Purification and characterization of chitinases from transformed callus suspension cultures of Trichosanthes kirilowii Maxim.

JOURNALOF FERMENTATION AND BIOENGINEERING Vol. 84, No. 1, 28-34. 1997

Purification and Characterization of Chitinases from Transformed Callus Suspension Cultures of Trichosanthes kirilowii Maxim. N.-J. REM1 SHIH AND KAREN A. MCDONALD* Department of Chemical Engineering and Materials Science, University of California at Davis, Davis, CA 95616, USA Received 21 November 1996/Accepted

27 May 1997

Three extracellular basic chitinases designated as TKC 15, TKC 28-1, and TKC 28-H were purified from Agrobacterium rhizogenes A4 transformed Trichosanthes kirilowii Maxim. callus suspension cultures using PerSeptive HS/M cation exchange and Sephadex G 75 S gel filtration chromatography. These chitinases exhibited maximal activity at pH 6 and temperature at 40-45”C. N-terminal analysis suggests that two chitinases (TKC 28-I and TKC 28-11) with indistinguishable molecular masses (28 kDa) belonged to the Class IH chitinase family. Another basic protein with a molecular mass of 15kDa (TKC 15) also possesses chitinase activity. Chitobiose was the major end product from chitin digested by TKC 28-I and TKC 28-H whereas TKC 15 released a mix of tetramers, trimers and dimers from chitin. Slow cleavage of chitotriose by TKC 28-I and 28-11 and no cleavage of tetramers and trimers by TKC 15 were observed. TKC 28-I cleaved tetramer faster than trimers, 1.1 x 10e4 M-h-l -N-l and 1.5 X 10m6M. h-l - ,q-l respectively. All chitinases showed inhibitory ability in a cell-free protein translation system but were far less potent than trichosanthin, a ribosome inactivating protein, found in the storage root tuber of T. kirhowii. The purified T. kirilowii chitinases did not show antifungal activity against Aspergillusflavus or Trichoderma viride. [Key words: chitinases, plant cell suspension cultures] families, which are induced under different environmental stimulation (14). A sugar beet acidic class III chitinase (SE) was strongly induced and expressed in transgenic tobacco during the infection with the fungus Cercospora beticol (16). However, a class III chitinase (IF3) was found in the healthy, nonstressed cell suspension cultures of healthy Lupinus albus. Thus, in addition to stress stimulation, plant chitinases may also exist as constitutive defense proteins against fungal pathogens (17). Trichosanthes kirifowii Maxim. (Cucurbitaceae) is a Chinese medicinal plant. Storage roots from T. kirilowii contain a bioactive protein, trichosanthin, a RIP that is the active constituent in an extract that was used to induce mid-term abortions in ancient China and is currently being studied due to its antiviral properties (18). RIPS are known to inhibit protein translation by deactivating ribosomes in a very specific manner (19). Savary and Flores (20) have studied the production of proteins from transformed hairy roots of T. kirilowii. Though transformed root cultures can produce some root-specific metabolites much more efficiently (21-26), few studies have dealt with transformed callus. Savary and Flores (20) found that transformation by Agrobacterium rhizogenes did not stimulate trichosanthin production, and they have identified three Class III chitinases in the transformed T. kirilowii Maxim. var joponicum hairy root. However, characteristics of chitinases secreted from T. kirilowii callus growing in suspension have never been fully studied. In this report, we describe the purification and some properties of basic chitinases secreted from A. rhizogenes A4 transformed T. kirilowii Maxim. callus grown in suspension.

