Eur. J. Biochem. 183,161 -167 (1989) 0 FEBS 1989

The purification and characterization of a dextranase from Lipomyces starkeyi David KOENIG and Dona1 DAY'

Department of Microbiology, Louisiana State University Audubon Sugar Institute, Louisiana Stata University Agricultural Center, Baton Rouge

(Received September 1, 1988/March 21, 198) ~ EJB 88 1040

Dextranase produced by Lipomyces starkeyi was purified 43-fold, by carboxymethyl-Sepharose chromatog- raphy followed by agarose gel-filtration chromatography. The purified enzyme showed four bands by SDS/ polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65 kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55"C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40 "C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto-oligosaccharides from glucose to isomaltotetrose.

Studies have indicated that dextranase ( u - D - ~ ,6-glucan 6- Enzyme and protein assay glucanohydrolase) can be useful in treating dextran-contami- nated sugar products [l -41. Industrial sources of dextranase are species of Penicillium [5] and Chuetomium [6]. Lipomyces starkeyi, an ascosporogenous yeast, also produces a dex- tranase [7,8]. In a previous paper we reported on the selection of a mutant of Lipomyces stuvkeyi (ATCC 20825) as a potent producer of dextranase [9]. This enzyme has been used to treat successfully a dextran-contaminated sugar process stream [lo].

The present report describes the purification and some of the enzymatic properties of the dextranase of L. starkeyi (ATCC 20825). These characteristics are compared to those of the Lipomyces dextranase reported by Webb and Spencer- Martins [8] and of the commercially available dextranases from Chaetomium [6] and Penicillium [S].

MATERIALS AND METHODS

Reagents

Dextrans T-2000, T-500, T-70, T-40 and T-10 (molecular masses 2 MDa, 500 kDa, 70 kDa, 40 kDa and 10 kDa, respec- tively) were purchased from Pharmacia Fine Chemicals AB, Sweden. The carboxymethyl-Sepharose was from the Sigma Chemical Co. (St Louis, MO) and Bio-Gel A-0.5 m agarose from Bio-Rad Corp. Dextran used in the production of en- zyme was industrial grade (molecular mass 5 - 40 MDa) from Sigma Chemical Company. The isomalto-oligosacharides used to calibrate the HPLC were a gift to Dr F. Paul, Bio- Europe, Ltd., Toulouse, France. All other reagents were of analytical grade.

Correspondence to D. Day, Audubon Sugar Institute, LSU Agri-

Enzymes. Dextranasc, 1,6-cl-~-glucdn 6-glucanohydrolase cultural Center, Baton Rouge, Louisiana, USA

(EC 3.2.1 .I 1 j ; p-D-galactoside galactohydrolase (EC 3.2.1.23).

Dextranase was assayed by a modification of the method of Webb and Spender-Martins [8]. Enzyme preparations were incubated with 2.0% (g/dl) Dextran T-2000 (Pharmacia) in 0.05 M citrate/phosphate, pH 5.5, at 50°C for 10-30 min. Activity was determined from the rate of increase in reducing sugar as measured by the 3,5-dinitrosalicylic acid method [l I]. 1 IU dextranase is defined as the amount of enzyme which liberates 1 pmol glucose equivalents/niin under the described conditions. 0.05 M sodium acetate, pH 5.5, replaced citrate/ phosphate buffer in experiments determining the effect of salts and inhibitors on activity. No difference was observed in activity between the two assay buffers. Protein was determined by the method of Lowry et al. [12].

Dextranuse purijkation

Lipomyces starkeyi (ATCC 20825) was grown as pre- viously described [9, 13, 141. Culture supernate was concen- trated from 20 1 to 500 ml using a 10-kDa cut-off stacked membrane (Pelican, Millipore, Co.). Protein and enzyme ac- tivity were monitored throughout the course of the purifi- cation.

A carboxymethyl-Sepharose column (75 cm x 1.5 cm) was prepared and equilibrated with 0.02 M potassium phosphate (pH 6.0). The crude concentrate (9 mg protein/ml) was ap- plied to the column. The enzymes was desorbed by elution with 0.5 M NaCl, and active fractions pooled. The pooled fractions were concentrated on a 10-kDa cut-off membrane in an ultrafiltration apparatus (Amicon Co.).

