Chitinases:

a new pesticide?

Litten, C., Liddle, G., Khanam, T., Lin, Z., Morter, R. & Holmes, E.

Why do we need

pesticides anyway??

• Pest control is vital to the

agricultural industry

• Current methods however

are harsh on the

environment

• Even banned substances

are still used by some

countries

• Lack of an alternative

currently

Effects to the Environment

• Harmful pesticides leach into soils, aquatics

systems and also hedgerows.

• Often they kill not only the pest but other

organisms associated with areas heavily

contaminated with insecticides.

• Neonicotinoids

• DDT

Effects to Wildlife and Humans

• Amphibians, bees and birds have all

been reported to be affected by

pesticides

o Malformations

o Immune suppression

• Human health can also be affected

o DDT

o Non-Hodgkin lymphoma

o Childhood brain tumours

References:

Gilliom, R. J., 2007, Pesticides in U.S. streams and groundwater:

Environmental Science & Technology, v. 41, p. 3407-3413.

Kabasenche, W. P., and M. K. Skinner, 2014, DDT, epigenetic harm, and

transgenerational environmental justice: Environmental Health, v. 13.

Nielsen, S. S., R. McKean-Cowdin, F. M. Farin, E. A. Holly, S. Preston-

Martin, and B. A. Mueller, 2010, Childhood brain tumors, residential

insecticide exposure, and pesticide metabolism genes: Environmental

Health Perspectives, v. 118, p. 144-149.

Schinasi, L., and M. E. Leon, 2014, Non-Hodgkin lymphoma and

occupational exposure to agricultural pesticide chemical groups and

active ingredients: A systematic review and meta-analysis:

International Journal of Environmental Research and Public Health, v.

11, p. 4449-4527.

Sparling, D. W., and G. M. Fellers, 2009, Toxicity of two insecticides to

california, usa, anurans and its relevance to declining amphibian

populations: Environmental Toxicology and Chemistry, v. 28, p. 1696-

1703.

Why do we need an alternative method?

Pesticide resistance is a huge issue in modern agriculture

Resistance to pesticides arises from natural selection, and beneficial

traits may be passed down from generation to generation, resulting in an

eventual population of resistant organisms

The Colorado potato beetle feeds on potato leaves, an adult Colorado

potato beetle can consume up to 10cm2/day

This beetle has developed resistance to 52 compounds from all major

insecticide classes

Huge burden to the US agricultural

industry, the state of Michigan loses

approximately $1.4 million per annum as a

result of this insecticide resistance

Fig. 1 Cumulative number of different active ingredients to which resistance has been reported in the Colorado potato beetle (Whalon et al. 2008)

Chitin

Chitin is the second most abundant natural polymer

A structural component of crustaceans and insects, forming

>80,000 metric tonnes of marine waste per annum

Comprises 22-44% of the cell wall component of most fungi

Chitin is isolated to a distinct group of organisms and is not

found in humans

An ideal target for biopesticide action

Chitinases

Chitinases are lytic enzymes with the ability to break down the

glycosidic bonds of chitin

Increasingly recognised importance for their biotechnological

applications

Naturally produced in organisms with a requirement to break down

chitin, for example Ophiocordyceps unilateralis, or the “zombie ant

fungus”

O. unilateralis uses chitinases to break down the exoskeleton of

carpenter ants and disseminate through their bodies, resulting in

altered behavioural patterns, and eventual death

Importance of Chitinases

Glycosyl Hydrolases are enzymes that are actively hydrolyze glycosidic bonds into

monomers

Chitinases can break down polymeric chitin by cleaving them into chio-

oligosaccharides

Further can be converted by N-acteylglucosamine to GlcNAc monomers

Optimal conditions for maximum effectiveness vary based on species (temperature,

pH, metal ion acquisition and incubation time)

Grouped into 45 different families

Family 18: Bacteria, Fungi, animals and some plant

Family 19: mainly plants (similar to lysozymes)

Arthropods: Chitinases are essential in molting for growth and digestion

Bacteria: recycling of nitrogen and carbon

Fungi: germination and growth

Chitin content vary in fungi regards to growth and lifestyle

Structural Activities

Fig. 2Adapted from: Neuhaus (2001)

Comparing the Enzymes

Fig. 3Adapted from: Withers and Williams (2013)

α-glycosidase

β-glycosidase

α-glycosidase

β-glycosidase

Types of Chitinases

Exochitinase

Endochitinase

Example: Streptomyces spp.

