CHAPter VII

APPlICAtIon of CHItInAse

CHAPTER VII Page 128

7.1 INTRODUCTION

Chitinases belongs to the class of hydrolytic enzymes with a potential to inhibit or

degrade the chitin containing pathogens like fungi, insects and their larva’s. The use of

chitinases as a bio control agent is one of the attractive and environmentally safe strategies.

Chitinolytic enzymes have various potential applications such as preparation of

chitooligosaccharides and N-acetyl-D-glucosamine which are known to have various

biological activities (antimicrobial, antifungal, immunoenhancers, antitumor, etc.) (Tsai et al.,

2000; Shen et al., 2009) with high interest in the pharmaceutical sector (Wen et al., 2002).

Moreover, chitinases can be used for the control of pathogenic fungi in agriculture (Dahiya et

al., 2005a) and the degradation of crustacean chitinous waste in sea food industry. These

enzymes are also useful for the preparation of single-cell protein, the isolation of protoplasts

from fungi and yeast, etc. (Dahiya et al., 2006).

Biological control, using microorganisms to suppress plant disease, offers powerful

and alternative to use of synthetic chemical. Chitinolytic bacteria such as Aeromonas

hydrophila, A. caviae, Pseudomonas maltophila, Bacillus licheniformis, B. circulans, Vibrio

furnissii, Xanthomonas sp. and Serratia marcescens have been reported and played important

role as biological control agents. Biological control using microorganism has been studied

intensifely since not many alternatives to control are available (Duffy et al. 1995).

Furthermore, chitinases together with proteases, glucanases and cellulases are

frequently considered critical in the biocontrol of phytopathogenic fungi (Gohel et al.,

2006a). Chitinases occupy a unique position in agricultural biotechnology because the lytic

activity inhibits fungal development by degrading chitin and glucan components of cell wall

and have proven potential as antifungal agents (Jung et al., 2003; Punja and Zhang, 1993).

Chitinases therefore play vital role in agricultural industries and medical fields, furthermore,

chitinases also play vital roles in sea food industry for crustacean chitinous waste

degradation. To accelerate identification of optimal chitinase formulations to function

broadly in a range of environments for the biocontrol of phytopathogenic fungi, for

cytochemical localization of chitin/chitosan using chitinase–chitosanase–gold complex, for

fungal protoplast technology, for preparation of chitooligosaccharides and for degradation of

chitinous waste (Vaidya et al., 2003b), we have gained a fundamental understanding of

behaviour of chitinase molecules in various conditions. Health, environmental concern,

development of resistance in target populations also contributes to developing biological

control using natural enemies (Martin and Loper, 1999). Nonetheless, the vast array of

CHAPTER VII Page 129

antimicrobial molecules produced by diverse soil microbes remains as a reservoir of new and

potentially safer biopesticides (Kang et al., 1998).

In recent years, there has been a constant increase in the exploitation of fish resources

and the estimated quantity used for human consumption (105.6 million tons) is globally 75 %

of the worldwide fish production. The remaining 25 % of the catch (34.8 million tons) are

considered as wastes (FAO, 2007). Furthermore, the commercial fish processing industry

generates large quantities of solid waste and wastewater. Solid waste which represents 20–60

% of the initial raw material contains various kinds of residues (whole waste fish, fish head,

viscera, skin, bones, blood, frame liver, gonads, guts, some muscle tissue, etc.) (Awarenet,

2004). In some countries, these discards are not utilized, but incinerated or dumped at sea

causing environmental problems (Bozzano and Sarda, 2002). Recently, environmental

regulations are becoming stricter, requiring new disposal methods based on the fact that fish

wastes (solid waste and wastewater) may considered as an important source of protein, lipids

and minerals with high biological value (Toppe et al., 2007; Kacem et al., 2011).

The disposal of wastes generated by fishery processing industries represents an increasing

environmental and health problem. However, these by-products have attracted considerable

attention as an alternative feedstock and energy source, since they are abundantly available.

