study on characterization of chitinase from streptomyces

Summary chitinase activities of 500 Streptomyces strains 42 Table 3.3.. Primary screening good Streptomyces strains for chitinase production .... Chitinase activities of 60 Streptomyce

Nguyen Thanh Huong

STUDY ON CHARACTERIZATION OF CHITINASE

***

Nguyen Thanh Huong

STUDY ON CHARACTERIZATION OF CHITINASE

FROM STREPTOMYCES

Speciality: Biotechnology Code: 60 42 80

MASTER THESIS MAJOR BIOTECHNOLOGY

SUPERVISOR: Dr DUONG VAN HOP

***

PCR - Polymerase chain reaction

PR - protein - pathogenesis-related proteins

rDNA - Ribosomal DNA

SEM - Scanning electron microscope

SDS - PAGE - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TLC - Thin layer chromatography

VTCC - Vietnam Type Culture Collection

YS - Yeast extract - starch

LIST OF FIGURES

Figure 2 Chemical structures of cellulose and chitin 6

Figure 4 Phylogenetic relationships of family 19 chitinases 17

Figure 5 Amino acid sequence of a chitinase from Streptomyces

erythraeus

18

Figure 6 Purification of the pea antifungal hydrolases 21

Figure 7 Production of recombinant chitinase from Trichoderma

virens UKM-1 in E.coli

23

Figure 8 Calibration curve of N - acetyl – Glucosamine 33

Figure 9 Clear zones’ diameters illustrated Chitinase activity of

Figure 11 Extraction of the total DNA (A) and amplification of 16S

rDNA (B) from strain VN08A-438

47

Figure 12 Phylogenetic tree contruction for VN08-A0438 strain 48

Figure 13 Effect of some parameters on chitinase activity of

Streptomyces VN08-A0438

50

Figure 14 Chromatographygram of chitinase enzyme on Sephadex

G100 (A) and bioassay of the active fraction (B)

52

Figure 15 Zymogram (A) and SDS-PAGE (B) of chitinase 53

Figure 16 Effect of temperature and pH on chitinase activity 54

Figure 17 TLC analyzing of final chitinase reaction products 55

LIST OF TABLES

Chapter 1

Table 1.2 Comparison of the characteristics of purified chitinase from

others reported Enterobacter sp

Table 3.2 Summary chitinase activities of 500 Streptomyces strains 42

Table 3.3 Chitinase avtivity of 60 selected Streptomyces strains 43

Table 3.4 Effect of sugar on the growth of strain VN08-A0438 46

TABLE OF CONTENTS

ABSTRACT 7

CHAPTER 1 INTRODUCTION 10

1.1 Chitin and application of chitin and chitinoligosaccharides 10

1.1.1 Application of chitin in Agriculture and Environment 12

1.1.2 Application of chitin in Medicine 14

1.1.3 Application of chitin in cosmetic and industry 15

1.2 Compositions and methods for producing chitin 17

1.3 Chitinase 20

1.3.1 Main chitinase sources 20

1.3.2 Chitinase from Streptomyces and other sources 22

1.3.3 Purification of chitinase 26

1.3.4 Recombinant chitinase 28

1.3.5 Diversity of chitinase 29

1.4 Potential of chitin product application in Vietnam 31

1.5 All domestic related studies 32

CHAPTER 2 MATERIALS AND METHODS 35

2.1 Analytical instruments 35

2.2 Microbes 35

2.3 Media 35

2.4 Methodology 36

2.4.1 Screening of chitinase-producing Streptomyces and culture conditions 36

2.4.2 Selecting good chitinese producers by chitinase activity assay 36

2.4.3 Identification of Streptomyces strain 39

2.4.4 Effect of culture conditions (temperature, pH, aeration, carbon, nitro sources) for chitinase

fermentation from Streptomyces 42

2.4.5 Purification of chitinase 43

2.4.6 SDS-PAGE and activity gel (zymogram) 44

2.4.7 Characterization of the partly purified chitinase 45

CHAPTER 3 RESULTS AND DISCUSSION 47

3.1 Screening of chitinase-producing Streptomyces 47

3.1.1 Primary screening good Streptomyces strains for chitinase production 47

3.1.2 Chitinase activities of 60 Streptomyces strains in liquid medium 48

3.2 Identification of Streptomyces strain VN08-A0438 50

3.2.1 Morphology of strain VN08-A0438 50

3.2.2 Studying carbon sources assimilation of the culture 51

3.2.3 Some physiological criteria of the culture 53

3.2.4 16S rDNA sequencing of Streptomyces VN08-A0438 53

3.3 Selecting medium and conditions for chitinase production 55

3.4 Purification of chitinase 57

3.5 Characterization of the partly purified chitinase 59

CONCLUSION 62

FURTHER STUDIES 62

BIBLIOGRAPHY 63

ABSTRACT

In this study, a total of 500 Streptomyces strains isolated from soil in Hoang

Lien Son national park (Sa Pa, Vietnam) were subjected to a screening for their

chitinase activities Through two screening steps, Streptomyces strain

VN08-A0438 had the highest chitinase activity so it was selected for next studies Taxonomical studies based on the morphology, physiological criteria and 16S rDNA gene sequencing indicated that strain VN08-A0438 was belonging genus

Streptomyces and was proposed as Streptomyces chromofuscus

Besides that, selecting conditions for chitinase production from strain A0438 were studied, focused on some key factors on chitinase production: optimum temperature, pH, aeration, fermentation time, carbon and nitrogen sources The culture grew well on medium with carbon source as glucose - 5 g, colloidol chitin 5 g and nitrogen source as (NH4)2SO4 - 2 g, at 35oC, pH 6.5 with shacking rate 200 rpm for 5 days