Fig. 1Adapted from: Sigma-Aldrich (2013)

Chitinolytic Activity of C. violaceum in-vitro

Colloidal chitin

supplemented on SM agar

showing

• Left : colloidal chitin

supplemented plate

• Right: colloidal chitin and

HHL supplemental plate

1 and 3: mutant strains

2 and 4: controls

_________________________

Mutant strain grown in two

separate liquid SM medium

• Left: with colloidal chitin

• Right: with HHL

HHL supplemented mutant

shows complete hydrolysis

whereas the other shows

visible non-hydrolysed

particles

Fig. 4 Adapted from: Chernin at al. (1998

Bacteria grown at for 72h in agitation (200rpm) at 28°C.

Plate incubated for 4 days at 28°C.

Chitinolytic Activity

Various filamentous fungi

are destined for autolysis

due to the mixture of

chitinolytic enzyme that is

present. Except for one

species of fungi,

Trichoderma atroviride.

Chitinases do not effect the

spore germination.

Fig. 5 Adapted from: Hartl and Zach (2012)

References:

Information:

Chernin, L. S., Winson, M. K., Thompson, J. M., Haran, S., Bycroft, B. W., Chet, I., Williams, P. and Gordon,

S. A. B. S & Stewart, G. S. (1998). Chitinolytic activity in Chromobacterium violaceum: substrate analysis

and regulation by quorum sensing. Journal of bacteriology, 180(17), 4435-4441.

Hartl, L., Zach, S., & Seidl-Seiboth, V. (2012). Fungal chitinases: diversity, mechanistic properties and

biotechnological potential. Applied microbiology and biotechnology, 93(2), 533-543.

Kuddus, M., & Ahmad, I. Z. (2013). Isolation of novel chitinolytic bacteria and production optimization of

extracellular chitinase. Journal of Genetic Engineering and Biotechnology, 11(1), 39-46.

Neiendam Nielsen, M., & Sørensen, J. (1999). Chitinolytic activity of Pseudomonas fluorescens isolates

from barley and sugar beet rhizosphere. FEMS microbiology ecology, 30(3), 217-227.

Saks, E., & Jankiewicz, U. (2009). [Chitinolytic activity of bacteria]. Postepy biochemii, 56(4), 427-434.

Withers, S. and WIlliams, S.. (2013). Glycoside hydrolases. [ONLINE] Available

at:http://www.cazypedia.org/index.php/Glycoside_hydrolases. [Accessed 16 October 14].

http://www.pnas.org/content/95/8/4276/F1.expansion.html

Images:

Figure 1. Sigma-Aldrich, (2013), Chitinase and Chitin [ONLINE]. Available at:

http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/carbohydrate-

analysis/carbohydrate-analysis-ii.html#Chitin [Accessed 17 October 14].

Figure 2. Neuhaus, Jean-Marc, (2001), Chitinases [ONLINE]. Available

at:http://wwwa.unine.ch/bota/bioch/chitinase.html [Accessed 15 October 14].

Figure 3. Withers, S. and Williams, S., (2013), Retaining and Inverting Glycosyl Hydrolases [ONLINE].

Available at:http://www.cazypedia.org/index.php/Glycoside_hydrolases [Accessed 16 October 14].

Figure 4. Chernin, L. S., Winson, M. K., Thompson, J. M., Haran, S., Bycroft, B. W., Chet, I., Williams, P.

and Gordon, S. A. B. S & Stewart, G. S, (1998), Assay of chitinolytic activity on plates with SM agar

medium supplemented with colloidal chitin (0.2% [wt/vol]) [ONLINE]. Available at:

http://jb.asm.org/content/180/17/4435.figures-only [Accessed 16 October 14].

Figure 5. Hartl, L., Zach, S., (2012), Effect of a chitinolytic enzyme mix from the autolytic phase of T.

atroviride cultures on germination of different fungi [ONLINE]. Available

at: http://link.springer.com/article/10.1007/s00253-011-3723-3/fulltext.html [Accessed 15 October 14].

Chitinases in biocontrol

1. A novel strain of Brevibacillus laterosporus produces

chitinases that contribute to its biocontrol potential

2. Chitinase in the Biocontrol of Sugarcane Red Rot Caused

by Colletotrichum falcatum Went

3. Use of Rhizosphere Chitinolytic Bacteria for Biological

Control of Fusarium oxysporum f. sp. dianthi in Carnation

A novel strain exhibiting entomopathogenic and chitinolytic activity was

isolated from mangrove marsh soil in India.