Various microbes are capable of using these substances as carbon and energy sources

beneficial in enzyme production process. A number of such substrates have been tested for

the cultivation of microorganisms to produce several enzymes (protease, lipase, chitinases,

peroxidases, laccases, oxidases, etc.). This may have numerous advantages for enzyme

production process, such as superior productivity, simpler techniques, reduced energy

requirements and reduced production costs. Generally, fish waste pre-treatments may be

necessary to maximize microbial growth and enzyme production. However, each microbial

strain has its own special conditions for maximum enzyme production. Therefore, it is of

great significance to optimize the medium composition, taking into consideration the

variability of fish waste composition, the nutrient requirements of microbial strain and

fermentation parameters (pH, temperature, aeration, agitation, etc.). Nevertheless, the

improvements in fish waste technology (pre-treatments, characterization, formulation, etc.)

are still necessary before large-scale application of this new strategy can be realized.

Microbial chitinase has been produced by liquid fermentation processes (batch,

continuous and fed-batch fermentation) and is commercially available at a high cost (Dahiya

et al. 2006). Generally, the production is controlled by physical factors (aeration, pH, and

incubation temperature) and by the growth media components (Miyashita et al., 1991). In

CHAPTER VII Page 130

order to increase the supply of active chitinase, it is necessary to reduce the production cost

by using wastes for microbial growth. In this perspective, various chitinous materials from

marine sources [shrimp shell powder (SSP), squid pen powder (SPP), shrimp and crab shell

powder (SCSP)] have been utilized for chitinolytic enzyme production as alternative to waste

disposal. The use chitinase for production of chitiooligosaccharide and NAG is one of the

major applications for human health care and biotechnological sector. There are few reports

on industrially available production of NAG from chitin by using chitinolytic enzymes since

the enzyme reaction on chitin progress is very slowly and also yield is very low from the

standpoint of practical use. Chitinase can also be employed for the biocontrol of plant

pathogens and for developing transgenic plants. Although biocontrol of plant pathogenic

fungi using chitinase producing microorganisms and plant has been studies extensively but

more work is required for studying microbes-microbes, microbes-plant and chitinase-microbe

interaction which would be of great help in using chitinase as a biocontrol agent. Developing

a bioprocess for production of N-acetyl –D-glucosamine from chitin will be a novel

approach.

CHAPTER VII Page 131

7.2 MATERIAL AND METHODS

7.2.1 Antifungal activity of crude chitinase

To evaluate the effect of culture filtrate on radial growth of fungi the experiment was

conducted according to the method of Prapagdee et al., (2007). Culture filtrate was added into

warm molten PDA (Appendix 1) (45ºC) to give final concentration at 10, 20 and 30% (V/V)

and placed until solidfied. The control plate was added equal volume of sterile distilled water

instead of culture filtrate. Each plate was seeded with 6mm diameter mycelial plugs taken

from the margin of 5 day old fungal plate. Inoculated plates were incubated at 28ºC and fungal

growth was recorded at every 24 hrs until those of the control plate reaching the edge of the

plate. The fungal growth inhibition was expressed as the percentage of inhibition of radial

growth relative to the control as shown in equation. Inhibition of radial growth (%) = Diameter of control sample - Diameter of test× 100

Diameter of control sample

7.2.2 Production of N-Acetyl-D-Glucosamine from the degradation of chitinous waste

7.2.2.1 Collection of Chitinous waste The shrimp shell wastes and prawn shells were collected from fish market, Lucknow,

India. These wastes were washed with tap water repeatedly, dried in room temperature and

crushed it for further experiments. Colloidal chitin was prepared from the chitin by the

modified method of Hsu and Lockwood, 1975. Chitin powdered was purchased from Hi

Media.

7.2.2.2 Pre-treatment of Shrimp Waste

A total of four pre-treatment methods and one control (untreated) were carried out to

investigate the efficiency of pre-treated prawn waste in fermentation process. Pre-treatment

method is described in Appendix 6.

