Biochimica et BiophysicaActa, 1074(1991) 1-5 © 1991 ElsevierScience PublishersB.V. 0304-4165/91/$03.50 ADONIS 030441659100143Y BBAGEN 23489

Studies on N-acetylglucosaminidase activity produced by Streptomyces hygroscopicus A . I r h u m a , J. G a l l a g h e r , T . J . H a c k e t t a n d A . P . M c H a l e

Department of Microbiology, MoyneInstitute, Trini(vCollege. Dublin(Republicof Ireland) (Received 10 May 1990) (Revised manuscript received8 November 1990) Key words: N-Acetylglucosaminidase;Chitobiase;Chitinase:(S. I[vgroscopicusl Following growth on chitin-containing media the actinomycete, Streptomyces hygroscopicus produces N-acetylglucosaminidase. When the organism was grown in submerged culture on chitin-containing media, at 3 0 ° C , production of the N-acetylglucosaminidase activity has been shown to increase to a maximum at around day 18. Following electrophoretic analysis of culture filtrates and subsequent zymogram staining with fluorescent substrate analogues, at least three general pools of N-acetylghicosaminidase activity were detected. These were named NAI, NA2 and NA3. In addition, a potential chitinase was also identified. The N-acetylghicosaminidases, referred to above as NAI and NA2, were separated and their enzymatic properties were investigated.

Introduction Chitinases, a group of enzymes capable of degrading chitin to low molecular weight products have been shown to be produced by a number of microorganisms and sources of some of the most studied to data include Serratia marcescens [1], Streptomyces antibioticus [2], Streptomyces griseus [3], Vibrio harveyi [4] and Talaromyces emersonii [5]. A more comprehensive study of chitinase sources is reviewed by Cabib [6]. Since chitin represents a vast renewable fermentation feedstock of both carbohydrate and nitrogen, enzymes capable of it's bioconversion to low molecular weight fermentable products are of potential commercial value. In addition, the unique properties of chitin and commercial interest in utilisation of chitin and its derivatives have demonstrated the need for inexpensive reliable sources of active and stable chitinase preparations [7]. In order to effect complete hydrolysis of chitin to N-acetylglucosamine, a number of enzymes are required. These include chitinases (EC 3.2.1.14) which are

Abbreviations: NA, N-acetylglucosaminidase;MUG,4-methylumbelliferyl derivativeof N-acetylglucosamine;MUC, 4-methylumbeUiferyl derivative of chitobiose; MUT, 4-methylumbelliferylderivative of chitotriose. Correspondence: A.P. McHale, Department of Microbiology, Moyne Institute, Trinity College, Dublin 2, Republicof Ireland.

capable of degrading the insoluble substrate to low molecular weight products and chitobiases (N-acetyl-flglucosaminidases, EC 3.2.1.30) which are capable of degrading the low molecular weight products to Nacetylglucosamine [4]. Another enzyme which may p!ay a rolc in chitin degradation is N-acetylhexosaminidase (EC 3.2.1.51). Chitin has been shown to be an excellent carbon and nitrogen source for many Streptomyces strains, and chitino!ytic systems from such strains have been the subject of several publications [8-11]. In this study we report the production of significant quantities of extracellular, thermostable N-acetylglucosaminidase activity following growth of the actinomycete, Streptomyces hygroscopicus on chitin-containing media. In addition, we demonstrate, using a variety of synthetic substrates, the presence of multiple forms of N-acetylglucosaminidase/chitobiase activity and we report enzymological studies on two general pools of this activity. Materials and Methods

Microorganism. The actinomycete, Streptomyces hygroscopicus was obtained from the National Collection of Industrial and Marine Bacteria, Aberdeen, U.K. and maintained on Lemco agar plates (Oxoid) at 30 o C. The organism was grown in submerged culture by inoculating flasks containing 0.5% (w/v) bacto-peptone (Oxoid), 0.5% (w/v) KH2PO 4, 1% (w/v) ammonium nitrate,

