Biosynthesis of cyclodextrin glucosyltransferase and β-cyclodextrin by Bacillus macerans 314 and properties of the crude enzyme

THE BIOCHEMICAL ENGINEERING JOURNAL ELSEVIER

The ChemicalEngineeringJournal 61 ( 1996) 247-253

Biosynthesis of cyclodextrin glucosyltransferase and /3-cyclodextrin bY Bacillus macerans 3 14 and properties of the crude enzyme Abdel-Mohsen

S. Ismail, Usama I. Sobieh, Ahmed F. Abdel-Fattah

Departmentof Naturaland MicrobialProductsChemistry,NationalResearchCentre,Dokki.Cairo,Egypt Received 14 September

1995; accepted 14 September 1995

Abstract Production of cyclodextrin glucosyltransferase (CGTase) enzymes by six bacterial cultures was investigated. Enzyme production was better in shaken than in surface cultures. Bacillus macerans 3 14 and B. amyloliquifaciens cultured on potato dextrose (PD) medium were the most potent strains used. The former strain which produced @cyclodextrin (P-CD) was chosen for further studies. Addition of corn-steep liquor to PD medium afforded the maximal CD yield; nevertheless, it was Q-CD instead of P-CD. Starch addition directed the organism to produce a mixture of (Y-and P-CD. 5% (by volume) inoculum, 72 h incubation period and 37 “C were the most applicable and led to maximal productivity of CGTase by B. macerans 3 14 cultured on PD medium supplemented with 0.3% CaCl,. The lyophilized crude CGTase exhibited maximal activity at an enzyme concentration of 1.65 mg ml - ‘, 2% soluble starch, 60 “C and pH of 6. Keywords:Biosynthesis;

Cyclodextrin

glucosyltransferase;

fi-Cyclodextrin;

1. Introduction Cyclodextrin glucosyltransferase (EC.32 1.19, CGTase) is the enzyme responsible for the production of cyclodextrins (CDs) from starch by a cyclization reaction. There are different types of CDs, i.e., LY-,p- and y-CDs, which are known as Schardinger dextrins. CDs are able to include various kinds of compounds inside their hydrophobic cavities. These inclusion compounds have completely new pharmacotechnical properties [ 1,2]. Thus CDs are used in the pharmaceutical industry as drug carriers and also in ink manufacture, copolymer preparations, emulsified cosmetics, toothpastes, fragrancy plastics and others. Also, they are used in food industries such as cheese making and citrus canning and in the form of foodstuffs [ 31. Chemical synthesis of CDs is more difficult and expensive. The present work is aimed at the fermentative production of CDs by some local and foreign bacterical strains. Production of CGTase enzymes was also attempted by whole immobilized cells of the chosen strain on different chromatographic supports. The general properties of the crude lyophilized CGTase enzyme were also studied. 2. Materials and methods 2. I. Microorganisms The bacterial strains used in the present work were obtained from the culture collection of the Centre of Cultures of the Elsevier Science S.A.

SSDIO923-0467(95)03055-7

Bacillusmaceram

National Research Centre, Cairo, Egypt, and from the US Department of Agriculture and Development Division, Peoria, IL 61604, USA (NRRL). The bacterial isolates, Bacillus macerans 3 14 and B. amyloliquifaciens 312, were kindly provided by Dr. Sanaa Omar, Botany Department, Faculty of Science, Alexandria University, Egypt, 2.2. Culture media The following culture media were used (grams per litre) Medium 1 (potato dextrose (PD) medium) : potato slices, 500; glucose, 20. Medium 2: this had the same composition as medium 1, but the pH was adjusted to 9.0, using 1 N NaOH. Medium 3 [ 41: soluble starch, 20; NH&l, 5; K,HPO*, 0.5; MgS0,.7H20, 0.025; CaCO,, 5. Medium 4 [ 51: soluble starch, 7; corn-steep liquor, 50 ml; K,HPO,, 1; MgSO,.7H,O, 0.2; Na2C03, 10. -.3. Cultivation Transfers were made from the subcultures to PD slopes or plates, which were then incubated at 30 ‘C for 48 h. Cultivation was carried out in 250 ml Ezlenmeyer flasks, each containing 50 ml of sterile medium. 1 ml of cell suspension was taken from 24 h old culture and used for inoculating each flask. In some cases, the culture flasks were agitated on a rotary shaker (200 rev min-‘) at 30 “C for the required