7.2.3Production of N-acetyl-D-glucosamine

7.2.3.1 Production of GlcNAc from bacterial degradation of different source of chitin

For the production of GlcNAc, both Aeromonas hydrophila and Aeromonas punctata

were grown in a production media inoculated with 1% of different chitin source in shaking

condition at 37ºC for 5 days and after every 24 hrs culture broth was centrifuged at 10,000

rpm for 20 min at 4ºC and the resultant supernatant was tested for estimation of NAG using

standard curve with N-acetyl-glucosamine as a standard.

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7.2.4 Experimental Design

The research was divided into two main parts, screening for the best pre-treatment of

prawn waste and optimization of chitinase production and NAG using pre-treatment prawn

waste. The first stage involved pre-treatment of prawn waste and inoculums preparation.

Fermentation process would be done to investigate the efficiency of each pre-treatment

method used. The pre-treatment which yield the highest degradation rate and chitinase

production was selected for further optimization study.

Chitin source stock culture of bacteria

Pretreatment culture on nutrient agar

Method

1. Chitin Colloidal treatment Production media preparation

2. Chitin (Hi-Media)

Prawn shells

• Colloidal treatment • Acid-base treatment • Oven dry • Boiling and crushing

Incubate up to 5days, centrifuge, and collect supernatnant

Test for NAG production, enzyme activity, protein estimation

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7.3 RESULTS AND DISCUSSION

7.3.1 Antifungal activity crude enzyme from Aeromonas hydrophila HS4

Chitinolytic enzymes are able to lyse the cell wall of many fungi. The microorganisms

that produce these chitinolytic enzymes are capable of eradicating fungal diseases that are a

problem for global agricultural production. Chitinase showed stronger inhibitory activity

(47.33%) towards potent phytopathogen, Shizophyllum commune in comparsion of control

(Fig 7.1). The purified chitinase from Aeromonas hydrophila HS4 was tested for antifungal

activity by their ability to inhibit hyphal extension growth of Shizophyllum commune.

A B

D C

Fig 7.1: Radial growth of Shizophyllum commune on PDA plate amended with culture filtrate of Aeromonas hydrophila in various concentration (A) 30% (B) 20% (C) 10% (D) Control

CHAPTER VII Page 134

Myc

elia

l Gro

wth

(cm

s)

7.3.1.2 Radial growth suppression by extracellular antifungal protein by

Aeromonas hydrophila HS4 Crude

From Figure 7.2, radial growth on Shizophyllum commune on PDA plate was

inhibited at concentration of 10 % in comparison of control. The percentage of radial growth

inhibition by culture filtrate of Aeromonas hydrophila was 23.33 after 3day of incubation and

36.48 after 7day incubation (Fig 7.2). The potential of antifungal activity increased when the

culture filtrate was increases from 10% to 30% (Table 7.1).

10

9 Control

8 10%

7 20%

6

5 30%

4

3

2

1

0

1 2 3 4 5 6 7 Time (Days)

Fig 7.2: Graphical representation of radial growth inhibition of Shizophyllum commune by Aeromonas hydrophila HS4

7.3.2 Antifungal activity of crude enzyme from Aeromonas hydrophila HS6

on Shizophyllum commune Chitinolytic enzymes are able to lyse the cell wall of many fungi. The microorganisms

that produce these chitinolytic enzymes are capable of eradicating fungal diseases that are a

problem for global agricultural production. Chitinase showed stronger inhibitory activity

(61.79%) towards potent phytopathogen, Shizophyllum commune in comparsion of control

(Fig 7.3). The purified chitinase from Aeromonas punctata was tested for antifungal activity

by their ability to inhibit hyphal extension growth of Shizophyllum commune.

CHAPTER VII Page 135

7.3.2.2 Radial growth suppression by extracellular antifungal protein by

Aeromonas punctata HS6 Crude:

From Figure 7.4, radial growth on Shizophyllum commune on PDA plate was

inhibited at concentration of 10 % in comparsion of control. The percentage of radial growth

inhibition by culture filtrate of Aeromonas punctata was 26.82 after 3day of incubation and

38.2 after 7day incubation (Fig 7.4). The potential of antifungal activity increased when the

culture filtrate was increases from 10% to 30% (Table 7.1).