VN08-Chitinase from Streptomyces sp VN08-A0438 was purified by ammonium

sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel filtration Treatment of chitinase (80% ammonium sulfate saturation) gave highest specific activity (40U/mg protein) The high chitinase activity was found in fractions from 45 to 70 The sample was concentrated by evaporation at room temperature to 10 folds, and loaded on SDS-PAGE and activity gel Characterization of the partly purified chitinase was also checked, including effect of pH, temperature, and Thin layer chromatography (TLC) for detecting the enzymatic product Enzyme was stable at pH 5-5.5 and 55oC The TLC chromatogram showed that there were a number of three enzymes involved: endochitinase with chitobias and chitinooligosacharide as the main products, exochitinase with N-acetyl glucosamine and chitinooligosaccharide as the main products, chitobiase with N-acetyl glucozamin as the final products

TÓM TẮT

Tên luận văn: Nghiên cứu đặc tính chitinase từ xạ khuẩn

Người hướng dẫn: TS Dương Văn Hợp

Viện Vi sinh vật và Công nghệ Sing học, Đại học Quốc gia Hà Nội

Ngành: Công nghệ sinh học Chuyên ngành: Công nghệ sinh học

Mã số: 60 42 80

Trong đề tài này, năm trăm chủng xạ khuẩn Streptomyces được phân lập từ

Vườn Quốc gia Hoàng Liên Sơn được tiến hành tuyển chọn và xác định hoạt tính chitinase Thông qua 2 quá trình sàng lọc cơ bản, chủng xạ khuẩn mang kí hiệu VN08-A0438 là chủng có hoạt tính sinh chitinase cao nhất, vì vậy chủng này được lựa chọn để phục vụ cho các mục đích tiếp theo của nghiên cứu này

Cũng trong nghiên cứu này, chủng VN08-A0438 được tiến hành định loại dựa trên đặc điểm hình thái, hóa sinh và giải trình tự 16S rDNA, kết quả cho thấy

xạ khuẩn phân lập được là chủng Streptomyces chromofuscus

Bên cạnh đó, chúng tôi cũng tiến hành các thí nghiệm xác định điều kiện tối

ưu cho sự phát triển và sinh chitinase của chủng Streptomyces chromofuscus

VN08-A0438 bao gồm các thiết lập về nhiệt độ, pH, chế độ thoáng khí, thời gian lên men, thử nghiệm các nguồn cácbon và nitơ khác nhau Kết quả phân tích cho thấy chủng này phát triển tốt ở môi trường có nguồn cacbon là glucoza (5g/l), colloidol chitin (5g/l) và (NH4)2SO4 (2 g/l) được sử dụng là nguồn cung cấp nitơ, điều kiện nhiệt độ 350C, pH = 6.5 và lắc 200 vòng/phút trong 5 ngày

Chitinase của chủng Streptomyces VN08-A0438 sau đó được tinh sạch sơ bộ

bằng kết tủa amôn sunphat Chitinase cũng được nghiên cứu đặc tính về độ bền nhiệt, pH và sắc ký bản mỏng (TLC) Kết quả phân tích cho thấy chitinase bền ở

pH 5.5 và nhiệt độ 550

C Kết quả phân tích TLC cho thấy sản phẩm tinh sạch có chứa toàn bộ mono-; di- và oligomers

Chitinases (EC 3.2.1.14) are glycosyl hydrolases group of enzymes that vary widely in size (20 kDa to about 90 kDa) Bacterial chitinases have a molecular weight range of ~20-60 kDa, which is similar to that of plant chitinases (~25-40 kDa) and are smaller than insect chitinases (~40-85 kDa) Chitinases can be

produced by many bacteria, including Aeromonas, Alteromonas, Bacillus, Serratia, Streptomyces, Enterobacter, Vibrio and Escherichia Chitinase-producing bacteria

were isolated from different environments including soil, garden and park waste compost and shellfish

During the last decade, chitinases have received increased attention because

of their wide range of applications The enzyme could either be used directly in the biological control on microorganisms

To contribute to purify chitinase from Streptomyces strains and detect their

characterization, we have implemented topic: “Study on characterization of

chitinase from Streptomyces”

CHAPTER 1 INTRODUCTION

1.1 Chitin and application of chitin and chitinoligosaccharides

Historically, chitin was first discovered in the sediment of a fungus extracted

by Braconnot in 1821 The substance is named "Fungine" to remember its origin

In 1823, Odier isolated a substance from the beetle, which he called chitin or

"chiton" (Greek meaning is armor) However, he did not detect the presence of nitrogen in this substance Both Odier and Braconnot eventually concluded that chitin has the same formula as cellulose [41]

Chitin (C8H13O5N)n is one of the natural polysaccharides including a copolymer of N-acetyl-D-glucosamine and D-glucosamine residues These two components are linked together by β-1,4 glycosidic bonds Chitin is popular and can be found in a variety of species such as in shells of crustaceans, in cuticles of insects or in the cell wall of fungi and some algae [15] Being an amorphous solid, chitin has typical properties of these groups like largely insoluble in water, dilute acids and alkali as well Although chitin is well known in numerous commercial uses, greater commercial benefits can be found by changing it into a deacetylated product named chitosan [7]

Figure 1 Natural sources of chitin [33],[36],[37],[40]

