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Indian Journal of Experimental Biology Vol. 42, May 2004, pp. 541-544 Characterization and identification of chitinase producing Streptomyces venezuelae P 10 G Mukherjee & S K Sen* Microbiology Laboratory, School of Life Sciences, Department of Botany, Visva-Bharati, Santiniketan 731235, India Received 5 November 2003; revised 19 December 2003 In an attempt to isolate chitinase producers from soil, a streptomycete strain was found potent using natural chitin as the substrate. Chitinolytic activity was tested directly on agar plates, also with crude enzyme. Chitinase assay showed that the isolate could produce 0.8 Vlml of the enzyme. The morphological, cultural, physiological and biochemical characters of the isolate P IO were studied, and identified as Streptomyces venezuelae P IO . Keywords: N-Acetylglucosamine, Chitin, Chitinase, Colloidal chitin, Streptomyces vellezuelae IPC Code: Int. Cl. 7 AOIN; C12N Chitin is a tough, pliable, widely distributed natural product resembling cellulose. It's fibrous structure is derived from long unbranched chains of N- acetylglucosamine (GlcNAc) units connected by glycosidic linkages. Chitin is extremely resistant to chemical reagents that requires strong mineral acids to dissolve 1 . In most organisms chitin is modified by forming linkages with other polymers like glucans, proteins etc. The complete enzymatic hydrolysis of chitin to free N-acetylglucosamine is performed by a chitinolytic system, the action of which is known to be synergistic and consecutive 2 - 5 . It is worthy to mention here that chitinase being inducible (adaptive) enzymes play a secondary nutritional role for carbon and nitrogen sources 6 . Studies have been made on the enzymes responsible for decomposition of chitin? Chitinases are found in a variety of living organisms such as snails, crustacea, Insects 8 - 9 , vertebrates 6 . 10 and bean seeds ll . But the most convenient source is the microorganisms 12-16. Chitinases play an important role in the carbon cycle of biosphere to maintain the ecosystem. The present communication deals with the selection of potent chitinase producer and it's enzyme assay. Media used--Glucose asparagine medium consisted *Correspondent author-E. mail. [email protected] of (gll)-glucose, 10.0; asparagine, 0.5; and K 2 HP04 0.5. Chitin asparagine medium consisted of (gIl)- colloidal chitin, 5; asparagine, 0.5; and K 2 HP0 4 , 0.5,.. Chitin mineral salt medium comprised (gIl)- colloidal chitin, 5.0; KCl, 0.5; K 2 HP0 4 , 1.2; MgS0 4 .7H 2 0, 0.5; and FeS04.7H20, 0.01. Chitin light agar medium having (g/lOO ml)-Colloidal chitin, 4.0; and agar, 1.3. Isolation-Isolation of chitin utilizers was done through enrichment in chitin asparagine medium. The enriched suspension was streaked in plates containing 20 ml chitin asparagine agar (1.5%) medium. By visual analyses, well-separated colonies were transferred to slants as pure culture. Later, streaking from the slant on the same medium and development of small isolated colonies confirmed the purity of the sample. Selection- The primarily selected isolates were further tested both in solid and liquid media with 0.5% of colloidal chitin (w/v), incubated for 4 to 6 days at 30° C under varying pH range from 5 to 8 separately. The selection of working strain was made on the basis of their chitinase activity determined qualitatively (measuring clearance zones) and quantitatively (chitinase assay). Characterization and identification of the selected isolate-- The selected isolate P 10 was characterized following the methods of Waksman and Henrici 17 , International Streptomyces Projed 8 , WillianlS et at 9 , Williams 20 , Bergey's Manual of Systematic Bacteriologl 1 . For determination of morphological characteristics, petriplate culture, cover glass culture 22 , phase contrast microscopic and scanning electron microscopic (SEMi 3 observations were performed. Preparation of colloidal chitin--Colloidal chitin was prepared from crab shell chitin (Analytical Grade, Sigma, USA) following Hsu and Lockwood 24 with modification. The recovered (85%) chitin was stored after sterilization. Chitinase assay--Extracellular chitinolytic activity of the cell supernatant was using swollen chitin as the substrate 7 at optimum pH of chitinolysis as determined by primary selection. The reaction mixture contained 2 ml of colloidal chitin (3 mg/ml) and 5 ml of crude enzyme was kept at 35°C under shake for 1 hr. The amount of reducing sugar formed was measured with dinitrosalicylic acid

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Indian Journal of Experimental Biology Vol. 42, May 2004, pp. 541-544

Characterization and identification of chitinase producing

Streptomyces venezuelae P 10

G Mukherjee & S K Sen*

Microbiology Laboratory, School of Life Sciences, Department of Botany, Visva-Bharati, Santiniketan 731235, India

Received 5 November 2003; revised 19 December 2003

In an attempt to isolate chitinase producers from soil, a streptomycete strain was found potent using natural chitin as the substrate. Chitinolytic activity was tested directly on agar plates, also with crude enzyme. Chitinase assay showed that the isolate could produce 0.8 Vlml of the enzyme. The morphological, cultural, physiological and biochemical characters of the isolate P IO were studied, and identified as Streptomyces venezuelae P IO.