Control 10%

20% 30%

Fig 7.3: Radial growth of Shizophyllum commune on PDA plate amended with culture Filtrate of Aeromonas punctata in various concentrations

CHAPTER VII Page 136

Myc

elia

l gro

wth

(cm

s)

10

9 control 8 10% 7 20%

6 30%

5

4

3

2

1

0

1 2 3 4 5 6 7 8 Time (days)

Fig 7.4: Graphical representation of radial growth inhibition of Shizophyllum commune by Aeromonas punctata HS6

Table 7.1: The percentage of radial growth inhibition of Shizophyllum commune by

culture filtrate of Aeromonas hydrophila HS4 and Aeromonas punctata HS6 after 3 and 7

day

Concentration of culture filtrate (V/V)

% Radial growth inhibition

HS4 HS6

3 day 7 day 3 day 7 day

10% 23.33 36.48 26.82 38.2

20% 33 42.03 36.58 45.05

30% 47.33 47.59 46.34 61.79

7.3.3 Standard curve of N-acetyl-D-glucosamine (GlcNAc)

The standard curve of GlcNAc was constructed using standard solution of GlcNAc at

concentration of .02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16 mg/ml (Fig 7.5). The 2ml of

potassium ferricyanide solution was added into 1.5ml of standard GlcNAc at various

concentrations. The mixture was mixed and incubated in boiling water for 15min. The color

development was measured for the absorbance at wavelength of 420nm using D.W as a

CHAPTER VII Page 137

O.D

(420

nm)

blank. A regression line was drowning between GlcNAc concentrations against their

absorbance value at 420nm.

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

y = 4.670x + 0.083 R² = 0.990

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

Conc. of NAG (mg/ml)

Fig 7.5: Standard curve for estimation of N-Acetyl-D-Glucosamine

7.3.4 Standard curve of Protein estimation

The standard curve of protein was constructed using standard solution of BSA at

concentration of 20, 40, 60, 80, 100, 120, 140, 160, 180µg/ml (Fig 7.6). The color

development was measured for the absorbance at wavelength of 660nm using D.W as a

blank. A regression line was drowning between BSA concentrations against their absorbance

value at 660nm.

Fig 7.6: Standard curve for the estimation of protein

CHAPTER VII Page 138

7.3.5 Production of N-acetyl-D-Glucosamine

7.3.5.1 Production of N-Acetyl-D-Glucosamine by the degradation of Biowaste from

Aeromonas hydrophila HS4

Prawn shells, fish shells was collected from the local fish market and used for the

production of NAG (Fig 7.7). Production was compared by using chitin and colloidal chitin

as control. From the result it is conformed that GlcNAc production was maximum to

colloidal chitin (0.257 mg/ml), but within biowaste prawn shells (0.2486 mg/ml) is best

substrate as compared to fish shell (0.233 mg/ml) with 89.40 U/ml enzyme activity (Table

7.2). So it can be used for the production of GlcNAc and is best way to degrade the fishery

waste and reduce environmental hazard. In previous studies N-acetyl D glucosamine

produced by the degradation of prawn shells waste and fungal biomass by the application of

partially purified chitinase from Trichoderma harzianum (Das et al., 2012).

Table 7.2: Production of N-acetyl-D-glucosamine by the degradation of biowaste

Period (days) GlcNAc (mg/ml)

Total activity (U/ml)

Total protein (µg/ml)

Specific Activity (U/mg protein)

