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Food Enzyme Technology Training

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                                 Date 2006     March             Valid 2008                        December
                                                                 until:


CONTACT DETAILS

Organisation       Centre of Biotechnology of Sfax
full name                                                                      Contact person:
Organisation       CBS                                         Title              Professor, Head of
acronym                                                                           laboratory
(Abbreviation)
Department /       Laboratory of Enzymes and                   First Name         Samir
Sector / Faculty   Metabolites from Prokaryotes (
                   LEMP
Address            Route de Sidi mansour Km 6                  Family Name        BEJAR
                   BP"K"
Postal code        3038                                        Telephone          74 216 74 440 451/ 216 98
                                                                                  550 009
City               Sfax                                        Fax                74 216 74 440 451
Country            Tunsia                                      E-mail             samir.bejar@cbs.rnrt.tn
www address        www.cbs.rnrt.tn

Former participation in EU research projects as Co-ordinator: YES                 NO

PPOJECT IDEA

 Title     Production and the study of some microbial enzymes and        Acronym
           biological active metabolites which can be used aspharma
           - ceuticals, agriculture and food additivecompounds.

 Project type: Specific Targeted Research Project

 CALL REFERENCE

 Call identification code:       KBBE-2007-2-3-03
 Topic addressed:        (Bio)- Technologies for the production of food additives, colorants and
 flavours

 Short description of the project idea:
 (Potential contribution of the Laboratory of Enzymes and Metabolites of Prokaryotes of the Centre of
 Biotechnology of Sfax – Tunisia,(LEMP-CBS-Tunisia)


 The research activities of the LEMP are focused especially on two topics. The first relates to the
 production and the study of some microbial enzymes of industrial interest, the second concerns
 biological active metabolites which can be used as pharmaceuticals, agriculture and food additives
 compounds. With these two major topics of research, the LEMP is very interested and can be
 contribute effectively to the proposed call related to the theme 2.2.3/KBBE-2007-2-3-03:”(Bio)
 Technologies for the production of food additives, colorants and flavours” of the FP7 Cooperation
 Work Programme. Hence, the research group of the LEMP is ready and hopes to participate in one or
 more of these following themes:

 I. Effects of the addition of enzymes on technological, nutritional and functional properties of baked
 goods
 (Main investigators: Prof. Samir BEJAR, Dr. Hichem Chouayakeh and Dr. Mamdouh Ben Ali)
 In recent years, we assist to an over increasing demand by consumers for baked goods more natural
 and with outstanding organoleptic and hygienic qualities. The return to traditional sourdough bread

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making in conjunction with the addition of innovative functional ingredients like enzymes (amylases,
phytases …) would bring a convenient solution.


I.1 Amylases used on the baking as an antirassissant agent
Amylases are now involved in practically all agro-food processes and in many other domains as the
pharmaceutical, laundering and animal feed industries. They exhibit potential applications notably in
the bioconversion of the starch for the production of syrups with high glucose content or maltodextrins
with high content in specific malto-saccharides. More selective amylases having a limited hydrolytic
action on the starch are rather used in bakery industry. In fact these later were reported to improve
baked product characteristics such as loaf volume and to delay the bread staling during storage.
Staling phenomenon, caused by the retrogradation of starch, refers to various undesirable changes,
such as the increase of crumb firmness, that occur in the loaf during storage, thereby lowering the
consumer’s acceptance.
Our group had already screened and studied many amylases useful for some industrial application.
The genes coding for these enzymes have been cloned, sequenced and analyzed and their structure-
function relationship has been studied.
In this project we plan to continue the screening and the study of some amylases as an antirassissant
agent to ameliorate the bread quality. Hence we will focus on the properties enhancement of
Paenibacillus pabuli US132 CGtase, which was demonstrated to improve significantly the loaf volume
and decreased the firmness of bread during storage. This study will also concern the maltogenic
amylase from new isolated Bacillus sp.US149.