Chitin has a fibrous shape and it is an extremely insoluble material With the exception of cellulose, chitin is the most abundant biopolymers globally with an

estimated yearly production account for 1010 to 1011 tons Structural similarities between chitin and cellulose are illustrated in Fig 1 Chitin occupies a large percentage in the structural component of most fungi and algae cell walls, insect exoskeletons, the shells of crustaceans, and the microfilarial sheath of nematodes Nevertheless, chitin is soluble in most of the organic solvents [41] In addition, chitins in animal tissues are frequently calcified, such as in the case of shellfish [16] In a detailed instance, the proportion of chitin from shrimp and crab are usually 0.06 and 0.17 g/ml respectively It means that chitin in shrimp is more porous than in crab Chitin in mollusks is 2.6 times as porous as in crab A thermal conductivity showed that the proportions of chitin and chitosan from crustaceans are very high (0.39g/cm3) In crustaceans, the proportion of commercial chitin and chitosan show some differences This may be due to the difference in crustacean species or processing methods In addition, the deacetyl level also increases their proportion [41] The content of crude chitin varies between species, as illustrated

in table 1 [16]

Table 1.1 Chitin content of some organisms

The amount of acetylation of the D-glucosamine (GlcN) residues in chitin made it notable Polymer consisting of 70% or higher acetylating are considered

chitin whistle those with less than 30% are called chitosan In fact, the vast majority of chitin produced annual in biosphere are degraded by chitinase [27]

Figure 2 Chemical structures of cellulose and chitin

1.1.1 Application of chitin in Agriculture and Environment

According to scientists, chitin is a useful substance that helps plants develop

It has been known to take part in a popular phenomenon named defense mechanisms in plants as an extreme good inducer Plants productivity and life expectancy also witnessed a remarkable increase by using Chitin as a specific fertilizer Chitin is also regulated in agriculture use within the USA by US Environmental Protection Agency Besides, in agriculture and horticulture, chitosan-a substance derived from chitin, can be used as a bio-control elictor [30]

Chitin oligosaccharides are also well-known by their abilities in “fast turning on” plant’s defense mechanisms against some invasion by fungi, therefore, enhance the plant disease resistance Similarly, in some symbioses such as beans and clover plants, symbiotic bacteria which usually live around plant’s root, can release chitin oligosaccharides in order to give a sign of root nodules formation, sites for nitrogen-fixation [31]

Chitosan is a form of de-acetylated chitin and have a better potential in biodegradation than chitin [38] It is known to offer a natural alternative to the use

of medical products Hence, environment and human can be protected better Chitosan can trigger plant defensive mechanisms as a vaccine in human, stimulate plant growth and induce unexpected effects of certain enzymes (synthesis of phytoalexins, chitinases, pectinases, glucanases, and lignin) The approach using

chitosan - an organic compound opens a real promising of bio-control chitosan

tool-In addition to the growth-stimulation properties and fungi, chitosans are used for: Seed-coating

Frost protection

Bloom and fruit-setting stimulation

Timed release of product into the soil (fertilizers, organic control agents, nutrients)

Protective coating for fruits and vegetables [39]

* The role of chitin in environment

Scientists believe that chitin is used for environmental treatment because of its features: natural origin and being biodegrable Most of physicochemical-type treatments result in environmental problems such as vulnerable and pollution, therefore, different approaches using “go green methods” are necessary Hence,

“chitosan method” can be the suitable choice for several points of view being indicated below

By integrating a natural polymer made of crustaceans into an existing system, two purposes would be achieved: (i) increasing the effectiveness of water treatment and (ii) reducing or eliminating harmful synthetic chemical compounds such as aluminum sulphate and synthetic polymers Some chitosan’s characteristics that can be enumerated for ecological solution are:

Natural and biodegradable

A powerful competitor for synthetic chemical products

Potentially reduces the use of alum by up to 60% and eliminates 100% of the polymers from the treated water

Improves system performance (suspended solids and chemical oxygen demand)

Significantly reduces odor [39]

1.1.2 Application of chitin in Medicine

Scientists estimated the extremely high cost of producing pure oligosaccharides in laboratories With the cost of $5 to $15 per milligram, a normal experiment taking place in a laboratory can cost many thousands of dollars Although chitin oligosaccharides may have potential use in human medicine, this costly experiment is considered as the main barrier to popularize knowledge about chitin oligosaccharides in medicine Fortunately, it is known that numerous bacteria species can easily transfer substances into others and chitin oligosaccharide is not an exception Thanks to natural enzymes, chitin oligosaccharides can be produced quickly and environmentally while, over a period of 30 years, in laboratories, this process requires intensive use of acid and bases [31]

Occupations associated with high environmental chitin levels, such as shellfish processors, are prone to high incidences of asthma Recent studies have suggested that chitin may play a role in a possible pathway in human allergic disease Specifically, mice treated with chitin develop an allergic response, characterized by a build-up of expressing innate immune cells In these treated mice, additional treatment with a chitinase enzyme abolishes the response [30]

* For biopharmaceutical uses

It is estimated that the number of chitosan applications in health fields is plentiful Such properties (bacteriostatic, immunologic, antitumoral, cicatrizant, hemostatic and anticoagulant) are of great value Take a human disease for example, because of its biocompatibility with human tissue, chitosan’s cicatrizant properties have illustrated its role as a component, notably in all types of dressings (artificial skin, corneal dressings, etc.), surgical sutures, dental implants, and in rebuilding bones and gums A specific technique that is developing nowadays is using chitosan instead of human or animal’s skin (artificial skin) and producing surgical sutures that can be absorbed after operations and corneal contact lenses Finally, chitosan can be used to delivers and time-releases drugs used to treat

animals and humans It is said that chitosan application in medicine can more and more developed unless having the human regulation in pharmaceutical-grade requirements Possible applications include:

Ointments for wounds

in dyes, fabrics and adhesives Industrial separation membranes and ion-exchange resins can be made from chitin In paper production, chitin is known to be a substance improving paper’s size and strength