PRAWN SHELLS 1 0.1273 80.13 2184.15 0.0366 2 0.1438 80.41 2594.47 0.0309 3 0.2486 89.40 2316.5 0.0385 4 0.1987 89.03 894 0.0995 5 0.1772 80.64 205 0.393 FISH SHELLS 1 0.2373 83.86 3053.25 0.0274 2 0.233 84.20 2835.12 0.0296 3 0.2233 84.43 2401.725 0.0351 4 0.155 83.60 1376.4 0.0607 5 0.126 72.52 1351.5 0.0536 CHITIN (HI-Media) 1 0.2552 72 1858.95 0.038 2 0.2439 80.89 1726.5 0.0468 3 0.206 84.97 1666 0.0510 4 0.201 73.34 917.5 0.0799 5 0.119 68.24 507.3 0.134 COLLODIAL CHITIN 1 0.257 69.37 1979.55 0.035 2 0.2392 73.37 2063.52 0.0355 3 0.2158 87.68 1569.22 0.0558 4 0.155 77.71 882.17 0.088

5 0.119 61.88 367 0.168

CHAPTER VII Page 139

O.D

(420

nm)

0.3

0.25

Prawn shells Fish shells

colloidal chitin chitin shells

0.2

0.15

0.1

0.05

0 1 2 3 4 5

Time (days)

Fig 7.7: Screening of substrate for N-Acetyl-D-Glucosamine by Aeromonas hydrophila

HS4

7.3.5.1.1 Screening for Suitable Pre-treatment Method for NAG production by

degradation of prawn shell by Aeromonas hydrophila HS4

The best substrate for the production of NAG by the degradation of pre-treated prawn

shells was observed that NAG production was maximum with colloidal treatment of prawn

shells (0.266 mg/ml) after third day of incubation for Aeromonas hydrophila, as indicated in

Table 7.3. This was then followed by colloidal chitin, Boiling and crushing, prawn oven dry

treatment, acid base treatment on the production of NAG (Fig 7.8).

Table 7.3: Effect of treatment on production of chitinase and NAG by using Aeromonas

hydrophila HS4

Period (days) GlcNAc (mg/ml)

Total activity (U/ml)

Total protein (µg/ml)

Specific Activity (U/mg protein)

PRAWN SHELLS (Oven dry/ PS)

1 0.1273 80.13 2184.15 0.0366 2 0.1438 80.41 2594.47 0.0309 3 0.233 89.40 2316.5 0.0385 4 0.1987 89.03 894 0.0995

CHAPTER VII Page 140

NAG

(mg/

ml)

5 0.1772 80.64 205 0.393 PRAWN SHELLS (colloidal treatment/ COP)

1 0.196 81.2 2345.12 0.0346 2 0.224 86.81 2632.1 0.0329 3 0.266 84.44 2567.11 0.0328 4 0.254 82.1 1309 0.0627 5 0.207 70 609 0.114 PRAWN SHELLS (Acid base treatment/ CP) 1 0.145 76.34 1987.56 0.0384 2 0.197 82.11 2234.12 0.0367 3 0.2074 70.12 2376.34 0.0295 4 0.187 69.11 987 0.0700 5 0.145 60.14 789 0.0762 PRAWN SHELLS (Boiling and Crushing/ BC) 1 0.2552 72 1858.95 0.038 2 0.2439 80.89 1726.5 0.0468 3 0.206 84.97 1666 0.0510 4 0.201 73.34 917.5 0.0799 5 0.119 68.24 507.3 0.134 COLLODIAL CHITIN (CC) 1 0.257 69.37 1979.55 0.035 2 0.2392 73.37 2063.52 0.0355 3 0.2158 87.68 1569.22 0.0558 4 0.155 77.71 882.17 0.088 5 0.119 61.88 367 0.168

0.3

PS COP CP BP CC

0.25

0.2

0.15

0.1

0.05

0

1 2 3 4 5 Day of incubation

Fig 7.8: Effect of different treatment of prawn shells on NAG production by Aeromonas

hydrophila HS4

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7.3.5.2 Production of N-Acetyl-D-Glucosamine by the degradation of Boiwaste from

Aeromonas punctata HS6

Screening of best substrate for the production of N-Acetyl-D-Glucosamine by the

degradation of biowaste was observed that colloidal chitin (0.257mg/ml) is the best substrate,

Among biowaste prawn shell and fish shell, prawn shell (0.254 mg/ml) enhanced the

production of N-Acetyl-D-Glucosamine after third day of incubation (Table 7.4, Fig 7.9).