248

A.-M..? Ismail et al. /The Chemical Engineering Journal 61 (1996) 247-253

incubation period. In other cases, surface performed at 30 ‘C. 2.4. Isolation of extracellular glucosyltransferase

cultures

were

cyclodextrin

enzyme

At the end of the incubation period, the culture broth was centrifuged (5000 rev min - ‘) in a refrigerated centrifuge. The transparent culture filtrate was taken for different investigations. 2.5. Protein estimation

2.8. Assay for P-cyclodextrin glucosyltransferase

activity

This method [ 111 is specific for P-CD forming enzyme activity measured by the reduction in the colour intensity of phenolphthalein after complex formation with P-CD. A reaction mixture containing 40 mg soluble starch in 1.0 ml 50 mM sodium phosphate buffer (pH 6.5) and 0.1 ml CGTase solution was incubated at 60 “C. The reaction was stopped by adding 3.5 ml 30 mM NaOH solution, and then 0.5 ml 0.02 wt.% phenolphthalein in 5 mM Na,CO, solution was added. After standing for 15 min at room temperature, the reduction in A,,, was measured. One unit of enzyme activity was defined as the amount of enzyme that forms 1 mg P-CD min-‘.

This was done by the method of Lowry et al. [ 61. 2.9. Assay for amylase activity 2.6. Immobilization of whole bacterial cells 2.6.1. Calcium alginate beads

The fermentative inoculum was prepared. Bacterial cells were entrapped in 2%, 2.5% and 3% calcium alginate gel beads as described by Woodward [ 71.

This was done according to Bergmann et al. [ 121 by estimating the released reducing sugars from 0.5% saline starch in 0.2 M phosphate buffer (pH 7.1) . Reducing sugars were determined by the method described by Nelson [ 131 and Somogyi [ 141. 2.10. Precipitation of cyclodextrin crystals

2.6.2. Agar beads Different concentrations of agar were prepared to give final agar concentrations from 1% to 3% after addition of inoculum. 2.6.3. Polyacrylamide beads Beads were preparedusing different concentrations ( 1.5%, 1.8% and 2%) of cross-linker (NJ-methylene bisacrylamide) according to Woodward [ 71. 2.6.4. Gelatin beads This was done according Dhulster et al. [ 81.

to the method

2.7. Assay for cyclodextrin glucosyltransferase

described

by

activity

This was done according to the method of Hale and Rawlins [ 91. The enzyme activity was determined as follows. A reaction mixture containing 4 ml enzyme solution, 1 ml 1 M Ca acetate-acetic acid buffer (pH 5.2) and 10 ml 2% soluble starch solution was incubated at 40 “C. After 5, 10 and 20 min, 0.5 ml of the digestion mixture was taken and added to a mixture of 5 ml 0.003 M I2 in 0.25 M KI and 0.2 ml of 2 N H,SO,. This mixture was diluted with 10 ml distilled water and light transmission of the diluted mixture was measured at 660 nm 3 min after the addition of iodine. The spectrophotometer scale is set so that the blank containing iodine, but no digest, gives 100% transmittance. One unit of CGTase activity is the amount of enzyme that produces a colour with 50% transmission in 10 min. This unit equals approximately 26 Tilden-Hudson units [ lo].

This was carried out by addition of n-propanol to a final concentration of 60% or a few drops of 0.1 N iodine to the culture filtrate or reaction mixture, and the precipitated CD crystals were examined microscopically.