In surgical thread, chitin with its strong and flexible properties is very favorable Its biodegradibility means it wears away with time as the wound heals Its unusual properties that accelerate healing of wounds in humans can make it

easy to produce artificial thread [30]

* In food

In Europe, United States and Japan, chitosan has widely used in food production, preservation and in diet diagrams Because of its “lipid trap”

properties, chitin acts as an important dietetic breakthrough When chitosan goes into human digestive system, that human body cannot digest this substance makes

it acts as a fiber, a crucial diet component Research has found that 20 to 30% of cholesterol can link with chitosan, hence, reduce the amount of cholesterol in human blood In fish sauce preparation, chitosan is used due to its thickening and stabilizing properties It is also known to be used in other dishes that hold their consistency well Finally, due to the flocculating property, chitosan acts as a flocculating agent that can be used to clarify beverages Moreover, chitosan is also phytosanitary and based on this property, human can use chitosan in food protection Chitosan can be changed into liquid forms and then sprayed in dilute form on foods such as fruits and vegetables, creating a protective, antibacterial, fungi static film This action is so popular that Japanese use it as an effective method of fruit protective measure There are many other applications in the areas

of nutraceutical and nutritional supplements, particularly for the broad range of chitosans that have been chemically or enzymatically modified

Principal commercial applications include:

Preservatives

Food stabilizers

Animal feed additives

Anti-cholesterol additives (fat traps) [39]

* In cosmetics

Chitosan is applied popularly in cosmetics Its abilities in skin treatment have been recorded Chitosan forms a protective, moisturizing and elastic film on the surface of the human skin, binds numerous failures, spots or ingredients on the skin Therefore, chitosan is applied in cosmetics in the name of formulating moisturizing agents such as sunscreens and organic acids protector… With these characteristics, chitosan can enhance skin bioactivity and effectiveness Besides, due to its antibacterial properties, chitosan is widely used in the composition of

skin-care creams, shampoos and hair spray It has been certified that there have been numerous patents registered and new applications are just beginning to appear including the most highly prized moisturizing and chitosan’s antibacterial properties Applications include:

Maintain skin moisture

Treat acne

Tone skin

Protect the epidermis

Reduce static electricity in hair

Fight dandruff

Improve suppleness of hair

Make hair softer [39]

1.2 Compositions and methods for producing chitin

Seafood has recently shown its potential value in contributing to biochemical sources With the percentage of 30%, edible proportions of seafood play an important part in human diet and become a delicacy praised by many gastronomists However, the remainder that counts for 70% (mostly include shells)

is an extremely waste As stated in recent researches, thanks to scientists’ efforts, numerous biochemical substances such as chitin - a natural biopolymer with unique properties, pigments, seafood peptones, etc., can be produced from seafood

Nevertheless, despite that chitin is a highly applicable polymer, it is still a relatively “new” polymer in research and food processing applications [16]

Chitin is common in most of flora and fauna species The traditional sources

of chitin can be found in shellfish waste from shrimp, Antarctic Krill, crab and lobster processing [5] It is also said that the amount of chitin in these species widely varies from trace quantities up to 40% of the body weight of the organism Among that, the crustacean waste is the most important chitin source for commercial use due to its high chitin content and ready availability [35]

Worldwide, ca 75,000 tons of dried shrimp shells are produced annually, and these could easily yield 3,000 tons of chitin [1]

* Chemical methods

It is almost easy and quick to produce chitin from shrimp waste by chemical methods A 4% NaOH solution which is used for deproteination and 4% HCl for demineralization can be used to isolate chitin in this treatment However, people believed that this process may not be considered as a good recovery option because

of expensive cost and non - environmental friendly This leads to other approaches

in producing chitin and biological method that uses the more environmental friendly technologies such as partial fermentation using lactic acid bacteria for the production of chitin [14]

* Physical methods

It can be stated that physical methods are of great value in producing chitin from seafood Researches demonstrated that shrimp waste contains about 23% chitin and annually, 80,000 tons of this waste was released in India Recently, scientists has tested and indicated that irradiation of shrimp shells with gamma irradiation dose of 25 kGy reduces the time of reaction of deproteinization from 8

hr to 1 hr, resulting in tremendous amount of energy and cost-saving in the process [27] Radiation method is illustrated in scheme 2

Scheme 1: Conventional method

Shrimp shells → Demineralization with 2 N HCl for 48 hr → Deproteinization with 1 N NaOH at 1000oC for 8 hours → Chitin

Scheme 2: Radiation method

Shrimp shells → Irradiated to 25 kGy with gamma radiation

→Demineralization with 2 N HCl for 48 hr → Deproteinization with 1 N NaOH at

1000oC for 1 hour → Chitin

Studies reported that in radiation process, chitin structure is changed and undergoes chain scission in a manner similar to cellulose as the backbones of these two substances are very similar

The only concern of chitin resulted from its insolubility property restricts its applications However, chitin can be easily converted into chitosan by using 50% w/w NaOH whereby acetyl group of chitin is converted into a free amino group Chitosan is readily soluble in acidic media Chemical structures of both chitin and chitosan are shown in Fig 3

Figure 3 Structure of chitin and chitosan [13]

Physical method in producing chitosan from chitin is more effective in comparison with chemical method because of its simple and fast features in degradation It is clear that radiation process can be operated in ambient temperature in either dry or solution form without any additive In physical method, not only contaminations of additive and thermally decomposed materials cannot be found, but also the molecular weight distribution is narrower than that of the conventional method [13]

* Biological methods (Enzymatic methods)