Chitinases (EC 3.2.1.14) are hydrases that cleave the (144)~P-N-acetyl-glucosamine linkage in chitin. On the basis of their amino acid sequence, plant chitinases have been classified into three classes. Class I consists of chitinases with a cysteine-rich domain near their aminoterminal and a catalytic site domain whereas class II chitinases only share the catalytic domain with class I chitinases. Class III are the isomers that are not homologous to Class I or Class II chitinases (1, 2). Chitinases produced by the higher plants are considered as protection agents for plants against fungal pathogens by degrading chitin, a major component of the cell wall in many fungi, or by inhibiting spore germination and mycelial growth of fungi (3-5). However, not every chitinase exhibits antifungal activity. Tobacco Class II chitinases (6) as well as chickpea Class III chitinases (7) lack antifungal activity. Some defense-related proteins such as p-1,3-glucanase and ribosome-inactivating proteins (RIPS) when mixed with purified chitinases exhibit high antifungal activity (4, 5, 8, 9). A class I chitinase (CBP20) isolated from tobacco was found to have a synergistic protective effect with j-1,3-glucanase against the fungi Fusarium solani and Alternaria radicina (10). Similarly, the combined expression of class II barley chitinase and P-1,3-glucanase in transgenic tobacco provided protection against Rhizoctonia solani (11). Also, some chitin-binding proteins that exhibited antifungal activity do not have any detectable chitinase activity (12, 13). In addition to the classification based on amino acid sequence, all chitinases can be also divided into two categories according to their isoelectric points, acidic and basic. Acidic chitinases are generally secreted extracellularly whereas basic chitinases accumulate in the central vacuole (14, 15). The acidic and basic isoforms of chitinases in higher plants are encoded in multigene

MATERIALS

AND METHODS

Materials Aspergillus flavus and Trichoderma viride were provided by Department of Food Science, University of Massachusetts, Amherst. Trichosanthin

* Corresponding author. 28

VOL. 84, 1997

was obtained from GeneLabs Inc. (Redwood City, CA, USA). The cell-free protein translation system was purchased from Promega (Madison, WI, USA). Chitin, chitinase, chitin azure, chitotriose and chitobiose, chitotetraose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Transformation Transformation procedures followed the method designed by Thorup et al. (27). Briefly, sterile stems of T. kirifowii Maxim. were treated with A. rhizogenes A4 (1.0x 10r°CFU.ml-l) for 10min and incubated at room temperature in a petri dish containing Linsmaier and Skoog (LS) medium plus 100 PM acetosyringone and cefotaxime (350 pg. ml-i) until the transformed root and tumor callus arose from the infection site. The transformed tumor calli were obtained by serial transfers of healthy calli to new LS medium and maintained at room temperature in the dark. Preparation of crude proteins Three to four grams of transformed T. kirilowii callus was transferred into 250 ml flasks containing 100 ml of LS medium. The cultures were maintained at room temperature with gentle shaking (150 rpm) for 21 d. The harvested extracellular fluid from five shake flasks was collected by filtration (Whatman no. 1 filter), dialyzed against 20mM phosphate buffer (pH 7.0) in a Amicon (Beverly, MA, USA) stirring cell with a YM 10 membrane and concentrated in the same cell at 4°C. The concentrated sample (0.81 mg protein.ml-‘) was stored at 4°C for further analysis. Protein purification Crude extracellular concentrate (8 mg total protein) was filtered through a 0.2 pm filter membrane before it was injected into a 10 x 100 mm (20(tm) PerSeptive HS/M cation exchange column (PerSeptive Biosystems Inc., Framingham, MA, USA) in a Hewlett Packard 1050 HPLC. The column was washed with mobile phase A (20mM phosphate buffer at pH 7.0) for 5 min and the proteins were eluted with a linear increase in mobile phase B (1 M NaCl in 20 mM phosphate buffer at pH 7.0) from 0 to 4% in 20 min and then an additional 5 min from 4 to 24%. The column was regenerated with 100% mobile phase B for 2 min followed by 100% mobile phase A for 5 min before the next sample was introduced. The flow rate was 5 ml. min l and protein detection was performed by a UV detector at 280nm. Fractions corresponding to chitinase activity from eight chromatography runs were collected and concentrated with an Amicon stirring cell to l-2ml before being injected into a Sephadex G-75 S column (2.6 x 74 cm) equilibrated with 20 mM phosphate buffer (pH 7.0). Proteins were eluted with the same buffer at a flow rate of 25 ml. h-l at 4°C and were stored in 200 mM NaCl solution at 4°C for further analysis . Protein conProtein analysis and electrophoresis centration was analyzed with the Bradford method using bovine serum albumin as a standard protein (28). SDSPAGE was performed in 13% polyacrylamide gels in a BioRad mini Protean II gel electrophoresis apparatus according to the manufacturer’s manual. The proteins in the polyacrylamide gel were visualized with silver staining (29). A BioRad glycoprotein detection kit (BioRad Laboratories, Hercules, CA, USA) was employed to detect glycoprotein. Proteins (5-6 pg) separated by Peptide mapping SDS-PAGE were digested in the 13% polyacrylamide gel with protease Lys C (0.5 pg) overnight at 30°C in 0.1 M