3. Results and discussion The results recorded in Tables 1 and 2 indicated that all the bacterial isolates investigated produced extracellular CGTase enzymes in both shaken and surface cultures. Generally, shaken cultures (200 rev min- ’ ) were more favourable for the production of active CGTase. For each bacterial isolate a parallel relationship existed between protein content of the filtrates and their CGTase activities. It was of extreme importance to find that the alkalinity of the culture filtrates was essential for the enzyme activity. Maximal activities were recorded at pH 9. On the contrary, culture filtrates of pH values below 7.0 possessed moderate or feeble CGTase activities. Adjustement of the initial pH of culture medium 1 to the value 9.0 (medium 2) led to remarkable improvement in the productivity of CGTase enzymes by most of the strains tested. Many investigators isolated highly active enzymes from alkalophilic bacilli strains [ 5,15-181. The use of culture media 3 and 4, recommended by Nicholas [4] and Shih [5] respectively, did not improve the enzyme productivity with any of the isolates tested in shaken or surface culture. It is worth mentioning that both media accommodate soluble starch instead of potato starch. Microscopic investigation of CD crystals precipitated by 60% n-propanol from the culture filtrates affirmed that each

A.-MS. Ismail et al. /The Chemical Engineering Journal 61 (1996) 247-253

Table 1 Production

of cyclodextrin

Bacterial strain

B. amyloliquifaciens

glucosyltransfemse

Type of culture

Shaken

(312) Surface

B. macerans

Shaken

(314) Surface

B. macerans

Shaken

(394) Surface

B. macerans (3185)

Shaken

Surface

B. macerans (3186)

Shaken

Surface

B. megaterium (NRC4)

Shaken

Surface

249

enzymes by bacterial strains in shaken and surface cultures using culture media 1 and 2 a

Period of incubation

Medium 1

(days)

Protein content of culture filtrate (mg ml-‘)

Medium 2 CGTase activity b (U ml-‘) for the following times of reaction

Protein content of culture filtrate

CGTase activity (UrnI-‘) forthe following times of reaction

(mg ml-‘) 5 min

10min

5min

10 min

1 2 3 2 3 4

3.07 5.18 6.20 2.70 4.60 6.70

1.58 1.85 1.85 0.76 0.83 1.09

0.97 1.12 1.24 0.52 0.73 0.86

1.91 2.48 2.56 1.71 2.12 2.44

0.86 2.26 4.06 0.82 1.19 1.39

0.28 3.32 5.00 1.61 0.68 0.48

1 2 3 2 3 4

3.18 4.79 5.60 2.99 5.00 5.42

1.02 1.43 2.46 0.50 0.58 0.66

0.81 0.84 1.29 0.44 0.58 0.69

1.66 3.07 2.84 1.57 2.55 2.61

0.81 1.57 1.76 0.67 0.95 1.32

0.54 0.85 1.01 0.70 0.69 0.69

1 2 3 2 3 4

2.51 2.60 2.20 1.43 1.92 2.21

0.92 0.97 0.98 0.64 0.68 0.81

0.47 0.54 0.53 0.33 0.36 0.43

2.64 3.08 3.25 2.51 2.80 2.91

1.18 1.50 1.67 0.47 0.64 0.64

0.60 0.79 1.14 0.34 0.37 0.39

1 2 3 2 3 4

2.5 1 2.60 2.00 2.61 2.86 2.58

0.90 0.95 0.96 0.56 0.68 0.88

0.46 0.50 0.52 0.30 0.43 0.45

2.62 3.15 3.45 2.30 2.61 2.85

1.06 1.20 1.24 0.58 0.97 1.08

0.55 0.76 0.99 0.37 0.59 0.59

1 2 3 2 3 4

1.78 1.78 1.64 1.65 1.79 2.05

0.78 0.92 1.11 0.53 0.79 0.81

0.45 0.52 0.59 0.29 0.42 0.45

2.57 3.11 3.40 2.25 2.64 2.15

1.15 1.31 2.02 0.63 0.93 1.21

0.60 1.03 1.14 0.36 0.57 0.63

1 2 3 2 3 4

3.50 3.98 4.91 2.75 3.39 4.79

0.32 0.72 0.71 0.64 0.67 0.88

0.27 0.38 0.50 0.45 0.48 0.57

1.85 2.62 2.76 1.15 1.70 2.37

0.82 1.10 1.25 0.54 0.84 0.65

0.53 0.68 0.72 0.48 0.49 0.35

a For compositions of media 1 and 2 see Section 2.2. b In this and Tables 2-5, the reaction mixture contained 4 ml enzyme + 1 ml of 1 M Ca acetate buffer + 10 ml of 2% soluble starch.