0.05% CaCI2, 0.05% (w/v) MgSO4, 0.1% (w/v) Na2SO4, 1% (w/v) chitin and 2% trace salts as described previously [12]. The pH of the medium was adjusted to 7.0 using NaOH. Inoculated flasks were incubated at 3 0 ° C in an orbital shaking incubator. Extracellular enzyme was recovered in the supernatant after eentrifugation at 5000 × g for 15 min at 4 ° C . N-acetylglucosaminidase activity. Activity was assayed for by incubating 2 mM p-nitrophenyl-fl-N-acetylglucosaminide (PNAG) (Sigma, U.K.) or p-nitrophenylfl-chitobioside (PNAC) (Sigma, U.K.) in 0.1 M phosphate/citrate buffer at pH 5.0 with 20 /~1 of culture f!ltrate for 15 rain a' 4 0 ° C . Reactions were terminated by the addition 9f 1 ml of 0.4 M glycine-NaOH buffer (pH 10.8) and the absorbance at 430 n m was determined. Activity was expressed as ttmol of pnitrophenol rel,~ased per rain per ml of enzyme.

cally active bands were visualised using an ultraviolet transilluminator.

Polyacrylamide gradient gel electrophoretic analysis of culture filtrates and zymogram staining for components of the chitinolytic system. Polyacrylamide gradient gel elec-

Results

trophoretic analysis of culture filtrates was carried out on samples of culture filtrate which were harvested following 20 days of growth and concentrated 20-fold using an Amicon ultrafiltration unit which contained a PM10 membrane. The apparatus was maintained at 4 ° C . Desalting was achieved by passage of distilled water through the concentrate in the Amicon apparatus. Approx. 25 p,g quantities were applied to each gel lane and electrophoretic conditions were as described previously [13]. Following eleetrophoresis, gels were stained for enzyme activities by direct immersion in 1.0 m M solutions of 4-methylumbelliferyl derivatives of Nacetylglucosamine, chitobiose and chitotriose (MUG, MUC, MUT) (Sigma, U.K.) in 0.15 M p h o s p h a t e / citrate buffer (pH 5.0). Incubation times at room temperature were dependent upon the amount of activity applied to the gels prior to electrophoresis. Enzymati-

0.20 0.18 0.16

~

o.14

"~

0.12 0.10

:.

0.08 0.06 0.04

~" ~

0.02 0.00

1~

1~

20

TIME (days) Fig. 1. Production of N-acetylglucosaminidaseby S. hygroscopicus during growth on chitin-containing medium. Culture supernatants were harvested at the times indicated and assayed for activity using PNAG as suhstrate.

Separation of N-acetylglucosaminidases, NA1 and NA2. In order to separate pools NA1 and NA2, described in the Results section and shown in Fig. 2 below, samples of culture filtrate were concentrated by ultra-filtration and electrophoresed on the polyacrylamide gradient gels as describe above. A section of each gel was then stained with the relevant substrate and this was used in order to locate the required enzyme activity on the unstained portion of the gels. Areas containing activities NA1 and NA2 were then excised from the unstained gels and these were then minced, separately. The minced gels were then placed in tubes containing 0.1 M citrate phosphate buffer (pH 5.0) and the relevant e,cti¥~ties were allowed to leach out overnight.

Production of N-acetylglucosaminidase by S. hygroscopicus following growth on chitin-containing media Results obtained following assaying" of culture supernatants for activities capable of degrading the pnitrophenyl substrate derivatives are shown in Fig. 1. It was found that the organism produced N-acetylglucosaminidase activity up to a maximum of 0.19 U (0.19 /xmol of p-nitrophenol released/rain) per ml at day 18, after which time activity production began to decrease. Since chitin was present in the medium at this stage it was deduced that production of enzyme ceased as a result of depletion of some other component in the medium.

Polyacrylamide gradient gel electrophoretic analysis of culture filtrates and zymogram staining for N-acetylglucosaminidase activity In order to determine whether or not this organism was capable of producing a number of different forms of N-acetylglueosaminidase, it was decided to analyse culture filtrates using polyacrylamide gradient gel eleetrophorcsis and zymogram staining using a variety of artificial substrates. When the gels were stained using 4-methyhimbelliferyl-fl-N-acetylglucosaminide (MUG), a smear of activity was evident on the gels (lane 1, Fig. 2) and no discrete bands could be detected. When the gels were stained using the chitobioside derivative (MUC), a diffuse area at the top of the gel stained for activity (lane 2, Fig. 2). In addition, two bands were also observed towards the bottom of the gel (lane 2, Fig. 2). The two bands at the bottom of lane 2, Fig. 2, were absent in lane 1 and these results suggest that, at least two functionally different forms of N-acetyiglucosaminidase are produced by S. hygroscopicus. It is also interesting to note that a discrete green fluorescent band was also observed at the centre of the gel. This is not an active band as it occurs in lanes which have not

W

TABLE I

Properties of the N-at'etylglucosaminidasecontained in pool NA 1

A3

Temp. opIim,im pH optimt~m t :, at 40°C ll,~ at 50°C tl~ at 30°C K.~ (mM) ~,.~ (U)

.e.