Proteases can be used for the deproteinization of crustacean shells for the production of chitin or chitosan Enzymes as tuna protease were used at pH 8.6 and 37.5oC, papain at pH 5.5-6.0 at 37 oC, or bacterial protease at pH 7.0 and 60oC for

over 60 hr After treatment with enzyme, the amount of protein still associate with

chitin was about 5% In 1988, Shimahara and Takiguchi used bacterial protease from Pseudomonas maltophilia in a culture medium with crustacean shell, and observed that after 24h, the protein content remaining in the shells was only about

1%

Chitin deacetylase, the enzyme responsible for the deacetylation of chitin to chitosan, shows a pH optimum at 5.5 and is markedly inhibited by acetate It showed no effect against chitin in its isolated form, but was active against a soluble glycol chitin substrate

According to Kauss and Bauch (1988), chitin deacetylase extracted from

Colletotrichum lindemuthianum exhibited similar properties but had a pH optimum

at 8.5 and was not inhibited by sodium acetate The enzyme is found in the cell extracts and also secreted by this plant pathogen, so it is more easily isolated from the culture filtrate

One important fact about chitin deacetylase is that it is ineffective against preformed chitin but it readily attacks nascent chitin chains In other words, chitosan is made by deacetylation of chitin provided that the deacetylation process occurs in tandem with chitin synthesis, those requirements being met when both chitin synthetase and chitin deacetylase are present simultaneousfy Seemingly, the

two enzymes operate in tandem, one polyrnerizing GlcNAc unit from GlcNAc, the other removing acetate moieties from the nascent chains (Bartnicki-Garcia, 1989) [16]

UDP-1.3 Chitinase

1.3.1 Main chitinase sources

In biotechnology, chitooligomers that are well-known for their biotechnological value were generated in a degradation process catalyzed by chitinase Chitinase (EC 3.2.1.14) is a member of the glycoside hydrolyse family, and are characterized by their ability to catalyze the hydrolytic cleavage of chitin [27] Chitinase cleaves a bond between the C1 of N-acetylmuramic acid and C4

of N-acetylglucosamine in the bacterial peptidoglycan (by displaying a more or less pronounced lysozyme activity EC 3.2.1.17) A chitinase was first described

in 1911 by Bernard, and after that, in 1929 Karrer and Hofmann found chitinase

in snail [4] Chitinase has been detected in a wide variety of organisms including organisms that do not contain chitin, such as bacteria, fungi, viruses, plants and insects [27] and play important physiological and ecological roles [34] such as energy extraction from the environment, modification of the chitin components in

fungi, anthropods and the stress response systems in plants [33]

The roles of chitinases in different organisms are diverse Bacteria produce chitinases to digest chitin for use as a carbon and energy sources, while fungi produce this enzyme to modify the important cell wall component chitin and invertebrates require chitinases for the partial degradation of old exoskeletons In plants, however, chitinases are part of the plants defense mechanisms against fungal pathogens [26]

The two types of chitinase families differ not only in 3D structure but also in their biochemical properties For instance, family 18 chitinases hydrolyse the glycosidic bond with retention of the anomeric configuration while family 19 chitinases hydrolyse with inversion Family 18 chitinases are sensitive to allosamidin, but a family 19 chitinases from higher plantshave been shown to be insensitive Family 18 chitinases hydrolyse GlcNAc- GlcNAc and GlcNAc-GlcN linkages, whereas family 19 chitinases hydrolyse GlcNAc-GlcNAc and GlcN-GlcNAc Because of their catalytic mechanisms, these differences are probably common among all members of the two families Substrate - assisted catalysis is the most widely accepted model for the catalytic mechanism of family 18 chitinases, whereas a general acid-and-base mechanism has been suggested to be the catalyticmechanism for family 19 chitinases [34]

Recently, chitinase can be produced from marine bacteria and fungi on a laboratory bioreactor and pilot plant scales based on three major modes of operation: batch, fed-batch and continous Batch growth refers to culturing in a vessel with an initial charge of medium that is not altered by further nutrient

addition or removal This form of cultivation is simple and widely used both in the laboratory and industrially In fed-batch culture, nutrients are continuously or semi-continuously fed, while effluent is removed discontinuously Fed-batch operation permits the substrate concentration to be maintained at some pre-determined level Continuous culture provides constant reactor conditions for growth and product formation and supplies uniform-quality products [20] Naturally, chitinases are produced by microorganisms and its production is influenced by environment conditions such as temperature, nutrients resources and soil pH [8]

1.3.2 Chitinase from Streptomyces and other sources

1.3.2.1 Chitinase from Streptomyces

Streptomyces species are important soil microorganisms Chitinase C from Streptomyces griseus HUT6037, described in1997, is the first family 19 chitinase found in an organismother than higher plants [34]

Family 19 chitinase was almost found in plant (plant class I, II, IV chitinase)

with among the aception, the Streptomyces griseus Chitinase C S griseus

chitinase C exhibited strong antifungal activity, whereas the other bacterial chitinases which belong to family 19 did not This notes that antifungal activity may be a general property of family 19 chitinases Two relationships between

Streptomyces family 19 chitinase and plant family 19 chitinase were constructed A

common ancestral chitinase was already present prior to divergence of plant and bacteria, and family 19 chitinase then evolved independently in plants and bacteria

It means that family 19 chitinase in Streptomyces and plant develop from an origin (figure 4) Some strains of Streptomyces are plant pathogens, and family 19 chitinase from Streptomyces can be acquired from plants by horizontal gene

transfer [34]