I.2. Phytases.
Phytase offers excellent possibilities as a bread-making improver, with two main advantages: first, the
nutritional improvements produced by decreasing phytates content and second, the activation of
endogenous alpha-amylase due to the release of calcium ions from calcium phytates complexes
catalysed by phytase activity which permits an increase in the specific bread volume and improvement
of the crumb texture.
The LEMP laboratory has recently developed a novel project which consists on the isolation of
microbial strains producing phytase activities and characterization of enzymes from the best producing
strains. Until now, a fungal phytase as well as a Bacillus phytase are under investigation. The enzyme
possessing desirable properties for application in bread making could be tested to demonstrate its
suitability.


I.3 Expertise and reference in this filed

*Expertise

Our research group has a good experience in:
         Isolation of microorganisms from original Tunisian biotope (soil of hot spring, desert, salt lake
etc…) having interesting activities.
         Enzymes Purification and characterisation of their pyhsico-chemical properties.
         Molecular cloning and sequencing of genes encoding corresponding enzymes.
         structure resolution by molecular modelling
         Structure-function relationship Studies
         Improvement of the enzymes performance regarding some specific application by rational
design and using genetic engineering (directed or random mutagenesis. This includes enhancement
of the activity and stability at low or high pH, improvement of thermoactivity, thermostability, resistance
to inhibitors, chelating and oxidant agents…
         Construction of recombinant strains over-expressing wild type or improved enzymes.
         Scale-up and application of enzyme technology

* Recent publications in this field

1.      BEN ALI M., MHIRI S., MEZGHANI M. AND BEJAR S. (2001). Purification and sequence
analysis of the atypical maltohexaose-forming -amylase of the B. stearothermophilus US100. Enzyme
and Microbial Technology 28:537-542.

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2.       MELLOULI L., KARRAY REBAI I. AND BEJAR S. (2002). Construction of alpha-amylase
producing strains not subject to carbon catabolic repression. FEMS Microbiology Letters 206:157-162
3.       BEN MESSAOUD E., BEN AMMAR Y., MELLOULI L. AND BEJAR S. (2002). Thermostable
pullulanase type I from new isolated Bacillus thermoleovorans US105, Cloning, Sequencing and
Expression of the gene in E. coli. Enzyme and Microbial Technology 31:827-832.
4.       BEN MESSAOUD E., BEN ALI M., ELLEUCH N., MASMOUDI-FORATI N. AND BEJAR S.
(2004). Purification and properties of a maltoheptaose and maltohexaose-forming amylase produced
by Bacillus subtilis US116. Enzyme and Microbial Technology, 34: 662-672.
5.       BEN ALI M., KHEMAKEM B., ROBERT X., HASER R. AND BEJAR S. (2006). Thermostability
enhancement and starch breakdown-profile change of the maltohexaose-forming-amylase of B.
stearothermophilus US100 strain. Biochemical Journal. 394: 51-56
6.       JEMLI S, BEN MESSAOUD E., AYADI-ZOUARI D., NAILI B, AND BEJAR S (2006). A                      -
cyclodextrin glycosyltransferase from a newly isolated Paenibacillus pabuli US132 strain: Purification,
properties and its potential use in bread-making Biochemical Engineering Journal (In press).
7.       KAMMOUN R., NAILI B. AND BEJAR S. Application of a statistical design to the optimization
of parameters and culture medium for -amylase production by Aspergillus oryzea CBS 819.72 grown
on gruel (wheat grinding by-product). Submitted to Bioresources Technology.
8.       BEN MESSAOUD E., BEN MABROUK S., JEMLI S. AND BEJAR S. (2006) Cloning and
sequencing of the alpha-amylase gene from Bacillus subtilis US 116 strain encoding an enzyme
closely identical to that from Bacillus amyloliquefaciens but distinct in thermal stability. Submitted to
Biochemical Engineering Journal.