The isolate was identified as Brevibacillus laterosporus by phenotypic

characterization

Chitinase activity was inhibited by allosamidin indicating that the enzymes

belong to the family 18 chitinases.

The enzymes exhibited antifungal activity against the phytopathogenic

fungus Fusarium equiseti.

Insect toxicity bioassays with larvae of diamondback

moths (Plutella xylostella), showed that addition

of chitinases reduced the time to reach 50 %

mortality upon infection with non-induced

B. laterosporus from 3.3 to 2.1 days.

Antifungal bioassay

The antifungal activity of the chitinase mixture obtained from B.

laterosporus by chitin affinity chromatography was tested using F. equiseti

as indicator strain. Figure below shows that the enzymes indeed inhibited

the fungus in a dose-dependent manner.

Method:

Antifungal assay showing antagonism

against F. equiseti. Paper discs were treated

with 100, 200, and 500 μl of the mixture of

purified chitinases with a protein

concentration of 64 μg/ml, in 10 mM sodium

phosphate, pH 6.0; the samples are marked 1

to 3, respectively. C indicates a control disc

Result:

The control disc is overgrown, disc1 (with

100 μl chitinase solution) and disc 2 (with

200 μl chitinase solution) seem less

overgrown, whereas disc 3

(500 μl) shows a clear zone of inhibition.

Insect toxicity bioassays

The effect of the chitinases on the insecticidal activity of B.laterosporus

Lak1210 against larvae of diamondback moth was studied by analyzing

larval survival on cabbage leaves that had been treated with non-induced

bacteria (i.e. no chitinases produced) alone or with non-induced bacteria

supplemented with chitinases from an induced culture。

a. instar larvae from the control

treatment (sterile water). b Dead

larvae showing discoloration

from green to brownish-black.

The larvae shown are from the

treatment with a suspension of

noninduced B. laterosporus

Lak1210 supplemented with 250

μl of supernatant from a

chitin-induced culture of

B. laterosporus Lak1210

Figure shows that supernatants from chitin-induced cultures increased the lethal

effect of B. laterosporus Lak1210 on the larvae in a dose-dependent manner.

Fitting of the experimental datapoints to the logistic model revealed clear dose–

response effects. Upon addition of 500 μl supernatant, 100 % mortality was

observed on day 5, compared to day 7 without this addition.

All treatments with B. laterosporus Lak1210 caused death of 100 % of the larvae

within 7 days.

Figure. Survival of larvae

on cabbage leaves treated

with sterile water or with

a suspension

of non-induced B.

laterosporus Lak1210

supplemented with 0–500

μl of

supernatant from a chitin-

induced culture of B.

laterosporus Lak1210.

Their ability to produce chitinase were proved under in vitro conditions.

Results from the dual culture study further confirm that the chitin has

influence on the activity of biocontrol agents in suppressing the pathogen

growth.

The native rhizospheric strains of fluorescent pseudomonads and T.

harzianum from sugarcane were able to protect the crop from soil borne

inoculum of red rot and the efficacy has positive correlation with soil

chitinase activity

Antibiosis mode of fungal (Trichoderma spp.) and

bacterial (Pseudomonas spp.) antagonistic strains

Efficacy of chitinases produced by the antagonistic strains was tested by using cell free culture

filtrate in different concentrations (1, 5 & 10%) in the pathogen growth medium (oat meal broth)

and their effect was studied on pathogen growth. To study their nature (proteinaceous/ non-

proteinaceous) they were used in autoclaved and unautoclaved conditions

Results on inhibitory effect of culture filtrate from antagonistic strains

revealed that the secondary metabolites in the filtrates were able to

inhibit the C. falcatum growth

the efficacy was increased with increase in concentration of the filtrate.

The efficacy was found to be high in unautoclaved condition than the

autoclaved condition, which indicates enzymes.

All the antagonistic strains were able to produce chitinase in the medium

their antagonistic activity was proved in terms of lytic zone in the chitin-

amended medium.