Table 7.4: Production of N-acetyl-D-glucosamine by the degradation of biowaste

Period (days) GlcNAc (mg/ml) Total activity

(U/ml) Total protein

(µg/ml) Specific Activity

(U/mg)

PRAWN SHELLS 1 0.244 89.16 4765.5 0.0187 2 0.248 93.86 4850.5 0.0189 3 0.254 79.56 1979.55 0.0401 4 0.238 69.41 1232.2 0.0563 5 0.222 60.51 1176.1 0.0514

FISH SHELLS 1 0.25 91.22 3028.47 0.03078 2 0.246 89.26 3473.4 0.0256

3 0.252 78.51 3838.5 0.0204 4 0.242 70.12 2899 0.0241 5 0.252 60.45 2655.95 0.0227

CHITIN (Hi-Media) 1 0..236 92.48 1954.72 0.0473 2 0.2528 87.45 1255.85 0.0696 3 0.248 80.54 1232.2 0.0653 4 0.244 74.19 1146.75 0.0646 5 0.238 65.11 1020.5 0.0638

COLLOIDAL CHITIN 1 0.244 92.98 3584.25 0.02594 2 0.257 84.43 2367.4 0.0356 3 0.25 81.83 2317.75 0.0353 4 0.2446 81.72 1788 0.0457 5 0.2368 72.11 1040.6 0.0692

CHAPTER VII Page 142

O.D

(420

nm)

0.26

Prawn shells Fish shells chitin colloidal chitin

0.25

0.24

0.23

0.22

0.21

0.2

1 2 3 4 5

Time (days)

Fig 7.9: Screening of substrate for N-Acetyl-D-Glucosamine by Aeromonas punctata

HS6

7.3.5.2.1 Screening for Suitable Pre-treatment Method for NAG production by

degradation of prawn shell by Aeromonas punctata HS6

The best substrate for the production of NAG by the degradation of pre treated prawn

shells with Aeromonas punctata was observed that NAG production was maximum with

colloidal treatment of prawn shells (0.28 mg/ml) after third day of incubation, as indicated in

Table 7.5. This was then followed by colloidal chitin, boiling and crushing, prawn oven dry

treatment, acid base treatment on the production of NAG (Fig 7.10).

Table 7.5: Effect of treatment on production of chitinase and NAG by using Aeromonas

punctata HS6

Period (hrs) GlcNAc

(mg/ml)

Total activity

(U/ml)

Total protein

(µg/ml)

Specific

Activity (U/mg

protein)

PRAWN SHELLS (Oven dry/ PS)

1 0.244 89.16 4765.5 0.0187

2 0.248 93.86 4850.5 0.0189

3 0.254 79.56 1979.55 0.0401

4 0.238 69.41 1232.2 0.0563

CHAPTER VII Page 143

5 0.222 60.51 1176.1 0.0514

PRAWN SHELLS (colloidal treatment/ COP) 1 0.15 74.67 4679 0.0159

2 0.32 80.23 5467.4 0.0146

3 0.28 86.34 1678.12 0.057

4 0.108 80.99 987 0.082

5 0.096 76.11 877 0.0867

PRAWN SHELLS (Acid-base treatment/CP)

1 0.146 56.12 1267 o.o44

2 0.186 64.13 3467.1 0.0184

3 0.25 72.33 4234.1 0.017

4 0.199 67.8 3216.6 0.0210

5 0.185 54.44 2467.1 0.022

PRAWN SHELLS (Bioling and Crushing/ BP)

1 0.236 92.48 1954.72 0.0473

2 0.2528 87.45 1255.85 0.0696

3 0.248 80.54 1232.2 0.0653

4 0.244 74.19 1146.75 0.0646

5 0.238 65.11 1020.5 0.0638

COLLOIDAL CHITIN (CC)