of the type, the shape and the size of crystals was essentially dependent on both bacterial strain and the culture medium used. B. macerans 3 14 produced needle-shaped o-CD when cultured on medium 4 and prismatic P-CD when cultured on media 1 and 2. In a comprehensive review, French [lo] reported that different starchy substrates with B. macerans resulted in different CD yields and different proportions of cy- and P-CD. Of the six isolates investigated, B. macerans 3 14 cultured on medium 1 was the most potent and produced high CGTase activity in 3 days shaken culture (2.5 U (ml enzyme) -I) and the CD crystals precipitated from the culture filtrate were

of the p type. On the contrary, B. amyloliquifaciens 312 produced more active CGTase enzyme in 3 days shaken culture on using medium 2 (5 U ml-‘), but the CD crystals furnished were of (Ytype (Table 1) . Both bacilli strains were chosen for the succeeding studies, using media 1 and 2 for 3 days at different incubation temperatures. From the data recorded in Table 3 it is obvious that in 3 days shaken cultures, B. macerans 3 14 cultivated on medium 1 at 37 “C was more potent (17.8 ‘tr ml-‘, after 20 min reaction) than B. amyloliquifaciens 312 cultivated on medium 2 at 33 “C (8.93 U ml-‘, after 10 min reaction). The optimum temperature for the production of CGTase by

A. -MS. Ismail et al. /The Chemical Engineering Journal 61(1996) 247-253

250

Table 2 Production

of cyclodextrin

Bacterial strain

glucosyltransferase Type of culture

B. amyloliquifaciens

enzymes by bacterial strains in shaken and surface cultures using culture media 3 and 4 a

Period of incubation

Medium 3

(days)

Protein content of culture filtrate (mg ml-‘)

Shaken

(312) Surface

B. macerans

2 3 4

Shaken

(314)

B. macerans

Shaken

(394)

2 Surface

2 4

B. macerans (3185)

Shaken 2 Surface

B. macerans (3186)

Shaken 2 Surface

B. megaterium (NRC4)

2 3 4

2 3 4

Shaken

Surface

’ For compositions

Medium 4 CGTase activity (U ml-‘) forthe following times of reaction 5 min

10 min

0.29 0.44 1.07 0.32 0.42 0.88

0.92 1.26 0.64 0.73 0.84

0.58 0.77 0.83 0.48 0.55 0.45

0.33 0.49 0.84 0.29 0.45 0.80

1.17 1.30 1.47 0.59 0.69 0.92

0.29 0.42 0.45 0.26 0.37 0.56

Protein content of culture filtrate (mg ml-‘)