Figure 4 Phylogenetic relationships of family 19 chitinases

Exo-and endoactivities were described in Streptomyces plicatus; Streptomyces erythraeus chitinase had a Mr of 30,000, a pI of 3.7 and showed optimal activity at pH 5.0 in the presence of a ≤ 0.2 M buffer Using chitooligosaccharides and their derivatives, the binding mode of the Streptomyces erythraeus chitinase to the substrate seems similar to that of hen egg white or Streptomyces erythraeus lysozymes The Streptomyces erythraeus chitinase

consisted of 290 amino acid residues (Mr = 30,000) and has two disulfide bridges

at Cys 45-Cys 49 and Cys 265-Cys 272 Chitinase from Streptomyces plicatus was cloned (figure 3) The partial DNA sequence showed that the protein possesses a

signal sequence of 30 amino acids This chitinase didn’t exhibit sequence

homology with the Streptomyces erythraeus chitinase [4]

Cellulose, chitin, xylan, for example, are the most abundant carbon polymers

on Earth and are major constitients of the soil The Streptomycetes depend on these subtrates for growth, and glucose kinase and not cAMP seems to be involved in

catabolite control in Streptomyces [3]

Figure 5 Amino acid sequence of a chitinase from Streptomyces erythraeus

1.3.2.2 Chitinase from other sources

* Plant chitinase

All plant chitinases are small proteins with Mr from 25.000 to 40.000 units Plant chitinases are classified in three classes: class I, II and III chitinase All three classes can be present in the same plant Many plant chitinase are PR-proteins They are induced in the presence of pathogens, or pathogen extracts, and also after

a stress Plant chitinases are potent inhibitors of fungal growth, but other enzymes are induced simultaneously [4]

* Fungal chitinase

Fungal chitinases could be involved in growth regulation For example, the chitinase from Saccharomyces cerevisiae was required for cell separation Chitinases could also be involved in the penetration into a host by mycoparasites of

by entomopathogenic fungi [4] Fungal chitinases played roles in autolytic,

nutritional, and morphogenetic functions [12]

* Bacterial chitinase

Bacterial chitinases were extracellular enzymes Bacteria played a large role

in chitin mineralization, for example in marine waters and sediments, but not all species were able to hydrolyse chitin [4]

* Insect chitinase

In insects, chitinolytic activities were found principally in the integument, moulting fluid, haemolymph and alimentary canal The molecular weights of chitinases and β - N - acetylglucosaminidases from invertebrates usually range between 40000 and 75000 and between 60000 and 150000 respectively Several insect chitinases are glycoproteins [4] Chitinases have important roles in postembryonic development and degradation of old exocuticle in molting [12]

* Fish chitinases

Chitinases have also been described in the digestive tract of some fishes feeding on invertebrates, such as antarctic fishes feeding on krill or in Dover sole

Chitinase was purified from the stomach of red sea bream Its molecular weight

was about 46000 bps, its pI 8.3, its optimal temperature and pH were 50oC and 5.5, respectively The activity was strongly inhibited by Hg2+, Fe 2+ and Sn2+ The final hydrolysis products of chitin were N - acetylglucosamine and chitobiose The chitinase was synthesized within the egg and could be induced during the larval period by the consumption of exogenous foods In Japanese eel, chitinase was found in the stomach However, the digestive tract of eel contained also chitin-decomposing bacteria [4]

* Role of chitinase in humans

Chitinases in humans play a role in defense against pathogens, which have chitinous structures [12]

1.3.3 Purification of chitinase

Chitinase is difficult to purify and modify chemically A comparison table of

the characteristics of purified chitinase from Enterobacter sp was established (table

2) [2] There are many methods for chitinase purification, depend on the sources

where chitinase was purified from

Plant chitinase is specific example for chitinase purification Chitinases can

be purified from a total homogenate, from the intercellular fruid or from latex Affinity chromatography, using chitin or colloidol chitin, is a successful method

for the purification of the chitinases from barley leaf intercellular fluid, soybean, bean, tobacco and wheat Chitinases have very basic or very acid isoelectric points

(and they are described as basic or acidic proteins) This characteristic has been

used to purify chitinase Figure 6 shows an example of the purification of pathogenesis-related proteins (PR-protein) [4]

Figure 6 Purification of the pea antifungal hydrolases

Table 1.2 Comparison of the characteristics of purified chitinase from others

(GlcNAc) (GlcNAc)2

Cu2+, Co2+, Ag+, Hg2+, NBS, DTNB,

Iodoacetamide

Enterobacte

sp G-1 Endochitinase 60 70 40

Colloidol chitin

(GlcNAc)2 (GlcNAc)3 (GlcNAc)4

1.3.4 Recombinant chitinase

Chitinase is known as a material for control of phytophathogenic fungi and insect pests Because of this reason, chitinase has attracted a lot of attention However, chitinase is uneconomic for commercialized due to the high cost in its productions The development in recombinant DNA technology has helped to reduce chitinase production cost and enhance its production Bacterial expression systems are the most common and preferred method for the over expression of recombinant proteins because of fast cell growth, inexpensive media, and

relatively simple gene manipulation Escherichia coli bacterial has been widely

studied in recombinant chitinase Method of recombinant chitinase from

Trichoderma virens UKM-1 in E.coli will be shown latter as a typical example Recently, chitinase genes isolated from Serratia marcescens was introduced into the endophytic bacterium Pseudomonas fluorescens to improve the control of Rhizoctonia solani on beans. Chitinases genes from rice have potential to enhance plant resistance against fungal disease in many plants including strawberry, cucumber and tobacco [8]

Figure 7 Production of recombinant chitinase from

Trichoderma virens UKM-1 in E.coli [8]