A descending flow (10.44 cm/h) Bio-Gel A-0.5 m agarose column (75 cm x 1.5 cm) was prepared and equilibrated with 0.05 M citrate/phosphate (pH 5.5) plus 0.15 M NaCl. The concentrated carboxymethyl-Sepharose fraction was applied to the column (1.6 mg protein/ml). The fractions with activity were pooled and concentrated. This was the preparation used for all studies reported in this paper.

162

For comparative purposes dextranase was also purified Lectin reactivity

The enzyme was tested for retention on a lectin support as described by Montreuil et al. [17]. Concanavalin-A- Seph- arose (Sigma Chemical), Lens-culinaris (Lentil)-agglutinin -

by the method of Webb and Spencer-Martins [8].

Gel electrouhoresis

Analytical slab electrophoresis by the method of Laemmli [15] was run in 7.6% SDS/polyacrylamide gels. At least 10 mg protein was added to each well. A standard mixture of pro- teins of known molecular mass (Sigma Chemical; SDS-6H containing carbonic anhydrase, egg albumin, bovine serum albumin, phosphorylase b from rabbit muscle, j-D-galactoside galactohydrolase from Escherichia coli, myosin from rabbit muscle) was processed in the same manner and loaded on to the gel as a reference for determination of the apparent molecular mass.

The gels were subject to electrophoresis at 60 V until the tracking dye migrated to the bottom of the stacking gel, volt- age was raised to 120 V and the electrophoresis was allowed to proceed until the tracking dye was within 3 mm of the bottom of the gel.

The gels were fixed in 25% (by vol.) isopropanol and 10% (by vol.) acetic acid for 2 h then stained with Coomassie blue R-250 for 18 h. The gels were destained electrophoretically and scanned with a Ephotec/Joyce Loebl densitometer at 530 nm, with an aperture setting 0.05 mm x 3.0 mm, to detect protein.

Isoelectric focusing

Isoelectric focusing was performed using precast IsoGel agarose isoelectric focusing plates, pH 5.0- 8.5 (FMC Bio- products, Rockland, Maine). Plates were prepared and pro- cessed as recommended by the manufacturer. A standard mixture of proteins (Serva Chemical, test mix 9) was applied in the lane next to each sample. Protein profile were quantified by densitometer scans as previously described. The enzyme activity of the gel was determined by slicing an unstained gel into 0.9-mm sections. Each section was placed in a test tube with 1 .O mlO.05 M citrate/phosphate (pH 5.5) buffer, allowed to elute overnight at 4 "C and assayed for enzyme activity.

Sepharose (Sigma Chemical), and wheat-germ-agglutinin - Sepharose (Sigma Chemical) were all tested. Each support was prepared and equilibrated as previously described [17]. Pure enzyme (500 IU) was applied to each column and activity in the void volume determined. Various elution conditions were used to remove the enzyme from the support. The ability of the enzyme to bind and the conditions required to desorb were compared for the three supports and the relative reac- tivities to the lectins were determined.

EJfect qf p H and temperature on enzyme activity and stability

The pH optimum was determined using the standard dextranase assay at 50°C with a 10-min incubation time. The pH of the substrate solutions covered a range from pH 2.5 - 7.0 in intervals of 0.5 pH units. The pH of the reaction mixture was measured before and after each assay and found not to change.

To determine the stability of dextranase, stock enzyme was diluted with 0.05 M citrate/phosphate and adjusted to the desired pH (between pH 2.5 and 8.0). The solutions were incubated at 25°C for 72 h. Every 24 h the activity of each reaction mixture was assayed. A plot of the relative activity at each pH versus the time of incubation was used to determine the pH at which the enzyme was most stable.

The temperature optimum was determined using the stan- dard dextranase assay run at various temperatures. A plot of relative activity versus incubation temperature was used to determine the temperature optimum.

Temperature stability was determined by incubating the enzyme at specific temperatures for specific time intervals (0 min, 5 min, 10 min and 20 min) then measuring dextranase activity. A plot of relative activity at each temperature versus time of incubation was used to determine the temperature at which the enzyme was the most stable.