CGTase activity (U ml-‘) for the following times of reaction 5min

10min

0.92 1.05 1.19 0.70 0.85 1.02

1.45 1.78 1.83 0.90 0.93 0.96

0.90 0.91 0.96 0.53 0.27 0.57

0.71 0.85 0.85 0.44 0.51 0.53

0.50 0.80 1.08 0.31 0.48 0.70

1.29 1.95 1.96 0.88 0.93 0.93

0.87 1.00 1.03 0.49 0.52 0.52

0.48 0.49 0.68 0.49 0.47 0.58

0.31 0.32 0.40 0.27 0.30 0.38

0.27 0.44 0.56 0.29 0.39 0.53

0.97 1.62 1.63 0.74 0.85 0.87

0.72 0.94 0.93 0.46 0.48 0.48

0.28 0.32 0.45 0.23 0.33 0.42

0.57 0.67 0.65 0.52 0.60 0.67

0.36 0.46 0.51 0.32 0.43 0.41

0.47 0.60 0.62 0.43 0.53 0.57

0.92 1.62 1.64 0.82 0.98 0.96

0.71 0.95 0.96 0.48 0.58 0.56

0.26 0.34 0.39 0.32 0.33 0.41

0.52 0.64 0.86 0.51 0.61 0.61

0.38 0.42 0.49 0.35 0.39 0.44

0.48 0.58 0.59 0.45 0.54 0.57

0.87 1.34 1.58 0.73 0.84 0.80

0.70 0.81 0.85 0.49 0.52 0.54

0.18 0.38 0.71 0.28 0.35 0.56

0.65 0.86 0.98 0.48 0.57 0.84

0.47 0.54 0.61 0.36 0.40 0.45

0.39 0.45 0.64 0.26 0.42 0.52

1.08 1.05 1.68 0.69 0.71 0.77

1.42

of media 3 and 4 see Section 2.2.

Klebsiellu oxytucu was 40 “C [ 191; also other thermophilic

bacilli strains were used for the production of CGTase enzymes [ 20,211. Table 4 shows nitrogen sources added to the basal medium 1 (PD) and their effects on the productivity of CGTase enzyme, CD yield and starch conversion in the culture filtrate of B. maceruns 314. Of these, only milk whey caused a slight increase in the three parameters mentioned. The use of comsteep liquor afforded the maximal CD yield; nevertheless, it was W-CD. The use of corn-steep liquor in a culture medium as the N source favoured (w-CD production more than P-CD by other bacilli strains [ 22,231.

Different concentrations of milk whey were tested (Table 4). Although a concentration of 0.005% N was the best, even this caused only a trivial increase in enzyme activity. Higher concentrations resulted in drop of the pH of the culture broth, which caused an adverse effect on CGTase stability as indicated before. On an equal C basis, glucose in PD medium was substituted with different carbon sources, i.e. soluble starch, wheat bran, sucrose, maltose, lactose, glycerol, glycogen, maize starch or insoluble starch. All these carbon sources had adverse effects on CGTase productivity; nevertheless, sucrose led to an increase in starch conversion to CDs. Sakai and Horie [ 241

A.-MS. Ismail et al. /The Chemical Engineering Journal 61(19%)

247-253

251

Table 3 Effect of different incubation temperatures on the production of cyclodextrin glucosyltransferase enzymes by B. amyloliquifaciens 312 (cultivated on medium 2) and B. mucerans 3 14 (cultivated on medium 1) in 3 days shaken and surface cultures Bacterial Strain

Incubation temperature (“C)

Surface

Shaken Protein content of culture filtrate (mg ml-‘)

CGTase activity (Uml-‘) forthe following times of reaction smin

10min

20 min

Protein content of culture filtrate (tug ml-‘)

CGTase activity (U ml-‘) forthe following times of reaction Smin

1Omin

20 min

B. amyloliquifaciens (312)

28 33 37 40 45

6.20 4.5 1 4.14 4.55 5.28

1.93 5.04 1.46 1.19 1.11

1.24 8.93 0.84 0.73 0.63

0.73 5.48 0.54 0.54 0.45

0.93 0.85 0.79 0.84 0.97

3.70 3.41 3.15 3.36 3.87

0.86 0.82 1.06 0.96 0.55

0.56 0.49 0.64 0.53 0.34

B. macerans (314)

28 33 37 40 45

4.09 4.20 4.10 3.72 3.80

2.46 2.58 11.79 7.11 3.49

1.29 7.21 17.10 6.93 1.92

0.70 5.83 17.78 4.94 1.20

0.18 0.22 0.23 0.21 0.23

0.72 0.87 0.90 0.82 0.91

0.69 0.70 0.95 0.97 0.66

0.36 0.50 0.60 0.59 0.39

Table 4 Effect of addition of nitrogen sources to the basal medium (medium 1) on the production of cyclodextrin glucosyltransferace enzyme by B. macerans 3 14 Nitrogen source added ’