NA 1

~2 1

2

3

zl.

Fig. 2. Zymogramstaining polyacrylamidegradient gels for N-acetylglucosaminidase activity using MUG (lane I). MUC (lane 2) and MUT (lane 3). Lane 4 is a lane which was unstained. Gels contained electrophoresed culture supernatent as described in the Materials and Methods.

been exposed to substrate (lane 4, Fig. 2). The function of this band is unknown and will not be referred to again in this paper. When the gels were stained using the chitotrioside derivative (MUT), staining appeared somewhat similar to that pattern obtained in lane 2 of Fig. 2 (lane 3, Fig. 2), with the -xception of the decrease in staining of the lower two bands observed in lane 2. Again staining in this lane appeared to be smeared although smearing was not as great as that obtained in lane 1, Fig. 2. It should be noted that the existence of this smearing phenomenon will be addressed in the Discussion below. On the basis of the results obtained above, two major pools of N-acetylghicosaminidase were chosen for further study and these are named NA1 and NA2. The pools represent the protein extracted from the gel areas between the arrows marked in Fig. 2. For the purposes of this study the pool NA3 was excluded on the basis that it could represent a chitinase as distinct from an N-acetylglucosaminidase. As shown in zymogram staining above, NA1 appeared to be solely specific for MUG and NA2 was solely specific for MUC. This difference in specificity was also evident when the p-nitrophenol substrate analogues were utilised. The pool containing NA1 was shown to be incapable of hydrolysing PNAC or chitin. The pool containing NA2 was shown to be incapable of hydrolysing PNAG or chitin.

Enzymological studies on N-acetylglucosaminidase, produced by S. hygroscopicus

NA 1,

Using the substrate PNAG It was found that NA1 exhibited conventional Michaelis-Menton kinetics, having a K m for this substrate of 0.12 mM and a Vm~~ of 0.058 U / m l (Table 1). It was found, however, that the

15-70° C 4.0-5.0 stable up to 21 h 18 h 2.5 rain 0.12 0.058

enzyme was subject to substrate inhibition at concentrations of substrate in excess of 2.0 mM (data not shown). This enzyme pool failed to degrade chitin. When the temperature optimum of the N-acetylghtcosaminidase. NA1, was examined it was found to be relatively broad, ranging from 4 5 - 7 5 ° C (Table I). In addition the enzyme activity was found to have a q/2 of 18 h at 50°C, 2.5 min at 70°C. There was no loss in activity over a period of 24 h at 4 0 ° C (Table I). The pH optimum for this activity ranged between 4-5 with approx. 60% of the activity remaining at pH 3.5.

Enzvmological analysis of N-acetylglucosammidase, produced by S. hygroscopicus

NAZ

When PNAC was utilised as substrate, this pool of activity had a K,,~ for that substrate of 0.76 mM and the enzyme had a Vmax of 0.8 U / m l (Table 11). As mentioned above this enzyme pool failed to hydrolyse PNAG or chitin. This activity, in contrast to that in pool NAI, had a sharp pH optimum at pH 5.0 and a sharp temperature optimum at 65nC ('fable II). With respect to thermal stability of this activity, pool NA2 was stable for 24 h at 4 0 ° C and had a tt/z of 16 h at 50°C. The enzyme was also shown to have a tt/2 of 5 min at 7 0 ° C (Table 11).

The effect of metal ions on N-acetylglucosaminidase activity in pools NA1 and NA2 in order to determine the effects of metal ions on the activity, enzyme was assayed for in the presence of a number of metal ions. The results obtained, are shown in Table 11I. In some cases a slight increase in activity in the presence of the metal ion was observed, e.g., calcium, potassium and manganese. Mercury was found TABLE II

Properties of N-acetylglucosaminidase in pool NA2 Temp. optimum pH optimum t~/2 at 40°C tl/2 at 50°C tl/z at 70°C Km(mM) Vma ~ (U)

65oC 5.0 stable for 24 h 16 h 5 rnin 0.76 0.80

TABLE 111 The effects of a variety of metals on N-acetylglueosaminidase activity in pools NAI and NA2 Activities were determined using the p-nitrophenylsubstrate derivatives and activities are expressedas a 70of the ~ntrol assayed under normal conditions as described in the Materials and Methods. Each salt was present at a concentration of 50 mM. Metal 14gCI~ CuSO4 CaCI2 MgSO4 KCI ZnSO4 MnSO4

NA1 NA2 (70of activity in control) 7 0 100 90 123 125 98 98 102 102 90 90 124 117

to be the most potent inhibitor of activity in both cases. It should be noted from Table III, that the inhibition/ activation patterns by the metals on both forms of activity were remarkably similar.