In further experiments, it was found that both strains were able to inhibit

the mycelial growth in the presence or absence of the chitin. However

addition of chitin resulted in the enhanced inhibition of mycelial growth by

ARR 1G, FP7 and VPT4 in P. fluorescens

Disease incidence in the non-treated control plants recorded 100 days

after treatment was 32 % as compared with 8 % in the inoculated plants

(Fig.). It increased continuously in the control plants reaching 62 and 96 %

after 130 and 150 days respectively, while treated plants were significantly

protected from the wik (15 and 21 % respectively).

Protection of carnation roots with chitinolytic bacteria

In summary

Those studies provide evidences for the presence of

inducible, extracellular chitinases that contribute to

the strain’s antifungal activity and insecticidal

activity

Confirm biocontrol potential of chitinase in plant

protection

The value of crop losses

20 million tonnes of cereal crops are produced in the

UK every year, the industry is worth £5bn to the UK

economy every year1,2

.

Losses in yield of up to 75% would be seen in some

crops such as potatoes without the use of pesticides3.

By 2020 the worldwide population will increase by

1.5bn, putting greater pressure on global food

supplies3.

The use of pesticides is vital in maintaining global food

security and economic stability.

Economics of chitinases

Difficult to get economic data on chitinases since they aren’t

currently in great use.

However production costs of conventional pesticides are very

cheap, approximately $32USD per acre of a cereal crop field4.

Chitinases would have to be produced for the same price, or

less than conventional pesticides to be economically viable.

It would be expected that production of chitinases en masse

would reduce the production costs involved.

References

1) Farming Statistics (2010). UK Agriculture. Accessed 18th

October 2014 from:

http://www.ukagriculture.com/statistics/farming_statistics.cfm?strsection=Total%20Cereals

2) Agriculture in the United Kingdom (2012). Department for Food, Rural Affairs and

Agriculture, UK. Accessed 18th

October 2014 from:

https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/208436/auk-2012-

25jun13.pdf

3) What are pesticides (2014). European Crop Protection. Accessed 18th

October 2014 from:

http://www.ecpa.eu/page/what-are-pesticides

4) Crop cost and return guide (2013). Purdue University. Accessed 18th

October 2014 from:

http://www.agecon.purdue.edu/extension/pubs/id166_2013%20_novembe.pdf

Are Chitinases worth it?

Environmentally safe and without creating any soil pollution

Successful biocontrol protection for plants

Optimal conditions for maximum effectiveness vary based on species –

adaptable to use on different crops

Already found naturally existing in many foods, such as: bananas,

chestnuts, kiwis, avocados, papaya and tomatoes – safe for

consumption.

Use of chitinases has been liked to a reduction in allergy levels in

conditions such as asthma

“The potential of chitinases is likely to be enhanced by combining

them with other bioactive peptides and lytic enzymes, such as

glucanases, as is found in natural systems”, (Herrera-Estrella & Chet,

1999).

Are Chitinases worth it?

Cost of production is not yet clear

As are the long term affects of wide usage

Requires more research into the full development of a wide-use

pesticide

May require the addition of other common pesticide components for

fully effective use – increase in costs and reduction in economical

safety?

In Conclusion:

Chitinases appear to be more beneficial to the environment as well as

being an effective pesticide than those currently in use

However, further research is required to produce a marketable

pesticide and calculate production/sale costs for its commercial and

agricultural use to be viable

Is it worth investing?

- chitinases are a potential highly successful pesticide of the near

future, with the required research already in progress; if production

costs are competitive enough with that of current pesticides it could

be both a highly successful financial investment as well as a highly

successful environmentally beneficial product.

References:

Herrera-Estrella, A. & Chet, I., ‘Chitinases in biological control’, EXS,

vol. 87 (1999), pp. 1710-1840.

Renkema, G.H., Boot, R.G., Muijsers, A.O., Donker-Koopman, W.E. &

Aerts, J.M., (1995), ‘Purification and Characterization of Human

Chitotriosidase, a Novel Member of the Chitinase Family of

Proteins’, The Journal of Biological Chemistry, vol. 270, issue 5, (Feb.

1995), pp. 2198–2202.

Salzer, P., Bonanomi, A., Beyer, K., Vögeli-Lange, R., Aeschbacher, R.A.,

Lange, J., Wiemken, A., Kim, D., Cook, D.R. & Boller, T., (2000),

‘Differential expression of eight chitinase genes in Medicago truncatula

roots during mycorrhiza formation, nodulation, and pathogen

infection’, Molecular Plant-Microbe Interactions, vol .13, issue 7, (July

2000), pp. 763–77.