Protein content of culture filtrate (mg ml-‘)

CGTase activity (U ml-‘) for the following times of reaction 10 mm

2omin

CD yield (mg per 50 ml culture filtrate)

Starch conversion (%)

None (control) Casein Corn-steep liquor soybean Peptone Wheat-bran extract Yeast extract Sodium nitrate

4.16 4.14 4.10 4.05 4.10 4.14 3.30 2.89

20.88 5.40 8.69 15.08 8.19 5.40 3.98 5.81

16.81 6.31 12.04 14.81 10.64 6.40 3.12 6.08

75.0 51.25 136.25 37.50 62.50 51.25 83.75 86.25

6.70 4.58 12.17 3.35 6.92 4.58 7.48 7.71

Milk whey O.O5gNl-’ 0.1 gNl-’ 0.15 g N 1-r 0.2gNl-’ 0.4gNl-’

4.21 3.94 2.97 3.42 4.16

21.92 21.22 6.89 6.86 1.29

17.88 18.13 5.35 6.11 0.83

91.25 93.75 62.25 58.50 33.25

8.15 8.37 5.56 5.22 2.97

’ 0.1 g N 1-l unless otherwise indicated.

reported that the addition of sucrose to the reaction mixture enhanced CD yield. On the contrary, Lee et al. [ 191 showed that mono-, disaccharides and amylose did not induce CGTase enzyme. It was also noticed that starch addition to medium 1 directed the organism to produce a mixture of LYand P-CD. In this respect, Dan et al. [25] reported that the ratio of U-CD increased with the increase in starch concentration to 3% and the formation of (Y-,p- and ?-CD from starch was in the ratio 61:32:7. The effect of addition of six metal ions, namely Ca*+ (as CaCl, and CaCO,), Na+, Fe3+, Mn*+, Mg*+ and Zn*+

(different concentrations), to the culture medium on CGTase productivity was studied. All metal ions added except Ca*’ had adverse effects on enzyme production at all concentrations used. Ca*+ when added as CaC12from 0.1% to 0.3% optimized both CGTase and P-CD productivities, above which both parameters decreased and this affirmed the stabilizing effect of Ca*+ on CGTase. However, CaC03 did not enhance the enzyme productivity as did CaC12.5 mM CaCl, was used by Rohrbach and Scherl [ 261 for enhancement of PCD production. On the contrary, 0.5% CaC03 has been recommended [ 25,27,28 J .

252

A.-MS. Ismail et al. /The Chemical Engineering Journal 61 (1996) 247-253

Table 5 Properties of the crude cyclodextrin macerans 3 14

glucosyltransferase

from B.

CGTase activity (l-l ml- ’ ) for the following times of reaction

Property

( I ) Enzyme protein concentration 0.25 0.33 0.55 0.71 0.99 1.65 2.48 3.30

10min

20 min

( mg ml - ’ ) 0.38 0.50 1.13 2.05 3.09 3.16 2.82 2.64

0.27 0.42 0.99 1.99 2.8 I 3.11 2.81 2.65

(2) Substrate (soluble starch) concentration (mg per reaction) 50 2.35 100 2.98 200 3.10 300 0.93 400 0.75 (3) Temperature 35 40 45 50 55 60 65 70 75