Keywords: N-Acetylglucosamine, Chitin, Chitinase, Colloidal chitin, Streptomyces vellezuelae

IPC Code: Int. Cl.7 AOIN; C12N

Chitin is a tough, pliable, widely distributed natural product resembling cellulose. It's fibrous structure is derived from long unbranched chains of N­acetylglucosamine (GlcNAc) units connected by ~-1,4-glycosidic linkages. Chitin is extremely resistant to chemical reagents that requires strong mineral acids to dissolve1. In most organisms chitin is modified by forming linkages with other polymers like glucans, proteins etc. The complete enzymatic hydrolysis of chitin to free N-acetylglucosamine is performed by a chitinolytic system, the action of which is known to be synergistic and consecutive2-5. It is worthy to mention here that chitinase being inducible (adaptive) enzymes play a secondary nutritional role for carbon and nitrogen sources6

. Studies have been made on the enzymes responsible for decomposition of chitin?

Chitinases are found in a variety of living organisms such as snails, crustacea, Insects8

-9

, vertebrates6.10 and

bean seeds ll. But the most convenient source is the

microorganisms 12-16. Chitinases play an important role in the carbon cycle of biosphere to maintain the ecosystem. The present communication deals with the selection of potent chitinase producer and it's enzyme assay.

Media used--Glucose asparagine medium consisted

*Correspondent author-E. mail. [email protected]

of (gll)-glucose, 10.0; asparagine, 0.5; and K2HP04 0.5. Chitin asparagine medium consisted of (gIl)­colloidal chitin, 5; asparagine, 0.5; and K2HP04, 0.5,.. Chitin mineral salt medium comprised (gIl)- colloidal chitin, 5.0; KCl, 0.5; K2HP04, 1.2; MgS04.7H20, 0.5; and FeS04.7H20, 0.01. Chitin light agar medium having (g/lOO ml)-Colloidal chitin, 4.0; and agar, 1.3.

Isolation-Isolation of chitin utilizers was done through enrichment in chitin asparagine medium. The enriched suspension was streaked in plates containing 20 ml chitin asparagine agar (1.5%) medium. By visual analyses, well-separated colonies were transferred to slants as pure culture. Later, streaking from the slant on the same medium and development of small isolated colonies confirmed the purity of the sample.

Selection-The primarily selected isolates were further tested both in solid and liquid media with 0.5% of colloidal chitin (w/v), incubated for 4 to 6 days at 30° C under varying pH range from 5 to 8 separately. The selection of working strain was made on the basis of their chitinase activity determined qualitatively (measuring clearance zones) and quantitatively (chitinase assay).

Characterization and identification of the selected isolate--The selected isolate P 10 was characterized following the methods of Waksman and Henrici 17

,

International Streptomyces Projed8, WillianlS et at9

,

Williams20, Bergey's Manual of Systematic Bacteriologl1. For determination of morphological characteristics, petriplate culture, cover glass culture22

,

phase contrast microscopic and scanning electron microscopic (SEMi3 observations were performed.

Preparation of colloidal chitin--Colloidal chitin was prepared from crab shell chitin (Analytical Grade, Sigma, USA) following Hsu and Lockwood24

with modification. The recovered (85%) chitin was stored after sterilization.

Chitinase assay--Extracellular chitinolytic activity of the cell supernatant was dc~ermined using swollen chitin as the substrate7 at optimum pH of chitinolysis as determined by primary selection.

The reaction mixture contained 2 ml of colloidal chitin (3 mg/ml) and 5 ml of crude enzyme was kept at 35°C under shake for 1 hr. The amount of reducing sugar formed was measured with dinitrosalicylic acid

542 INDIAN J EXP BIOL, MAY 2004

(DNSA) using a standard curve? of N­acetylglucosamine. The unit (V) of chitinase activity is defined as the amount of enzyme requires to produce 1.0 IJ.M of N-acetylglucosamine per min. Qualitative assay was also done by chitinase light agar-cup assa/5

.