T. KIRILO

WII CHITINASE

29

Tris-HCl (pH 9.0), 0.05% SDS. Peptides were extracted with water and 70% acetonitrile-0.1% triflouroacetic acid (TFA) two times. Extracted peptides were alkylated with iodoacetamide in 6 M Guanidine HCl-0.4 M TrisHCl (pH 8.0). Alkylated peptides were then digested with trypsin overnight in 1.5 M Guanidine HCl, I M Tris, pH 8.0. The digested peptides were injected into an HLPC equipped with a reversed-phase Cl8 column (1 mm x 25 cm). After injection of the sample, the column was washed with 100% solvent A (0.1% TFA in water) for 15 min and then with a gradient of 0% to 5% solvent B (70% acetonitrile-0.075% TFA) for 5 min. Peptides adhering to the column were then eluted with a linear gradient of 5% to 60% solvent B over 70min. The flow rate was 75 pl-min-’ and the peptides were detected by a UV detector at 210 nm. Chitinase activity was determined Chitinase activity by the method of Pedraza-Reyes and Lopez-Romero (30). Briefly, 0.5 ml of protein solution was mixed with 1 mg chitin azure in 0.5 ml of 50 mM phosphate buffer (pH 6.5) with 1OmM MgC&. The reaction mixture was incubated at 30°C with gentle shaking for 48 h followed by centrifugation at 14,000 x g for 4 min. The absorbance at 515 nm of supernatant fluid was then examined. Chitinase activity is defined as the amount of protein that results in an increase of 0.01 A5,* units under the above conditions. Protein translation inhibitory activity determination A cell-free luciferase translation system (Promega) was employed to examine the protein translation inhibitory ability of the tested protein. The protein sample (2.5 ~1) was mixed with 5 ~1 of rabbit reticulocyte lysate for 15 min at room temperature followed by luciferase translation carried out according to the manufacture’s manual. One ~1 of protein translation cocktail (a mix of 10 ~1 luciferase mRNA, 30~1 amino acid mixture and 5 /*l RNAsin in a final volume of 45 ,ul) was introduced to the treated rabbit reticulocyte lysate. The whole reaction mixture was incubated at 30°C for 2 h and the translated luciferase activity was detected by a luminometer. The protein translation inhibitory concentration (ICso) is thus defined as the concentration of protein in the tested sample solution that causes 50% reduction of luciferase activity compared with the control (20mM phosphate buffer, pH 7.0, instead of tested protein sample). In-vitro antifungal assay A chitin containing fungus, A. flavus, and a plant-pathogenic fungus, T. viride, were used in the antifungal assays. A loopful of growing test fungus was plated in the center of a petri dish containing nutrient agar. Sterile filter paper discs containing 1Opg of trichosanthin (at a concentration of 400 pg. ml-l) were laid on the agar surface around the fungus and 35 /*l of chitinase (20-300 pg.ml-‘) were applied to the discs. The plates were then incubated at room temperature. A clear boundary of fungal growth will be seen on the petri dish if the test sample is antifungal (9). To examine the effect of chitinases on spore germination, chitinase (5 pg) was incubated with a spore suspension in 100 ~1 of 1.5 malt extract (pH 6.0) at 25°C for 24 h. The percentage of spore germination was calculated by counting 200 individual spores under a phase-contrast microscope. Detection of chitinase digestion mixture The hydrolyzed products of chitinase digestion were analyzed by thin layer chromatography (TLC) (31), or by HPLC equiped with a BioRad Aminex@ HPX-42A column (300 X

30

J . FERMENT. BIOENG.,

SHIH AND MCDONALD 1.2

250

1.0

200 s 150

0.8

g i 100 8

fl 0.6 x 0.4

0

Fl F2

-I

kDa

8

-F3

--

:

:

:

:

7 -g

:

65 5s 4 .g 3:

,...

t 2 .c .Y 1 0 r 0

_... ,,....‘.