1.3.5 Diversity of chitinase

There are four forms of chitinase: endochitinase was defined as an enzyme

splitting within the chitin polymer, exochitinase (EC 3.2.1.14) as an enzyme releasing chitobiose, β-N-acetylglucosaminidase releases N-acetylglucosamine

monomers from chitin and chitobiase hydrolyses chitobiose [4] Endochitinases

(EC 3.2.1.14) cleaved chitin polymers at random internal sites, whereas exochitinases cleaved chitin progressively starting at the non-reducing ends of the chains and release N-acetyl-D-glucosamine (GlcNAc) monomers or

Further incubate at 18, 20, 22, and 30°C.Flasks were removed every 2 h

Harvest cells by centrifugation at 8000g, 4°C for 10 min Pellet was taken

Cell disruption by Lysozyme

Determination chitinase activity by using DNS method

E.coli cells were cultivated in 10 ml LB medium with ampicilin (16-18 hours at 37°C ,

200 rpm)

10% of culture of E coli was grown in 250 ml flask containing of 50 ml LB medium

The culture was incubated at 370C with shaking until A600 of the bacterial culture

was 0.4-0.5

IPTG was induced to a final concentration of 1.0 mM

diacetylchitobiose by b-(1,4)-N-acetylglucosaminidase activity (EC 3.2.1.30) or 1,4-b-chitobiosidase activity (EC 3.2.1.29), respectively [12]

Chitinases from all organisms were classified into two phylogenetically distinct families 18 and 19 among the 87 families of glycosyl hydrolases based on amino acid sequence of their catalytic domains Family 18 could be found in a variety of species, including bacteria, fungi, plants (classes III and V), mammals, and viruses whistle family 19 chitinases (class I, II, IV and V) was almost common only in plants and some bacterial strains [27] Class I chitinases were endochitinases, whereas Class II chitinases were exochitinases Class III chitinases showed any similarity to Class I and II chitinases Although Class IV chitinases showed similar characteristics as Class I chitinases, but they were smaller than enzymes Class I Class V chitinases were nettle lectin precursor that showed two chitin binding domain in tandem whereas, Class VI chitinases were all chitinases that no included in Class I, II, III, IV and V chitinases [8] Chitinase specifically cleaveed the N-acetyl-D-glucosaminidic bonds [27] The vast majority of bacterial chitinases fall within family 18, which was subdivided into three groups, ChiA, ChiB, and ChiC, based on the differences in the amino acid sequences of their catalytic domains ChiA and ChiB were processive chitinases that degraded chitin chains in opposite directions, while ChiC was a nonprocessive endochitinase ChiA has been used for studying the diversity and distribution of chitinolytic bacteria in terrestrial systems as well as aquatic environment [13]

Chitinase genes have been cloned from several Streptomyces species, soil bacteria known as major decomposers of chitin in soil: chi63 in S plicatus; chiA, B and C in S lividans; chiO1 and 92 in S olivaceoviridis; chiC, I, II, and III in S griseus; and chiA, B, C, D, E, F, G, and H in S coelicolor A3(2) The chitinases of Streptomyces species seemed suitable for detailed analysis with regard to their

molecular evolution [18]

1.4 Potential of chitin product application in Vietnam

Crustacean is an abundant aquatic product source accounting for one - third

of total fisheries production in Vietnam In seafood processing industry for export, the amount of freeze - dried crustacean accounts for 70 - 80% of processing capacity Processing factories discharge relatively large amount of crustacean wastes, estimated for about 70,000 tons annually The production of chitosan from shrimp shells can bring high economic benefit With the chitin and chitosan’s broad applications, their production has been studied in many countries, including Vietnam [41]

Annually, amount of chitin synthesized from water and marine ecosystems accounts for about 600 and 1600 million tons respectively With the annual yield of 29.9, 1.4 and 0.7 million tons, the best chitin’s source is shellfish (like shrimp, crab, lobster and krill), oysters and squid The proportion of chitin in shellfish’s shell varies from 13 to 42% depending on variety, nutritional conditions and seasonal harvest Besides that, protein (30-40%), minerals (30-50%) and carotenoids (with a very small amount) are also presented in in raw materials for chitin production By-products of fishing and seafood processing are used for the production of chitin and chitosan, making benefits not only for economy but also environment [41]

Chitin and chitosan are also found in the fibers of some fungi such as

Allomyces, Asperillus, Penicillium, Mucor, Rhiropus, Phytomyces Chitosan and

other polysaccharides are presented on the cells of these fiber systems Despite that this source mightbe of minor interest at present, it would be very beneficial in future because of the rapid growth of fungi, suitable with the speed of industry development Besides, these fiber systems contain enzymes, minerals, antibiotics and other hormones that would be useful for human [41]

1.5 All domestic related studies

Three bio-procedures based on the use of enzyme proteolytic was tested and used to extract chitin from shrimp’s head and shell at Vietnam Academy of Science and Technology These procedures included:

(i) Procedure using enzyme bromelain in pineapple extracted solution

(ii) Fermentation of the bacteria producing proteinase;

(iii) Natural fermentation: Enzyme technologies were used to extract chitin

in semi pilot-scale The volume was at 50 kg of raw material (fresh shrimp’s head and shell) per extraction Materials for glucosamine were acquired This research has gained significant outcomes, including:

(i) Microorganism strains that produce chitinase and chitosanse were isolated

(ii) Procedures converting chitin/chitosan into glucosamine have been constructed by acid hydrolysis method

(iii) First step in constructing enzyme technological processes that hydrolyze chitin/chitosan by enzyme chitinase, chitosanase, cellulase and lysozyme

(iv) Sulphate process converting glucosamine into glucosamine sulphate has been constructed

(v) Materials has been screened and trichosanthes kirilowi Maxim that has the strongest activity in inhibiting ß- glucosidase enzyme has been recorded [32]