Chitin is abundantly found in soil and water, the recycling of which is essential for maintenance of ecological niche. Soils of shrimp waste disposal area were plated for screening of chitinolytic microorganisms. Out of 55 isolates, 5 isolates showed excellent growth on modified mineral salt medium where chitin was the sole source of carbon and nitrogen.

The primarily selected isolates were again tested in the modified mineral salt medium, both in solid and liquid for different time period. All the isolates showed good visible growth and extracellular chitinase production (Table O. The detectable enzyme production (showing clearance zone) was observed on day 3 for PIO (Fig. 1) and day 4 for CM2, MCr!. MCr2, and day 6 for PB3. The isolate PIO produced chitinase maximally when colloidal chitin was used instead of chitin flake or chitin powder. Maximum chitinase production (0.8 Vim I) was observed at day 4 of incubation.

The morphological, micromorphological, cultural, physiological and biochemical characteristics of PIO were--rectiflexible yellow spore chain, smooth spore surface, production of diffusible pigment, melanin production on tyrosine agar, nitrate reduction, use of phenylalanine and no groWt:l at 45°C, thus indicating that the isolate Pro belongs to genus, Streptomyces. It resembled the species group spirales, grey series having rectiflexible spore chains of smooth (Fig. 3),

yellow spores. The species identification, supported by the probability matrix study, confirmed it as Streptomyces venezuelae (MTCC 4218) belongs to Type 126.

To test the suitability of the nature of chitin for enzyme production by the isolate P IO, the experiment was continued for 8 days. It was found (Fig. 2) that the enzyme production was maximum on day 4 of incubation, though all types of chitin could well support the production. It is also found that chitinase production decreased with the 1l1crease In

fermentation period accompanied by a decrease In

specific activity irrespective of protein contene. Soil organisms are subjected to selection pressure

leads to the enrichment of particular group of organisms and as a consequence of competition, the surviving groups become adaptive by expressing certain phenotypes directed by the genetic make-up that confers advantage over others. Streptomycetes have been reported as one of the major contributor to chitin hydrolysis5

, with chitinase activity 9.6 Vlml? to 0.2 Vlml'3. In this respect, the present experiment

Fig. I-Showing clearance zone on chitin agar plate after streaking (A) and agar cup assay with crude enzyme (B) by the isolate PIO'

Table I-Assay of chitinase by the selected isolates (CM2, MCrl MCr2, P IO and PB3)

Incubation CM2 MCrl MCr2 PIO PB3

period (days) a b c a b c a b c a b c a b c

3 1.15 0.30 1.10 0.20 10 4.15 0.70 4 7 4.00 0.65 5 4.00 0.60 6 4.00 0.65 14 4.45 0.80 5 8 ' 4.15 0.70 6 4.00 0.65 7 4.00 0.70 15 4.45 0.80 1.45 0.30 6 9 4.25 0.75 7 4.00 0.65 8 4.05 0.70 15 4.45 0.80 6 4.00 0.60 7 9 4.15 0.70 8 4.15 0.70 8 4.05 0.70 15 4.25 0.75 7 4.15 0.65 8 10 4.15 0.70 8 4.15 0.70 8 4.05 0.70 15 4.15 0.70 8 4.20 0.70 9 10 4.15 0.70 9 4.15 0.70 8 4.10 0.70 15 4.00 0.60 8 4.20 0.70 10 10 4.15 0.70 9 4.15 0.70 9 4.10 0.70 15 4.00 0.60 9 4.20 0.70 11 12 4.25 0.75 10 4.15 0.70 9 4.00 0.65 15 3.75 0.55 9 4.20 0.70 12 12 4.15 0.65 10 3.50 0.50 9 2.50 0.45 15 3.70 0.50 13 4.20 0.70 13 12 4.15 0.65 10 3.00 0.40 9 2.00 0.30 15 3.45 0.45 13 4.15 0.65

a - chitinase clearance zone diam. (mm), b ~ specific activity, c - chitinase activity (U/m)

NOTES 543

Fig. 3--Scanning electron micrograph of isolate P IO showing (A) aerial hyphae (x 6000); and (B) spore chain (x 8000).

0.9

Co 0.11

~ ::) OJ --C 0 0& :a ::I

~ 05

Go

a 0.4

I'IJ C 03 iii .t= 0 02

0.1 I

__ CoIo.1 CMin ··0·· ClUlpowd. ___ Ciltin Iltk'

'2 3 • 5 6 7 8 , 10 11 12 13 14

Days

Fig. 2-Effect of nature of chitin for chitinase production of by the isolate PJO.

suggested that S. venezuelae P IO was a potent chitinase producer. There is no available data on chitinase production by Streptomyces venezuelae prior to this report.

Author (SKS) is thankful to the University Grants Commission, New Delhi, for financial support.

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