:,A,: O.O 8

18.4-

10 12 14 16 18 20 22 24 26 28

12.4-

Time (min)

FIG. 1. Chromatogram of extracellular protein concentrate from transformed T. kirilowii callus tissue culture on PerSeptive HS/M cation exchange column. Symbol and lines are: 0, chitinase activity; -9 Azsa; .--‘.-, NaCl.

7.8 mm). The HPLC column was held at 85°C and the mobile phase is water with a flow rate of 0.3 ml.min-l. RESULTS Five hundred ml of Purification of basic chitinases extracellular fluid was collected from five shake flasks containing the transformed T. kirilowii callus cultures. The elution profile of the crude extract from a PerSeptive HS/M cation exchange column is shown in Fig. 1. Chitinase activity was found in three fractions designated as Fl, F2, and F3. The Fl fraction contains TKC 281 as the major protein, F2 contains TKC 15, and F3 contains TKC 28-11. Each fraction was collected and the minor protein impurities were further removed by Sephadex G-75 S chromatography. Final yields of 33, 6, and 30% based on the total activity were recovered during the purification procedure and 1.5, 4.6, and 4.7 fold purifications based on the increased specific activity of each protein were achieved for TKC 15, 28-1, and 28-11 respectively (Table 1). The three basic T. kirilowii chitinases obtained by gel permeation chromatography were examined by silver stained SDS-PAGE and each revealed a single band of protein (Fig. 2). The molecular weights of purified T. kirilowii chitinases were determined using matrix assisted laser desorption mass-spectrophotometry. TKC 28-I and TKC 28-11 are proteins with 28 kDa molecular mass (28,345+92 Da and 28,540&68 Da respectively) whereas TKC 15 is a 15 kDa protein (15,214 Da&S). None of the three chitinases (up TABLE 1.

28-l

Specific activity (U .mg-I)

Yield (%)

Fold purification

33.70 32.00

100.0 93.1

1 0.9

105.24 155.34

33.5 30.5

3.1 4.6

46.20 50.99

10.9 6.7

1.4 1.5

108.76 158.87

39.2 33.6

3.2 4.7

15

284

FIG. 2. SDS-PAGE of purified T. kirilowii chitinases. The proteins (1 ,ng) were developed in a 13% polyacrylamide gel and visualized with silver staining.

to 2.5 ,ug of total protein tested) were found to be glycosylated using the BioRad glycoprotein detection kit (data not shown). Characterization of purified basic proteins The three basic T. kirilowii chitinases exhibited hydrolytic activities in the range of pH 5-7 with a maximal activity at pH 6 under our assay conditions (Fig. 3). Maximal chitinase activity was observed at 40°C for TKC 15 and at 45°C for TKC 28-I and TKC 28-11. TKC 28-I and TKC 28-11 retained more than 60% of their maximal activity at 60°C under our assay conditions. All three T. kirilowii chitinases are basic proteins existing as monomers with p1 values greater than 8.5 (data not shown). Hydrolysis action of basic T. kirihwii chitinases Hydrolysis of chitin by T. kirilowii chitinase revealed that chitobiose was the major end product of TKC 28-I and TKC 28-11 whereas TKC 15 released a mix of dimers, trimers and tetramers (Fig. 4). No hydrolysis of chitotriose nor chitotetraose by TKC 15 was observed. TKC 28-I and TKC 28-B hydrolyzed chitotetraose faster than chitotriose (Fig. 5). The hydrolysis rate of chitotetraose and chitotriose by TKC 28-I is 1.1 X lop4

T*-

1008

80 .S

Purification of extracellular basic chitinases from transformed T. kirilowii callus culture

Total activity (U) Harvest medium fluid 1229 Ultrafiltration 1144 TKC 28-I PerSeptive HWM 412 Sephadex G 75 S 374 TKC 15 PerSeptive HS/M 133 Sephadex G 15 S 82 TKC 28-D PerSeptive HWM 481 Sephadex G 15 S 412 Step