Discussion In a number of previous studies it has been demonstrated that Streptomyces strains are capable of utilising chitin as a source of carbon and nitrogen [9,10]. Many of these strains have been shown to produce various forms of chitinolytic activity [11]. In this study it has been demonstrated that the actinomycete, S. hygroscopicus has the ability to grow on chitin-containlng media, hence one might presume that the organism was czpable of producing a chitinolytic system. Using PNAG as a substrate in order to assay culture supernatants, it has been demonstrated that the organism was capable of producing N-acetylghicosaminidase activity and enzyme production peaked at around day 18-20. When culture ,~iltrates were assayed for chitinase activity using chitin as the substrate, no activity was detected. However, when culture filtrates were concentrated and then assayed, chi,inase activity was detected (data not shown) although the amounts of activity were extremely low. The low levels of chitinase activity, may explain why it takes up to 20 days for N-acetylglucosaminidase production to peak. In the past, the use of polyacrylamide gradient gel electrophoresis in combination with zymogram staining has facilitated the resolution and identification of a number of fungal and bacterial carbohydrase systems [12,13]. Robbins et al. [11] have demonstrated that methylumbelliferyl substrate analogues could be utilised in order to facilitate ~'hc molecular cloning of Streptomyces chitinase genes. Wortman et al. [14] have demonstrated that methyhimbelliferyl N-acetylglucosaminide and chitobioside could be utilised in order to

detect expression of chitobiase/N-acetylglucosaminidase encoding genes. In this study we have utilised MUG, MUC and MUT as probes for N-acetylglucosaminidase and possibly chitinase activity on electrophoresed, nonde~aaturing polyaerylamide gradient gels. As shown in the results section, at least two general pools of Nacetylglucosaminidase activity were detected and differentiated between, using the MUG and MUC and these were named lqA1 and NA2. A third p0ol was identified which had the ability to degrade MUT and this was named NA3. Because of the ability of this pool to degrade the MUT, we believe that this may possibly be a chitinase containing pool. This pool was relatively incapable of degrading either the MUG or the MUC. The reason why the two bands in pool NA2 were totally specific for MUC and the activity in pool NA1 was solely specific for MUG is as yet unknown. It should be mentioned that Streptomyces plicatus has been reported to produce a "chitinase-63' which has high activity when MUT is used as t~z substrate and has relatively less activity when MUC is used as the substrate [11]. In addition, this organism produces a 'ehitinase-47' which exhibits low activity when the MUT was used as substrate and has a 10-fold higher activity when the MUC is used as substrate [11]. This situation is somewhat similar to our results although we would like to emphasise that enzyme in pool NA1 exhibited no activity when either MUC or PNAC were used as substrate and enzyme in pool NA2 exhibited no activity when either MUG or PNAG were used as substrates. It is obvious from the patterns obtaine2 following zymogram staining of gels that a significant degree of smearing occurs. It was initially felt that this may have been the result of proteolytie degradation since the samples were harvested at a relatively late stage during fermentation. However, when samples were harvested at 7 days this phenomenon was also evident. Reducing the staining time also failed to yield sharper patterns. Microheterogeneity resulting from some form of covalent modification such as glycosylation of extracellular proteins produced by eukaryotes, does, on occasion, result in smearing of proteins on some electrophoretic systems, although this is somewhat unlikely in our case. As far as we are aware there is no precedent for this result in the literature. In this study, enzymologicai analysis of the electrophoretically separated pools NA1 and NA2 were performed using PNAG and PNAC. Enzyme in pool NA1 had a Km for PNAG of 0.12 mM and failed to hydrolyse PNAC. This Km was significantly lower than values reported for other enzymes in the literature, with a reported K m of 0.45 mM for the N-acetylglucosaminidase from the chitinolytic fungus Pycnoporus cinnobarinus [4] and a reported K m of 0.25 mM for a N-acetylhexosaminidase from Vibrio harveyi [15]. Enzyme in pool NA2 had a K m of 0.76 mM for PNAC and failed to hydrolyse PNAG. This differs significantly