Determination of the molecular mass Ejject of carbohydrates, salts and inhibitors on activity

A down-flow gel-filtration column (75 cm x 1.5 cm) was prepared of Bio-Gel A-0.5 m (agarose) containing a total bed volume of 115 ml. The gel was equilibrated with 0.02 M pot- assium phosphate buffer (pH 7.0) plus 0.17 M NaCl. The flow rate was 0.38 ml/min. The column was standardized with the use of a Bio-Rad gel-filtration standard containing the follow- ing proteins: thyroglobulin, y-globulin, ovalbumin, myo- globin, and vitamin B-12 (total protein 18 mg). Using the absorbance at 280 nm to identify the protein peaks, a plot of AZ8, , versus fraction number was used to calculate the elution volume of the different protein species. The dextranase peak was identified by using the previously described enzyme assay. The apparent molecular mass was also determined by SDS/ PAGE as already described.

Determination of carbohydrate content

Enzyme (1 mg) was dialyzed overnight against 500 m16 M urea followed by three changes (4 I each) of deionized water. The carbohydrate was determined by the phenol/sulfuric-acid method [16] and the protein by the method of Lowry et al. [I 21.

Up to 50 mM of various carbohydrates (Sigma Chemical) was included in the standard assay buffer to tcst for their affect upon activity. Stock solutions of selected salts and enzyme inhibitors were made in 0.05 M sodium acetate (pH 5.5). A 100-in1 aliquot of suitably diluted test solution was added to 5 ml enzyme containing 850 ml 0.05 M sodium acetate (pH 5.5) at 50°C and incubated for 5 min, after which time 1.0 in1 4.0% dextran T-2000 in 0.05 M sodium acetate (pH 5.9, preincubated at 5 0 T , was added. Activity was de- termined as previously described.

End product analysis

Enzyme (200 IU) was incubated with 400 ml 2% dextran T-2000 at 4 0 T , pH 5.5. Samples (10 ml) were taken at various time intervals and boiled for 5 min to stop the reaction. Samples were then filtered through a 0.22-mm membrane and assayed by HPLC for the oligosaccharide content.

The high-performance liquid chromatograph used was a Varian Vista 5500 with a refractive index detector linked to a Varian 4270 integrator for data acquisition. The column employed for separations was a Rainin NH,-Dynamax 60A.

163

.

Table 1. Purijkation oflipomyces starkeyi dextranuse All steps were carried out as described in Materials and Methods. CM-Sepharose, carboxymethyl-Sepharose

Step Procedure Total Total Activity Specific Purification no. protein activity yield activity

mg IU O/o IU/mg -fold 1 Clarified broth 1740 55600 32

3 CM-Sepharose 148 55 044 99.0 372 11.6 4 Concentration 148 55044 99.0 372 11.6

Dialysis 148 29 524 53.1 199 6.2

2 Concentration 1718 55375 99.6 32 1 .o

5 Bio-Gel A-0.5 m 28 38 761 70.0 1385 43.3

Isopropanol precipitation 378 5502s 99.0 146 4.5

m The HPLC conditions were as follows. Solvent, acetonitrile/ water (1 : 1); column temperature, 25°C; flow rate, 1.00 ml/ min; column pressure, 5.27 x lo6 Pa with a 20-ml sample in- jected. The malto-oligosaccharide series DP 1-DP 7 (Sigma Chemical) were used as external calibration standards. A com- parison of the malto series to the isomalto series was performed. No differences were observed in detector re- sponses.

RESULTS

Enzyme purification

A five-step purification protocol (Table 1) was required to purify dextranase from Lipomyces starkeyi (ATCC 20725). The concentration steps, 2 and 4, were conducted for ease of handling. Two other steps (not numbered) are shown in Table 1, one which involves the dialysis of the carboxymethyl- Sepharose fraction (step 3) and another which uses isopro- panol precipitation to purify crude enzyme from step two, a technique described by Webb and Spencer-Martins [8].