1 0.244 92.98 3584.25 0.02594

2 0.257 84.43 2367.4 0.0356

3 0.25 81.83 2317.75 0.0353

4 0.2446 81.72 1788 0.0457

5 0.2368 72.11 1040.6 0.0692

CHAPTER VII Page 144

NAG

(µg/

ml)

0.35

0.3

PS COP CP BP CC

0.25

0.2

0.15

0.1

0.05

0

1 2 3 4 5

Time of incubation (days)

Fig 7.10: Effect of different treatment of prawn on NAG production by Aeromonas

punctata HS6 7.4. CONCLUSIONS

Protection of plants from disease produced by phytopathogenic fungi is one of the

most important challenges in agriculture. The total losses as results of plant diseases reach

almost 50% of the crop in developing countries. One–third of this is a consequence of fungal

infections. Therefore finding biological products that could be used for biological fighting is

very important in agriculture. Recent studies demonstrated that chitinase from plants and

microorganisms are able to inhibit the fungal growth.

In this study, a native chitinase with antifungal activity against a wide range of

phytopathogens was isolated from Aeromonas hydrophila and Aeromonas punctata, which is

important since not all Chitinases have antifungal activity. Furthermore, isolated chitinase in

this study may have important implications.

Chitinase plays an important role in the decomposition of chitin and potentially in the

utilization of chitin as a renewable resource. The result concluded that HS4 and HS6 are

mesophilic bacterial strain that have ability to produced huge amount of chitinase in short

cultivation time. The utilization of different chitin substrate by HS4 and HS6 for growth and

production of enzyme and treatment of prawn-shells on production of chitinase and NAG was

investigated. This study highlights the possibility of using HS4 and HS6 to deal with agro-

CHAPTER VII Page 145

industrial wastes to produce NAG. It was concluded that out of all the four different substrate

investigated, Prawn shell was found to be the most suitable substrate for NAG by both HS4

and HS6.

The strains has proved to be a valuable enzyme source for selective production of

NAG which has generated interest as a new functional material with high potential uses in

various pharmaceutical and medical fields. So it may be concluded that colloidal treatment is

the best treatment for HS4 and HS6 for NAG production.

Moreover, these strains are particularly interesting because of;

(1) The large quantities and variety of extracellular metabolites it produces

(2) The possibility of controlling its growth conditions using a variety of substrates for

production of enzymes,

(3) Having unique properties likewise antimicrobial, bioflocculant or even amino-sugar

(NAG) production that have great importance in medical and pharmaceutical fields.

These bacterial strains have adopted special metabolic pathways to survive in extreme

conditions and so has better capacity to produce special bioactive compounds.

Significance of the study

The study showed that soil from rhizosphere is the best source for chitinase producing microorganisms.

Aeromonas hydrophila HS4 and Aeromonas punctata HS6 are novel bacterial strains that have ability to produce large amount of chitinase in short time.

This study highlights the possibility of using Aeromonas hydrophila HS4 and Aeromonas punctata HS6 to deal with agro-industrial wastes to produce chitinase and NAG.

Prawn shell was found to be the most suitable substrate for chitinase production by both

the strains. Thus, it is an important element in utilizing prawn shell waste that not only to

solve environmental problems but also decreases the production cost of microbial

chitinases.

Both strains of Aeromonas sp. have the ability to produce chitinase between temperature

22 and 40 °C which is the field temperature for the cultivation of most of the crops in

India. So it may be applied to field condition against plant pathogens that are the major

problem for agricultural food production.

CHAPTER VII Page 146

Chitinase from Aeromonas hydrophila HS4 showed pH optima and stability in alkaline

range that has great potential in biological pest control because the peritrophic gut lining

of insects is chitinous and has an alkaline pH.

Chitinase from Aeromonas punctata HS6 showed pH optima in acidic range (pH 5) that could be exploited for control of fungal plant pathogens.

The strains have proved to be a valuable enzyme source with high potential in various

pharmaceutical and medical fields. Also, due to their antifungal properties, as proved by

suppressing growth of Shizophyllum commune; these strains may be used as biocontrol

agents.

CHAPTER VII Page 147