from the Km reported for enzyme in pool N A I using PNAG as substrate. Other major differences between enzyme in both pools includes the difference in p t t optima, with the optimum range for activity in pool NA1 being broad, while the optimum profile for enzyme in pool NA2 was very sharp. The optima for both enzymes, however, compared favourably with the optimum reported for the N-acetylglucosaminidase produced by P. cinnobarinus. The pH optimum for that enzyme was reported to be 5.4 [4]. On the other hand, however, the N-acetylglucosaminidase produced by V. harveyi had a pH optimum ranging between 6 - 9 [15]. It would appear that the thermal stability data obtained in this study for enzyme in pools N A ! and NA2 were remarkably similar. Both forms of activity have half-lives of 16 and 18 h at 5 0 ° C , while the N-acetylglucosaminidase from P. cinnobarinus was shown to be 'unstable above 5 0 ° C ' [4]. In comparison with other thermal stability studies on similar enzymes from th~ thermophilic fungus, T. emersonii [5], the enzymes in pools NA1 and NA2 are surprisingly thermostable. Enzyme activities in pools NA1 and NA2 also differed with respect to the temperature optimum profiles in that enzyme in pool NA1 had a very broad optimum range, while enzyme in pool NA2 had a very sharp optimum at 65 °C. The activities in both pool NA1 and NA2 were extremely sensitive to the presence of mercury and this is in contrast to results obtained with the N-acetylglucosaminidase produced by P. cinnobarinus which demonstrated only slight inhibition in the presence of that metal ion [4]. in the studies presented here many of the metals had very little effect on both activities and this has also been reported to be the case with the enzyme produced by V. harveyi [15]. It was interesting to note that the minor effects of the metals on each activity were relatively similar. It is also interesting to note that both calcium and manganese had considerable stimulatory effects on bottl activities. Since only very low levels of true chitinase activity were detected in culture filtrates it is possible that this organism utilises the N-acetylglucosaminidase activities in order to remove small quantities of relatively accessi-

ble chitooligosaccharides from the solid chitin in the medium. It is our intention to continue our studies with the enzymes characterised in this study. Such continuation will involve the isolation of genes encoding the relevant activities with a view towards understanding the mechanisms by which these enzymes are expressed by the organism at a molecular genetic level. In addition, it is our intention to characterise the enzyme in pool NA3 which apparently is capable of degrading the MUT ~ubstrate. Acknowledgements The authors wish to acknowledge funding for this study by the Libyan Government. A. Ihruma is in receipt of a Student's Wife Scholarship from the Libyan Government. Rcfere_nees 1 Monreal.J. and Reese, E.T. (1969)Can. J. Microbio! 15, 689-695. 2 Jeurtiaux, C. (1966) Methods Enzymol.8, 644-651. 3 Berfer, L.R. and Reynolds, D.M. (19581 Biochim. Biophys. Acta 29, 522-526. 40htakara, A. (1988) Methods Enzymol. 161,462-470. 5 Hendy, L., Gallagher.J., Winters, A., Hackett, T.J., McHale, L. and McHale, A.P. (1990) Biotechnol. Lens. 12, 673-678. 6 Cabib, C.P. (1987) Adv. Enzymol.59, 59-101. 7 Wadsworth, S.A, and Zikakis`J.P. (1984) J. Food Sci. 43, 11581164. 8 Beyer. M. and Diekmann, H. (1985) Appl. Microhiol. Biotechnol. 23, 140-146. 9 Trimble, R.B., Tarentino, A.L., Evans,G. and Maley, F. (1979)J, Biol. Chem. 254, 9708-9713. 10 Abeles,F.B., Bossart, R.P., Forrence, L.E. and Habig,W.H. (1970) Plant Physiol.47,129-34. ll Robbins`P.W., Albright,C. and Benfield, B. (1988)J. Biol. Chem. 47, 443-447. 12 McHale, A.P. and Morrison, J. (1986) Enz. Microbiol.Technol. 9, 749-745. 13 Bunni, L., MeHale, L. and McHale, A.P. (1989) Enz. MierobioL Technol. 11,370-375. 14 Wortman, A.T., Sommetwille,C.C. and ColwelLR.R. (1986) Appl. Env. Microbiol.52, 142-145. 15 Soto-Gill,R.W., Childers, L.C., Huisman, N.H., Daphris, S., Jannatipour, M., Hedjran. F. and Zyskind, J.W. (1988) Methods Enzymol. 161,524-529,