The crude enzyme fraction (step 1) showed a single activity peak, as determined by Bio-Gel A-0.5 m column chromatog- raphy, with an estimated molecular mass of 68 kDa. Five protein peaks were resolved by Bio-Gel A-0.5 m chro- matography. Cation-exchange chromatography (step 3), gave a 12-fold purification with no appreciable loss of enzyme. Fig. 1 depicts the Bio-Gel A-0.5 m profile for the carboxy- methyl-Sepharose fraction. Subsequent dialysis of this frac- tion against 0.02 M potassium phosphate produced a shift of both protein and activity (Fig. 2) profiles as separated by the Bio-Gel A-0.5 m column. The enzyme was found in numerous peaks ranging from 68 kDa to the void volume (> 700 kDa). The enzyme found in the void volume (> 700 kDa) appeared to be an aggregate. It was only possible to dissociate this aggregate with SDS treatment. SDS dissociated the aggregate to the 68-kDa form but irreversibly inactivated the enzyme.

A single protein and activity peak were obtained by frac- tionation of the carboxymethyl-Sepharose preparation on a Bio-Gel A-0.5 m gel-filtration column. This was the enzyme fraction used for all the kinetic and physical protein determi- nations.

Isopropanol precipitation by the method of Webb and Spencer-Martins [S] was used to purify dextranase from ATCC 20825 but produced only a fivefold increase in specific activity. This preparation was not electrophoretically pure (data not shown). The apparent molecular mass, estimated by gel chromatography of the active fraction, was the same as ob- served previously (68 kDa).

c 4 0.2 4

U

e t! 8 0.1

C .r( al U

Ir 0.0

I0 20 30 40 50 60 70

Fraction number

Fig. 1. Gel-filtrution projile f o r carhoxymethyl-Sepharose-purqied dextranase. Protein (opcn boxes) and enzyme activity (closed dia- monds) arc plotted against fraction number

60 70 I0 20 30 40 50

Fraction number

Fig. 2. Gel-filtration profile fo r carhoxymethyl-Sepharose-purified, fhen dialyzed, dextranase. Protein (open boxes) and enzyme activity (closed diamonds) arc plotted against fraction number

Molecular muss determination

The molecular mass of purified dextranase, by gel chromatography (Bio-Gel A-0.5 m), was calculated to be 68 kDa. The dextranase on SDSjPAGE (7.6%) fractionated into four protein bands with molecular mass 74 kDa, 71 kDa, 68 kDa and 65 kDa (Fig. 3). Lipomyces dextranase, in the native, nondenatured state, interacted with polyacrylamide, making normal acrylamide electrophoresis unreliable. Reacti- vation of the SDS treated enzyme was not possible.

Isoelectric ,focusing

To demonstrate that the four bands appearing on SDS/ PAGE were dextranase, the purified enzyme was fractionated

164

60 U U 56 61 10 12 14 16 18 M Distance from top of gel (am)

Fig. 3. Densitometer scan of7.6% SDSIPAGEfractionation ofpurified clexrrunuse. Molecular mass in kDa is noled on the figure

I 2 3 4 5 6 7 8

PH Fig. 5. EfTect q f p H o n dextranase activity. The unit of relative activity is YO

Distance from cathode (mu)

Fig. 4. Densitometer scan (if izn isoeIc.crricfocusing gel fractionation of purified dexiranasc. The isoelectric values arc shown o n the figure

by agarose isoelectric focusing. This method separated the protein mixture into five isoelectric bands (Fig. 4). All five forms were found to have dextranase activity and exhibited the same K,,, values.

Curbohydrate composition

The purified dcxtranase was found to contain 8% (by mass) carbohydrate. This glycoprotein wa5 tested for its reac- tivity to lectins and was found to bind very strongly to concanavalin A, weakly to Lens culinaris agglutinin and not at all to wheat germ agglutinin.

Elfect of p H and temperature on activity and enzyme stability

Dextranase was found to exhibit its highest activity at pH 5.0 (Fig. 5). The enzyme was very stable between pH 2.5 and 6.0. There was a dramatic loss of activity after 30 h of exposure to a pH above 7.0 (Fig. 6).

This enzyme was found to exhibit optimum activity at 50°C (Fig. 7). Dextranase was very stable between 30°C and 40°C. All activity was lost after 5 min exposure to 60°C (Fig. 8).