MW

-

60

-

40 20

-0

I I

3 4

I

I

I

I

I

5 6 7 8 9 PH

30

I

I

I

40

50

60

” ?J al p d

’

Temp (“C)

Effect of pH and temperature on the chitinase activity of purified basic T. kirilowii callus chitinases. Symbols are: 0 , TKC 15; 0, TKC 28-I; and, a, TKC 28-H. Fifty /Ig of purified chitinase was mixed with 1 mg chitin azure in 1 ml of 1OOmM buffer at 30°C for 48 h. Buffer systems are: pH 3-4, sodium acetate; pH 5-7, sodium phosphate, pH 8-9, Tris-HCl. For the effect of temperature on the T. kirilowii chitinases, 50 ,ug chitinase was incubated with 1 mg chitin azure in 1ml of 50 mM phosphate buffer containing 10 mM MgCll (pH 6.5) for 48 h. FIG.

3.

VOL. 84, 1997 60 TKC 28-11

50

TABLE 2. In vitro protein translation inhibition of proteins isolated from transformed T. kirilowii callus 2

Protein

40 ZJ 2

31

T. KIRILO WI1 CHITINASE

ICso (M) 3.7io.5 x 10-g 5.4k1.5 x 10-7 6.010.9x lo-’ 8.6Fl.l x 10-7

Trichosanthin TKC 28-I TKC 15 TKC 28-11

30 20 3

10 :: 0 28

32

36

28

32

36

28

32

36

Time (min) FIG. 4. HPLC elution profile of chitin digestion by T. kirilowii chitinases. Chitin (1 mg) was digested with 25 pg of T. kirilowii chitinase for 48 h at 30°C in 200 ~1 of 50 mM phosphate buffer @H 6.5). 2, Chitobiose; 3, chitotriose; 4, chitotetraose.

other plant Class III chitinases such as Azuki bean, cucumber, rubber tree, pokeweed, sugar beet, and sweet orange (1, 15, 16, 32-34) (Fig. 6). The digestion of TKC 28-I and TKC 28-11 by protease Lys C and trypsin gave similar peptide elution patterns on the Cl8 reverse phased HPLC except peptides 13 and 14 were missing in TKC 28-I whereas peptides 11 and 12 were missing in TKC 2811 (Fig. 7). DISCUSSION

M.h-~*./lggI and 1.5~10-~M.h-~./lg-~ respectively. TKC 28-I and 28-11 hydrolyzed tetramers into dimer and trimers into dimers and monomers (data not shown). Effect of basic chitinases on in vitro protein translation All three T. kirilowii chitinases had ICJOs of 5.5 to 8.5 x 10.. 7 M in the in vitro protein translation assay. Compared to trichosanthin, a ribosome inactivating protein with an I&, of 3.7 x 10 -g M, they are considerably less potent (Table 2). However, they inhibited protein translation much more strongly than BSA, which had shown no inhibitory effect up to 8 X 1O-6 M (data not shown). Inhibition of fungal growth by chitinases and TrichoAll three basic T. kirifowii chitinases failed santhin to inhibit mycelium growth of A. flavus and T. viride up to a concentration of 300 pg. ml-l when combined with 101-18 trichosanthin. The three T. kirilowii basic chitinases also had no effect on germination of A. Jlavus (p>O.16). Although only 15-20% of A. Javus spores germinated in presence of chitinase, while 19.6% of the spores germinated in the untreated control. Distinguishing characteristics of the chitinase isomers Amino-terminal sequences for a number of chitinases are given in Fig. 6. TKC 28-I and TKC 28-11 exhibited the same amino acid sequence for the first 12 residues. The same 12 amino sequence was also found in the Class III acidic chitinases isolated from T. kirifowii transformed hairy root and cucumber. TKC 28-I and TKC 28-11 also showed N-terminal amino acid sequences similar to 100

0

12

24

38

48

0

12

24

36

48

0

12

24

36

48

Time (h)

FIG. 5. Hydrolysis of chitotetraose and chitotriose by T. kirilowii chitinase. Substrates (1 mg) were digested with 25 ,ug T. kirilowii chitinase in 200 ,uI of 50 rnM phosphate buffer (pH 6.5) at 30°C. Symbols are: 0, chitotetraose; q , chitotriose.

