Process Biochemistry 2X (1993) 539-542

Thermostable a-Amylase from Derepressed Bacillus Zkheniformis Produced in High Yields from Glucose

Alexandra Tonkova, Radosvet Manolov & Elena Dobreva

Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

(Received 5 August 1992; revised version received 3 October 1992; accepted 1 November 1992)

High yields of thermostable a-umylase was produced by Bacillus licheniformis

44MB82-G, resistant to glucose catabolite repression, on the basis of inexpensive

raw materials and glucose as a main carbon source. The optimal parameters for

the a-amylase production were an agitation rate of 500 rpm, constant air-fLow rate (1 vvm) and cultivation iemperature 40°C. An enzyme activity of 4800-5M.W U/mi culture medium was reached in 96-120 h. The a-amylase preparation had the following characteristics: a-amylase activity 5.5 000 U/ml, high thermostability

(98% residual a-amyhtsr activity after 10 min treatment at OO”C), protein content

88 mg/ml and dty substances 30%.

INTRODUCTION

Induction and/or derepression control the expres- sion of many extracellular enzymes.’ The use of enzyme substrates which by themselves do not induce the synthesis of these enzymes, provides a pronounced selective stress for constitutive syn- thesis.2 On the one hand, some strains of Bucillus

lichenifotmis show constitutive a-amylase syn- thesis which is not dependent upon exogenous a-glucans for induction. 3,4 It is possible to isolate mutants resistant to catabolite repression by selecting colonies which are capable of forming ‘halos’ of degradation of screening agar containing a high concentration of g1ucose.5’6,7 Using this method we have succeeded in deriving the mutant Bacillus licheniformis 44MB82-G which synthe- sized a-amylase in the presence of 6% glucose.’

Corresponding author: A. Tonkova. Telephone: 3592 713 3163; Fax: 3592 700 109.

Catabolite repression-resistant mutants are valuable since they often synthesize and secrete extracellular enzymes at an earlier stage of the growth cycle in a batch culture than do the wild- type strains, thus increasing the effectivity of pro- duction. Furthermore, these mutants usually secrete a higher amount of enzymes and allow the use of inexpensive fermentation media, often con- taining a high concentration of glucose.

Thermostable a-amylase finds applications in a number of industrial processes, for example in the starch-glucose industry, the textile industry, brew- ing and so on, which explains the interest in research on the effective production of this enzyme.9, “7 1 ’

The purpose of the present work was to demonstrate the possibility of a high production of thermostable a-amylase using inexpensive raw materials and glucose as a main carbon source by Bacillus licheniformis 44MB82-G.

539 Process Biochemistry 032-9592/93/$6.00 0 1993 Elsevier Science Publishers Ltd, England

540 Alexandra Tonkova, Radosvet Manolov, Elena Dohreva

MATERIALS AND METHODS

Bacterial strain and growth conditions The organism used was Bacillus licheniformis 44MB82-G which is resistant to glucose cata- bolite repression.’

The bacteria were cultivated in a complex fer- mentation medium containing (g/litre): glucose (60), soybean flour (34), K2HP0, (10*4), corn- steep liquor (66), CaCl, (l.l), antifoam caralin (5). Sterilization was accomplished at 110°C for 30 min and the initial pH after sterilization was 7.0-7.2. Batch culture experiments were carried out in a laboratory fermenter (Bioflo model C-32, New Brunswick Scientific Co. Inc., Edison, NJ, USA) with a 2 litre fermenter vessel and a work- ing volume of 1.2 litres.

Bacteria grown in nutrient broth with 1% soluble starch and 2% glucose for lo-16 h were used as inoculum. They were transferred into the fermentation medium in a concentration of 0.07 X 1O’l cells/ml (15% (v/v)).

Fermentation conditions were: a constant air- flow rate (1 vol/vol/min) at a varying agitation rate from 300 to 600 rpm and temperature of cultiva- tion 40°C. The dissolved oxygen (DO) was deter- mined continuously by a standard electrode and DO-analyzer (New Brunswick, Sci., model DO- 50).

Liquid a-amylase preparation After the end of the fermentation process, 1.1% CaCl, was added to the culture medium. The pH was adjusted to 7.5-8.0 and the residue obtained was removed by centrifugation. The supernatant obtained underwent ultrafiltration (for reduction of the volume and partial purification) through membrane PM 10 using membrane filtration sys- tem Thomapor ND-400 (Reichelt, Heidelberg, Germany) and vacuum evaporation at 70°C for inactivation of the attendant unthermostable pro- teinase and additional concentration.

Analytical methods a-Amylase activity was assayed according to the method of Pantschev et al. I2 A unit of a-amylase activity was defined as the amount of enzyme that hydrolyses 0.162 mg of starch to dextrins at 3O”C, pH 6.5 in 1 min.

For measurement of thermostability, the sam- ples were treated at 90°C for 10 min and the residual a-amylase activity was determined.

Glucose was measured by the method of Somogyi13 and protein content by the Lowry et al.14 method. Dry substances were determined with a Carl Zeiss refractometer (Jena, Germany).