Effect ojcarhohydrates, salts and mzyrne inhibitors on activity

None of the carbohydrates tested had any effect on enzyme activity. The following carbohydrates were added to the assay

100

80 h r( ... U 0

60 : 4 U

d 0 e: a

40

20

\\ 0 20 40 60 80

Time (h)

Fig. 6. E@ct o f p H on dextranase .stability. pH 2.5 (open boxes), 3.0 (closed diamonds), 4.0 (closed syuarcs), 5.0 (open diamonds), 6.0 (closed rectangles), 7.0 (open boxes), 8.0 (closed triangles). The unit of relative activity is %

20 30 40 50 60 70 80

Fig. 7. Eflect oftemperature on dextrunase activity. The unit of relative activity is %

mixture at a concentration of 10 mM: glucose, 2-deoxy- glucose, a-methyl-D-glucopyranoside, P-methyla-gluco- pyranoside, maltose, maltotriose, maltotetraose, isomal- tose, isomaltotriose, mannose, a-methyb-mannoside and stachyose.

Various salts were included in the assay mixture to deter- mine their effects on activity (Table 3). The citrate/phosphate

165

100

80

x U .A > U 60 m

o ! 0 100 200 300

Time b i n )

Fig. 8. Ejyect of temperature on dextranuse stability. Temperature: 30°C (open boxes with dot), 40°C (closed diamonds), 50°C (closed boxes), 60°C (open diamonds). The unit of the relative activity is %

Table 2. Apparent K, vulues f o r native, aggregated and IGC 4047 dextranase IGC 4047 dextranase data was taken from [8]

Dextrdn type Native Aggregate IGC 4047

mM

T-2000 0.004 0.003 0.003 T-500 0.01 8 0.017 0.01 3 T-70 0.094 0.096 0.143 T-40 0.190 0.173 0.171 T-10 0.651 0.449 0.456

Table 3. effect ofsalts on dextranase activity Incubation was carried out as described in Materials and Methods. Dextrdnase activity is reported as percentage recovery after incu- bation of the enzyme with the salt at 50°C for 5 min

Salt Concentration Relative activity

BaCI2

CaCI,

CdCI2

COCIZ

CUSO4 FeCI,

mM 11.40 0.11

13.90 0.14 1.39 0.14 1.10 0.1 1 0.13 1.45 0.14 1.36 0.14 1 .00

15.00 0.15

15.00 0.15

11.10 0.1 1 1.24 0.12

%

51 101 48 83 20 78 60 89 7

19 30 0

30 0

66 96 66 95 14 89 8

52

Table 4. Efect of enzyme inhihitors on dextranuse activity Results are reported as percentage activity recovered aftcr incubation of enzyme with inhibitor at 50°C for 5 min

Inhibitor Concentration Relative activity

YO Control 100 EDTA 5 mM 103 Dithiothrcitol 5 mM 100 2-Mercap toethanol 5 Yo 103 HgC1/2-mercaptoethanol 1.0 mM/5% 131 Glutathione 125 mM 100 Guanidine/HCL 547mM 13

274 mM 61 50 mM 103

Urea 547mM 36 274mM 77 50 mM 99

Sodium m-periodate 5 mM 15 SDS 0.5% 0

0.05% 46 Triton X-I00 0.25% 99

0.025 Yo 100 SDS/Triton X-100 0.5 % 10.25 Yo 80

0.05%/0.025% 122

Table 5. Endproduct anulysis Carbohydrates quantified by HPLC, arter 26.5 h incubation of 2.0% T-2000 with 0.7 IU/ml, 40"C, pH 5.5

Carboh ydratc Relative amount

Glucosc lsolamtose Isomaltotriosc lsomaltotetrose Isomaltopentosc Higher isomal to-oligosaccharides

YO 0.8

41.5 34.9 2.7 0.0

20.3

buffer was replaced by sodium acetate to prevent chelation of the ions; otherwise the assays were conducted as described in the Materials and Methods. The following salts showed no inhibition at the 15mM concentration used; NH,Cl, KCl, NaCl and NaF. Mercuric chloride (1 mM) completely inacti- vated dextranase (Table 3). 2-Mercaptoethanol(5%) restored enzyme activity (Table 4).