We have purified three basic chitinases from the transformed T. kirilowii Maxim. callus suspension cultures by a two-step purification procedure. The most effective step in our purification method is the PerSeptive HS/M cation exchange step. Among the three T. kirilowii Maxim. chitinases we purified, two (TKC 28-I and TKC 28-11) were very similar in their molecular mass (28 kDa) and chitinase activities although they differed with regard to their elution behavior and in vitro protein translation inhibition activity. Peptide mapping indicates that these two proteins (TKC 28-I and TKC 28-11) share similar peptide structures. Thus, they might differ by a slight variation in their amino acid sequence. By using an internal amino acid sequence of one chitinase (Chit A) as a probe, Huynh et al. (14) have identified two chitinase isomers (Chit A and Chit B) from maize seed with 87% homology in their amino acid sequence, although they exhibited different antifungal activities. Plant chitinase isomers with the same molecular weight but with different isoelectric points were also observed in Wasabia japonica (35). Since TKC 28-I and TKC 28-11 Trichosanthes, callus, TKC 28-I Trichosanthes, callus, TKC 28-11

AGIAIYWGQN AGIAIYWGQN

Trichosanthes, hairy root (EAChi)

.......... .. . . . . . . . . . . y.

Trichosanthes, hairy root (EBChi) Trichosanthes, hairy root (IBChi) Arabidopsisb Azuki bean’ Cucumberd Lupin (IF3) Pokeweed (PCL-B)’ Rubber Treeg Sugar Beet (SE2)h Sweet Orange (AZ-CFl) Tobacco-A’ Tobacco-B’

AG..H.R...’ G......... G..S”..... . ... .. . ... A..“...... G......... G......,.. SQ.”

. . . . . .

GN GN

* .. .. . . . .G . . l

.D

G”.S...... . . G,,.” . . . . . . . . GD*VV*.**D VG

FIG. 6. Comparison of the N terminal amino acid sequence of T. kirifowii transformed callus chitinase TKC 28-I and TKC 28-11 with other plant class III chitinases. Amino acids identical to those in TKC 28-I and TKC 28-11 are indicated by dots. a, T. kirilowii Maxim. var japonicum transformed hairy root (20); b, Arabidopsis thaliana (2); c, Vigna angularis (32); d, Cucumis sativus (1); e, Lupinus albus (17); f, Phytolacca americana (15); g, Hevea brasiliensis (33); h, Beta vulgaris (16); i, Citrus sine&s (34); and j, Nicotiana tabacum (45).

.J. FERMENT. BIOENG.,

SHIH AND MCDONALD

32

6

I

b

6

28-h

ii5

I

2

I

I

100

I

1

1

I

I

80

60

40

20

0

Time (min)

FIG. 7. Peptide mapping of TKC 28-I and TKC 28-H. The protein was digested with protease Lys C and trypsin. Peptides were separated by Cl8 reverse phased HPLC. Peak 6 is the proteases Lys C and trypsin residue.

are not glycosylated, we believe that glycosylation or post translation modifications do not account for the difference between TKC 28-I and TKC 28-11, and that they are probably produced from a multigene family of chitinases. Although plant chitinases are believed to be a part of the plant’s defense system against fungi, not every chitinase reported has significant antifungal activity against all fungi containing chitin in their cell walls. Jai and Kuc (36) have purified three chitinases from the leaves of cucumber (Cucumis sativus L.) and found that only one chitinase has significant antifungal activity against Colletotrichum lagenarium germination and fungal growth. Also, chitinases differ with respect to their antifungal activity depending on the particular fungal species. For example Huynh et al. (14) purified two basic chitinases from maize seed that inhibited growth of pathogenic fungi Fusarium oxysporum and Alternaria solani and the nonpathogenic fungi Trichoderma reesei but had no effect on two other pathogenic fungi (Gaeumannomyces graminis and Sclerotinia sclerotiorum) that contained chitin their cell walls. Our preliminary data for A. j?avus and T. viride fungi do not show significant antifungal activities, however, this does not mean that these enzymes lack antifungal activity in general. In addition, the ability to inhibit protein translation in vitro by these T. kirilowii chitinases (though less effective than trichosanthin) might indicate their potential roles in the plant defense system. Protein translation inhibition is a typical characteristic property of ribosome inactivating proteins (RIPS). Some plant RIPS have functioned as defense proteins against fungi in plants (8, 9, 37, 38). Even in the fungal kingdom, fungal ribosome inactivating proteins are regarded as antibiotic agents against other fungi (39). Thus, a protein with both chitinase and RIP activity has much potential. However, it should also be noted that the in-vitro protein translation assay is a very sensitive assay (ICsO’s using the rabbit reticulocyte system for known RIPS are

