter- mine an equation to express the relationship between the rate of substrate consumption and CA and Cc. The fermentat ion was partially growth-dependent , and glu- cose supported both cell growth and enzyme produc- tion at similar times. It was found that this particular enzyme seemed to be affected by feedback repression because product rates decreased when product started to accumulate.

F rom the different data accumulated in this re- search, there was a strong indication that the addition of suitable carbohydrate-l ike maltose during the period of highest enzyme product ion might increase the yield of the process. This might occur if the process is not affected by feedback repression. If so, the use of a system that integrates product ion and recovery of the

miehei acid protease: J, Escobar and S. M. Barnett

enzyme, such as a membrane bioreactor , might be an interesting possibility to be explored for a continuous fermentation.

N o m e n c l a t u r e

CA Cp c~ /x Qp F A

YCP/CA YCC/CA

concentrat ion of substrate (g 1-1) concentrat ion of product (SU ml-1) concentrat ion of cells or biomass (g 1-l) specific cell growth (h-1) specific rate of product formation (SU-1/ml-g) rate of substrate consumption (g 1- = h - =) substrate conversion yield to product (SU g-~) Substrate conversion yield to cells (g g-1)

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10 Lasure, L. I. and Ingle, M. B. Some effects of temperature on zygospore formation in Mucor miehei. Mycol. 1976, 68, 1145-1151

11 Streets, B. W. and Ingle, M. B. Can. J. Microbiol. 1972, 18, 975-979

12 Dziedzic, S. Z. and Ireland, P. A. Analysis of Food Carbohy- drates (Birch, G. G., ed.). Elsevier Applied Science Publishers, London, 1985, p. 274

13 Erstrom, C. A. Heterogeneity of crystalline rennin. J. Dairy Sci. 1958, 41, 1663-1670

14 Rand, A. G. and Ernstrom, C. A. Effect of pH and NaCI on activation of Prorennin. J. Dairy Sci. 1964, 47, 1181-1187

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