Enzyme inhibitors were tested at different concentrations, and their effects are listed in Table 5. EDTA, dithiothreitol, 2-mercaptoethanol, glutathione and polyethylene ester (Tri- ton X-I 00) had no effect on enzyme activity. Sodium dodecyl sulfate inhibited the enzyme. The combination of Triton X- 100 and SDS overcame the inactivation. Addition of Triton X-I00 after SDS exposure did not reactivate the enzyme. Sodium m-periodate also inactivated the enzyme.

Reaction end products

The end products of the reaction were found to be typical for an endodextranase. The reaction products observed after 26.5 h of incubation of dextran with the enzyme are given in Table 5. The progression of reaction products are shown in

166

is significant difference between the enzymes' molecular mass, there does not appear to be catalytic difference between the two.

If the dextranase eluted from a carboxymethyl-Sepharose column was desalted by dialysis, an active enzyme aggregate was formed. This aggregate does not readily dissociate and exhibits a molecular mass of over 700 kDa. The aggregate will not dissociate upon the addition of salt. It will dissociate upon the addition of SDS, although the dissociated enzyme is not active. The aggregate form does not show any difference in apparent K , compared with the native (68 kDa) form.

The ATCC 20825 dextranase was a glycoprotein contain- ing 8% sugar. This sugar was determined to contain signifi- cant amounts of mannose by lectin reactivity. Differing substi- tutions of the carbohydrate portion of this enzyme may be an explanation for the multiple molecular mass and isoelectric forms observed. Penicillium [20] and Chaetomium [I 81 dextra- nases are both glycoproteins. The Penicillium enzyme also has been shown to contain a large amount of manose in its carbohydrate moiety [20].

Purified Lipomyces dextranase is completely inactivated by exposure to mercuric chloride. It was possible to reactivate the enzyme after exposure to the mercuric salt by addition of 2-mercaptoethanol. The Penicillium enzyme also has a re- quirement of a sulfhydryl group for activity [19]. Mercuric chloride had the same effect on the dextranase from Chuetomium [18].

The specificity of the endodextranase of L. starkeyi seems very similar to those of Penicillium [19], Aspergillus [21], Chuetomium [18] and the Lipomyces enzyme reported by Webb and Spencer-Martins [8]. Following the progression of reac- tion products with time, it is apparent that the minimum glucan chain length that will be hydrolyzed by the enzyme is four, although isomaltoterose was not hydrolyzed as quickly as was isomaltopentose.

This Lipomyces dextranase shows the same general prop- erties as those shown by other fungal dextranases, making it a potentially viable commercial enzyme.

0 I00 200 300 400

T i m e (mfn)

Fig. 9. Time course of' reaction product formation. Carbohydrate: isomaltose (open boxes), isomaltotriose (closed diamonds), isomalto- tetraose (closed boxes), isomaltopentose (open diamonds)

Fig. 9. Isomaltotetrose was more recalcitrant to enzymatic hydrolysis than was isomaltopentose. The final products of the reaction were isomaltotriose and isomaltose. Very little glucose was formed.

DISCUSSION

Dextranase produced by Lipomyces starkeyi (ATCC 20825) was purified in a five-step procedure that gave a 43.3- fold increase in specific activity. The molecular mass of the purified enzyme by gel filtration was found to be 68 kDa. SDSjPAGE separated the dextranase into four proteins with apparent molecular mass in the range 74 - 65 kDa. These values differ considerably from the value of 23 kDa reported by Webb and Spencer-Martins [8] for their Lipomyces dextranase. Webb and Spencer-Martins [8] also stated that the dextranase they purified showed a single band by SDS/PAGE, and they were able to separate their dextranase on a native nondenaturing PAGE to prove purity [8]. The dextranase from Lipomyces (ATCC 20825) did not migrate uniformly in a native PAGE. Separation by the technique of Webb and Spencer-Martins [8] gave an active protein smear throughout the gel. This sort of interaction has also been reported for the Chaetomium dextranase [18].