preparation

of the reaction

(4) pH of the reaction ’ 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

a 2.23 2.83 2.95 0.90 0.73

(“C) ’ 2.48 3.08 2.68 2.58 2.75 3.25 3.05 3.00 2.05

2.53 2.80 2.45 2.43 2.68 3.13 2.88 2.83 1.98

0.83 2.98 3.03 3.15 3.20 3.28 3.13 2.60

0.80 2.95 3.00 3.15 3.15 3.25 3.03 2.45

Some properties of the crude lyophilized CGTase enzyme produced by B. macerans 3 14 were studied and are summarized in Table 5. The data demonstrated that a parallel relationship existed between the enzyme concentration and CGTase activity up to a concentration of 1.65 mg ml- ‘, above which the enzyme concentration ceased to be a limiting factor controlling the enzyme action. Using 2% soluble starch (200 mg per 15 ml of reaction mixture) at pH 5.2 and 40 “C, the CGTase enzyme ( 1.65 mg ml-‘) exhibited its maximal activity. It is worth noting that at low substrate concentrations, from 2 to 20 mg per 15 ml of reaction mixture, all starch was completely consumed after the first few minutes. Also, it can be concluded that all the enzyme active sites became saturated by substrate at concentrations higher than 200 mg per 15 ml of reaction mixture. Su and Yang [29] obtained 70% CD yield from 1 wt.% liquified starch; 3% soluble starch in the reaction mixture was the proper choice according to Jeang and Sung [ 301. The crude CGTase exhibited two temperature optima, one at 40 “C and the other at 60 “C. Amylase activity was measured at both 40 “C and 60 “C, to find which is related to CGTase. Amylase activity was 1.83 U ml-’ and 0.24 U ml-’ enzyme at 40 “C and 60 “C respectively. Consequently, 60 “C was the optimum temperature for the crude CGTase. These results confirmed the presence of amylases with CGTase enzyme and also substantiated the thermostability of B. macerans 314 CGTase. Many investigators reported high optimum temperature (55-65 “C) for crude CGTase enzymes from other bacilli strains [ 5,15,31-331. The crude enzyme exhibited a pH optimum of 6 at 60 “C on using citrate buffer. Accordingly, the concentration of ionic form of CGTase must be maximal at a pH of 6.0. This optimum value is similar to those reported for other CGTase enzymes from bacilli strains [ 15,34-361.

References [l] J. Cohen and J.L. Lath,

a Enzyme protein h Enzyme protein mg per reaction. c Enzyme protein mg per reaction;

concentration, concentration,

1.65 mg ml-‘. 1.65 mg ml- ‘; substrate concentration,

concentration, 1.65 mg ml- ‘; substrate concentration, temperature, 60 “C.

200

[2]

200

The data also showed that an inoculum of 5% (by volume) and a 72 h incubation period were the most applicable and led to the maximal productivity. Production of active CGTase enzymes by B. macerans 314 immobilized cells on different supports, i.e. Ca alginate, agar, polyacylamide and gelatin (different concentrations), was attempted. None of the supports used for immobilization of B. macerans whole cells succeeded in the production of highly active CGTase enzymes as free cells did. Furthermore, immobilized cells caused liquefaction of gelatin beads at all gelatin concentrations used. It is worth mentioning that alkaline protease activities were recorded in the culture filtrates.

[3] [4] [5] [6]

[7] [8]

[9]

Interaction of pharmaceuticals with Schardinger dextrins, J. Pharm. Sci., 52 (1963) 132. M. Komiyama, H. Yamamoto and H. Hirai, Complex formation of modified cyclodexttins with organic solvents, Chem. L&t., (1984) 1081. J. Szejtli and L. Szente, Interaction between indomethacin and P_ cyclodextrin, Pharmazie, 36 ( 198 1) 694. D.P. Nicholas, Food Processing Enzymes, Noyes, Park Ridge, NJ, 1979,pp. 111-113. P. Shih, A new method for the production of cyclodextrin by Bacillus, Yu Fa Hsiao Kung Yeh, (6) ( 1986) 1. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement withtbeFolinphenolreagent,J. Biol. Chem., 193 ( 1951) 265. J. Woodward, Immobilized Cells and Enzymes: a Practical Approach, IRL, Oxford, 1985, p. 44. P. Dhulster, P. Parascandola and V. Scardi, Improved method for immobilizing inverstase-active whole cells of Saccharomyces cervisiae, Enzyme Microbial. Biotechnol., 27 ( 1983) 443. W.S. Hale and L.C. Rawlins, Amylase of Bacillus macerans. Cereal Chem., 28 (1951) 19.

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[ 111

[ 121

[ 131

[ 141 [ 15 J [ 161

[ 171

[ 181

[ 191

[20]

[21]

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