RESULTS AND DISCUSSION

Production of thermostable a-amylase by batch culture of Bacillus lichen$mmis 44MB82-G A comparison of the data obtained by applying different stirring rates (Fig. l(A), (B), (C), (D)) indicated an increase in a-amylase activity of the culture liquid (from 1400 to 5000 U/ml) with an increase of the agitation rate from 300 to 500 rpm. However, the further enhancement of the agitation rate to 600 rpm had a negative effect on a-amylase synthesis and the maximum a-amylase activity was 2000 U/ml. In fact, the very low (300 rpm) and the very high (600 rpm) stirring rates led to comparatively low a-amylase production, i.e. oxygen insufficiency and oxygen excess were both adverse factors on a-amylase production.

The total growth of the culture increased with an increase in agitation rate (the number of cells after 20 h was 2.8 x lo”, 3.4 X lo”, 3.6 X 10” and 3-9 X lo’*, under the growth conditions of Fig. l(A), (B), (C), (D), respectively). The transi- tion from exponential to stationary phase was also more rapid at high agitation rates. The conversion of the vegetative cells to forespores and the observed lysis of the culture also occurred earlier. During the stationary phase, the cell concentra- tion remained almost constant to the end of the process (Fig. 1 (A)) or for about 24 h (Fig. l(B), (C), (D)) and then began to decrease gradually.

The low stirring rate (300 rpm) was unfavour- able for a-amylase production but led to a rapid consumption of the readily metabolized carbon- source glucose (at the 20th hour only 2.3 mg/ml remained from an initial glucose concentration of 60 mg/ml) because of the pronoucned oxygen insufficiency. Higher agitation rates (400, 500 or 600 rpm) decreased the glucose consumption during the first 20 h of the process. Over the next few hours glucose was almost complete exhausted in all cases (Fig. l(A), (B), (C), (D)).

The change in pH-value of the culture medium during the fermentation process was different at different agitation rates. In experiments with low a-amylase production, the final pH-value was lower than in cases of high a-amylase production.

Thermostable a-amylasefrom derepressed Bacillus licheniformis 541

a 100.-y 5 0

! I 0 80 ’ . : c‘

0 20 40 60 80 100 120 0 20 40 60 80 100 120

Time (h) Time (h)

0 20 40 60 80 100 120 0 20 40 60 80 100 120

Time (h) Time (h)

Fig. 1. Thermostable a-amylase production by Bacillus lichetufomzis 44MB82-G at different agitation rates. A - 300 rpm; B - 400 rpm; C - 500 rpm; D - 600 rpm. 0, amylase activity (U/ml); A, glucose (mg/ml); - --, dissolved oxygen (%); n , number of cells ( X 10’ ‘/ml); 0, pH.

The optimal conditions for a-amylase produc- tion were established at an agitation rate of 500 rpm, a constant air-flow rate of 1 vvm and a culti- vation temperature of 40°C. An enzyme activity of 4800-5000 U/ml culture liquid (equal to 11520-12 000 Novo U/ml) was achieved for 96-120 h.

These results showed a 3.5-fold increase of a-amylase production by the batch culture of strain Bacillu lic~eniformis 44MB82-G in a com- plex fermentation medium with 6% glucose, in comparison to the parent catabolite sensitive strain 44MB82.15 These studies confirmed that catabolite repression-resistant strains secrete a higher amount of enzyme than the parent strains.l In addition, the absence of starch in the nutrient medium as a substrate for a-amylase synthesis added to the usefulness of the process. Thus the viscosity of the medium was decreased and the problems connected with starch sterilization

avoided (i.e. the formation of starch conglomera- tions, which were sources of a contamination in the first 24 h of the fermentation process). This fact is of a great significance for industrial prac- tice.

Liquid a-amylase preparation Using the procedure described in Materials and Methods, an 18-fold concentration and 2-l-fold purification of the culture supernatant (after CaCl,-precipitation and cell separation) was achieved with a negligible loss of a-amylase activ- ity (Table 1).

The partially purified a-amylase preparation had the following characteristics: a-amylase activ- ity 55 000 U/ml (equal to 132 000 Novo U/ml) a high thermostability, namely 98% residual a- amylase activity after treatment at 90°C for 10 min; protein content 88 mg/ml and dry substances 30%. Data obtained showed that the derived pre-

542 Alexandra Tonkova, Radosvet Manolov, Elena Dobreva

Table 1. Liquid a-amylase preparation

Step Volume (ml)

DrY substances

(W

Protein content (m&l) U/ml

a-amyhzse activity

Toial Yield Specific Pm&cation activity W activity factor

W Wlm@

Supernatant after CaCI, precipitation

Ultrafiltration concentrate

750 6 10 3 000 2 250 000 100 300 1.00

135 9 26 16 400 2214000 98 630 2.10

Ultrafiltration permeate

Vacuum evaporation concentrate

615 5 5 65 40 000 1.8 13 -

40 30 88 55 000 2 200 000 97.8 625 2.08

paration was similar to the well-known com- mercial preparation Termamyl 120L (Nova Industry, Bagsvaerd, Denmark).

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