To prove purity, the protein mixture was separated on an agarose isoelectric focusing gel. This separation resolved five protein bands, all of which had dextranase activity. All the isoelectric forms were kinetically similar, exhibiting the same K,. Therefore the differences in physical structure seemed to have no effect on the gross catalytic characteristics.

All carbohydrates tested had no effect on enzyme activity. This means that this enzyme is not regulated by product inhibition as was the Lipomyces dextranase reported by Webb and Spencer-Martins [8].

The temperature and pH profiles of Lipomyces (ATCC 20825) dextranase mirrored the profiles reported by Webb and Spencer-Martins [8]. Although the stability of the ATCC 20825 dextranase at 60°C was poorer than the IGC 4047 enzyme [8]. The characteristics of the Lipomyces (ATCC 20825) dextranase with regard to the effects of pH and tem- perature on activity and stability are very similar to that of the Chaetomium enzyme [I81 and the Penicillium enzyme [19].

The purified enzyme showed the same K , values as report- ed for the IGC 4047 dextranase [8]. Both exhibited catalytic constants consistent with an endodextranase. Although there

REFERENCES 1. Barfoed, S. & Mollgaard, A. (1987) Zuckerindustrie (Berl.) 112,

391 -395. 2. Fulcher, R. P. & Inkerman, P. A. (1976) Proc. Queens Suc. Sugar

Cane Technol. 44, 295 - 305. 3. Inkerman, P. A. & Riddell, L. (1977) Proc. Queens Soc. Sugar

Cane Technot. 43, 21 5 - 223. 4. Inkerman, P. A. &James, C. P. (1976) Proc. Queens Soc. Sugar

Cane Technol. 43, 307 ~ 31 5. 5. Novo (1977) Information 12b-GB, pp. 1-7, Novo enzyme Di-

vision, Bagsvaerd, Denmark. 6. Miles (1983) Technical information L-1475, pp. 1 - 11, Miles Lab-

oratories, Elkhart, Indiana, USA. 7. Emeis, C. (1970) Federal Republic of Germany 0f;fi.n-

legungsschrift, patent no. 1 908 225, August 20, pp. 1 - 17. 8. Webb, E. & Spencer-Martins, I . (1983) Can. J . Microhid. 29.

1029-1095. 9. Koenig, D. W. & Day, D. F. (1988) Ann. N . Y. Acad. Sci. 542,

10. Koenig, D. W. & Day, D. F. (1988) J . Am. Soc. S q u r Cane

11. Sumner, J . B. (1924) J . B id . Chem. 62, 287-290. 12. Lowry, 0. H., Rosenbrough, J . , Farr, A. L. & Randall, R. J.

13. Day, D. F. & Koenig, D. W. (1988) Louisiany State University,

111 -114.

Technol. 8, 112-116.

(1951) J . B i d . Chem. 193, 265-275.

US patent no. 4,732,854, March 22, pp. 1-9.

167

14. Koenig, D. W. & Day, D. F. (1988) Biotechnol. Lett. 10, 127-

15. Laemmli, U. K. (1970) Nature 227, 680-685. 16. Dubois, M., Gilles, K . A., Hamilton, J. K., Rebers, P. A. &

Smith, F. (1956) Anal. Chem. 28, 350-356. 17. Montreil, J., Bouquelet, S., Debray, H. Y., Fournet, B., Spik,

G. & Strecker, G. (1986) in Carhohydrate analysis: apracticul approach (Chaplin & Kennedy, eds) pp. 143-204, IRL Press, Washington, DC.

122. 18. Hattori, A, , Ishibashi, K. & Minato, S. (1981) Agric. Biol. C'hem.

19. Chaiet, L., Kemf, A. J., Harman, R., Kaczka, E., Weston, R., Nollstadt, K. & Wolf, F. J. (1970) Appl. Microbiol. 20, 421 - 426.

20. Danilova, T. I., Maksimov, V. I., Chukhrova, A. I. & Molodova, G. A. (1983) Prikl. Biokhim. Mikrohiol. 19, 104-110.

21. Hiraoka, N., Tisuji, H., Funkumoto, J., Yamamoto, T. & Tsuru, D. (1973) J . Peptide Protein Res. 5, 161 -170.

45,2409 - 241 6.