in the range of 0.002-4nM (40) and it is possible that a copurifying protein, present at an undetectable level on a silver stained gel, might be responsible for the effect on protein translation. However, it is unlikely that this copurifying protein would appear in all fractions collected during the purification process. Based on a comparison of their N-terminal amino acid sequences with other plant Class III chitinases, we conclude that TKC 28-I and 28-11 are Class III chitinases (Fig. 4). TKC 28-I isolated from the transformed T. kirilowii Maxim. callus may be similar, but not identical, to the chitinases identified by Savary and Flores (20) in transformed T. kirilowii Maxim. var japonicum hairy root. The basic chitinases (EBChi and IBChi) reported by Savary and Flores (20) do not share the identical N-terminal 12-amino acid sequence with either TKC 28-I or TKC 28-11. Though EAChi has the identical 12amino acid residue sequence with TKC 28-I and TKC 28-11, it is reported as an acidic chitinase. The difference between the T. kiriiowii chitinases found in our samples and in the transformed hairy root of Savary and Flores (20) may be due to the variation of the plant subspecies. In addition, we have identified a similar 28 kDa protein from the storage root of T. kirilowii Maxim. which also possesses the same N-terminal sequence as TKC 28-I and TKC 28-11 up to 12 amino acid residue sequence (data not shown). We have previously reported the extracellular accumulation (estimated to reach 1% w . w-l on a dry weight basis) of a basic, 29 kDa protein in the broth of transformed T. kirilowii cultures (27). Partially purified samples containing this protein as a major component caused in-vitro protein translation inhibition. However, the 29 kDa protein was not sequenced, mapped or tested for chitinase activity. So, it is difficult to assess the relationship between it and TKC 28-I or TKC 28-11. The TKC 15 protein is less well characterized than TKC 28-I or TKC 28-11. We were unable to determine the N terminal amino acid sequence of this protein due to a block on its N terminus. However, it exhibited less chitin hydrolysis activity than TKC 28-I or TKC 28-11 under the same assay conditions (Fig. 3). Digestion of chitin by the chitinases (TKC 28-I and 28-11) yielded chitobiose as the major product, although trace amounts of trimer can also be found in the digest. On the other hand, TKC 15 release a mixture of dimers, trimers and tetramers from chitin. In additional to its slow reactions on the chitin, TKC 15 didn’t cleave trimer nor tetramer. Slow hydrolysis of chitotriose was observed whereas chitotetraose was much rapidly (about 100 times) digested into chitobiose by TKC 28-I and 2811. Our results suggest that the recognition of the three chitose unit is crucial to T. kirilowii Maxim. chitinase activity. Most plant chitinases reported in the literature are endochitinases releasing mainly dimer, trimer and tetramer moieties during hydrolytic action on chitin. These include class I, II and III chitinases isolated from a number of plant species and tissues like carrot, pea, chickpea, potato and rye (7, 41-44).

ACKNOWLEDGMENTS

The authors would like to thank Dr. Abhaya Dandekar in the Department of Pomology, UC Davis for experimental design assistance, Dr. A. Dan Jones for mass-spectrometry analysis, Nicole Dennis, Rachael BaIog and Mussie Futur for their assistance on this project, Dr. Y. M. Lee in the Protein Structure Laboratory at

VOL. 84, 1997

T. KIRILO WII CHITINASE

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