In a research at Vietnam National Cancer Hospital in 2003, sixty patients from ages 35 to 76 was recorded in chitosan treatment The results proved chitosan supported effectively in cancer treatments Another research that give an 100mg

kg-1 chitosan injection on brown rat skin then caused inflammation by canageenin showed that chitosan has ability in anti-inflammatory on strong tissues

In medical-pharmaceutical, Vietnamese scientists produced Glusivac – a specific medicine for osteoarthritis treatment Besides, there are some weight loss pills made from Chitozan [29]

Vietnamese scientists have gained success in constructing chitin and chitosan production techonology from seafood’s shell (shrimp, crab, shell, squid) for health and food sectors There are numerous types of chitin/chitosan that has been used in many fields, including:

+ Agriculture

+ Industry (waste water treatment and clean drinking water)

+ Health and food sectors

* Chitosan membrane:

Chitosan and their derivatives have antibacterial properties, such as to inhibit

the activity of certain bacteria like E Coli, strawberries - killed fungi, carrots, peas

Chitosan is able to protect and conserve hard-shelled fruits and vegetables

In Vietnam, chitosan from shrimp shells has been used instead of borax in steamed rolled rice pancake and yellow colored wedding pasty manufacturings Recent studies in Vietnam showed that people have successfully completed the chitosan application for preservation of fresh food, perishables such as fish, meat, vegetables Chitosan film is almost tough, difficult to tear and has equivalent durability to some plastics used as packaging… Chitosan film slows down the process of intensive horticulture Harvested vegetables will eventually break, reduce their qualities and value Vegetables are bruised by the fermentation processes that create oquinon polymerized products Thanks to chitosan film, oxidant activity of polyphenols can be inhibited This film acts as part of anthocyamin, flavonoids and total amount of limit - changed phenolic compounds Therefore, it keeps vegetables fresh longer [38]

According to Dr Trang Si Trung, head of Department of Biochemistry - Food Microbiology, Nha Trang University, the addition of enzyme flavourzyme in chitin-chitosan production processes (from shrimp head waste bark) will not only reduces the use of chemical compounds but also obtains two major products (one more in comparison with the conventional process)

Beside chitin-chitosan, we also recovered a mixture of protein and astaxanthin - a pigment with high biological activity - which are widely used in aquaculture and functional foods was also recovered

Seafood by-products (especially shrimp wastes) can cause serious environmental pollution if they are not properly handled Many researches on the extraction from shrimp waste of chitin/chitosan - bio-polymers have significant applications in industries have been taken in Vietnam However, current manufacturing processes of chitin-chitosan in Vietnam are mainly based on chemical Moreover, because of the use of strong chemicals in the extraction process, chitin-chitosan obtained is very little and it usually comes with impurities

On the other hand, these processes mainly focus on chitin-chitosan acquisition Therefore, other products of shrimp waste such as proteins, pigments are wasted Chemicals and organic matters which are not completely received can cause environmental pollution [28]

In this thesis, the aim of study was screened strains that are capable of producing chitinase Since then, isolated strains were identified Chitinase from that strains were purified for the determination of its characterizations

CHAPTER 2 MATERIALS AND METHODS

2.1 Analytical instruments

Table 2.1 List of instruments

Biological safety cabin Nuaire (USA)

Digital weighting scale XT 2200C Pressisa (Switzerland)

2.2 Microbes

The soil samples used for this experiment were collected from Hoang Lien

Son national park A number of 500 strains of Streptomyces isolated from these

samples were kept in VTCC and used for the study on screening of chitinase

2.3 Media

* Agar medium (yeast extract - starch YS) was used for cultivation and

maintenance of Streptomyces: (g/l) soluble starch: 10; Yeast extract: 4;

MgSO4.7H2O: 0.5; KH2PO4: 0.5

* Medium 1 (M1) (culture fermentation): (g/l) Colloidol chitin: 5; Pepton: 3; KNO3: 3; K2HPO4: 0.7; MgSO4.7H2O: 0.5; KCl: 1.0; 50ml medium in 250ml flask

* Medium ISP4 (g/l): Soluble starch: 10; K2HPO4: 1; MgSO4: 1; NaCl: 1; (NH4)2SO4: 2; CaCO3: 2; FeSO4: 1 (mg/l); MnCl2: 1 (mg/l); ZnSO4: 1 (mg/l)

* Medium 2 (M2) (nitrogen source selection) (g/l): colloidol chitin: 5; yeast extract: 1,0; K2HPO4: 0.7; MgSO4.7H2O: 0.5; KCl: 1,0;

2.4 Methodology

2.4.1 Screening of chitinase-producing Streptomyces and culture conditions 2.4.1.1 Preparing culture broth of Streptomyces

A volume of 50 ml medium M1 kept in 250 ml flask and autoclaved at

121oC for 15 minutes This medium was cooled down to room temperature It was inoculated with 1 ml culture previously grown on YS medium for 48 hours The all flasks were shaken at 30oC for five days at 150 rpm The broth was obtained by

centrifuged at 10000 rpm for 10 minutes

2.4.1.2 Primary screening on agar Petri dish

+ Preparing the agar substrate Petri dishes

Plates for the tests were prepared by dispensing 25 ml of sterile agar containing 1% chitinase into (90 x 12 mm) sterile Petri dishes with aseptically wells,kept at 4oCbefore using

+ Screening chitinase activity

Amount of 50 µl broth will be added to the well on the above-prepared agar chitin Petri dishes All were kept at 4oC for 2 hours then transferred to 37oC, incubated overnight Finally, the clear zones were observed by adding Congo red

solution on the dishes The clear zone diameters were measured and recorded [6]

2.4.2 Selecting good chitinese producers by chitinase activity assay