*Main International Projects

-        European Community: Contract CII*-CT94-021; with IGM, Orsay-FRANCE, 96-99: New
amylolytics activities:
-        Collaborative Project with Novozymes Compagny 1999-2000: Amylolytics enzymes and
Glucose isomerase
-        Collaborative Project with Lesaffre-Développement 2002-2004
- ICGEB: Collaborative project CRP/TUN00-02, (2001-2003): Correlation between the aa sequence
and the Physico-chemicals Properties of three Enzymes involved in the Glucose and fructose
Production.
- Projet CMCU N° 04/0905: S.BEJAR :R.HASER : Criblage, clonage et étude de la structure-fonction
de quelques enzymes d'intérêt industriel : Grants for small and long period fellowship
- International Fondation for Sciences (IFS N) E4126-1) : Expression of L-arabinose isomerase
activities in the yoghurt lactic acid bacteria



II. Sweeteners and low caloric substrate useful for diabetic and to combat obesity. (Main
investigators: Prof. Samir BEJAR, Dr. Hichem Chouayakeh, Dr. Mamdouh Ben Ali)


This concerns mainly the D-tagatose and L-fructose. D-tagatose is a rare natural ketohexose having a
taste and physical properties similar to sucrose and it is an anti-hyperglycemiant factor, a very low
calorie carbohydrate and bulking agent. It was the subject of recent interests in food and drug industry
and is considered as a safe and low calorific substrate in the United States and in Europe. This natural
suguar could be obtained by isomerisation in vitro the D-galactose using the L-arabinose isomerase
(EC 5.3.1.4).
The D-fructose ketohexose is twice as sweet as D-glucose playing an important role as a diabetic
sweetener according to its slow intestinal absorption. Therefore it does not influence the glucose level
in blood. D-fructose production cost, in HFCS using xylose isomerase, was 10 to 20 % lower than that
of sucrose. Additionally, this natural sweetener is preferred in the food industry since it does not
provoke the crystallisation problem as it is the case with sucrose.
In this project, we will focus mainly on three enzymes already studied by our group in addition of novel
ones that will be screened along this project.
The first enzyme is an original Glucose isomerase useful for an industrial application having a very
efficient catalytic properties acting at low pH and high temperature. The importance of the residue Ala
103 on the useful properties of the GISG was also largely demonstrated. We planed to achieve this
study by crystallization and the determination of the structure of this enzyme. By another way we plan
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to study the best potentiality and conditions for using this enzyme (alone or in combination with others
enzymes) in order to produce high fructose syrup in one hand and D-tagatose and fructose syrup from
lactoserum hydrolysis in another hand.
The second enzyme is an L arabinose isomerase studied from Bacillus stearothermophilus US100.
This enzyme is distinguishing by its low need of metallic ions for its thermoactivity and thermostability,
and has very efficient catalytic properties. The study of structure function relationship is under
investigation. By another way we are in progress concerning the application of this enzyme to obtain
D-tagatose syrup or D-tagatose and fructose syrup from lactoserum hydrolysis.
A second L-arabinose isomerase was studied from Lactobacillus plantarum NC8. This enzyme was
distinguishable by its high activity and stability at acidic pH. Indeed, it exhibited 68% of its maximal
activity at pH 5.5 and retained 89% of activity after 24 h incubation at pH 5. The study of structure
function relationship is under investigation.
By another way, thess LAIs, enjoying the ability to convert D-galactose to D-tagatose, could be used
for the production of lower caloric fermented products. Hence we are in progress on the study of the
impact of the expression of these L-AI activities on the ability of the galactose negative yoghurt
starters Lactobacillus bulgaricus and Streptococcus thermophilus to convert to D-tagatose the
unconsumed D-galactose produced during milk fermentation.


Expertise and references in this filed

*Expertise
Our research group has a good experience in:
         Isolation of microorganisms from original Tunisian biotope (soil of hot spring, desert, salt lake
etc…) having interesting activities.
         Enzyme Purification and characterisation of their pyhsico-chemical properties.
         Molecular cloning and sequencing of genes encoding enzymes.
         structure resolution by molecular modelling
         Structure-function relationship Studies
         Improvement of the enzymes performance regarding some specific application by rational
design and using genetic engineering (directed or random mutagenesis This include enhancement of
the activity and stability at low or high pH, improvement of thermoactivity, thermostability, resistance to
inhibitors, chelating and oxidant agents…
         Construction of recombinant strains over-expressing enzymes.
         Scale-up and application of enzyme technology in collaboration with national and international
industrial partners.

* Recent publications in this field

1.       BORGI M.A., SRIH-BELGUITH K., BEN ALI M., MEZGHANI M., TRANIER M., HASER R.
AND BEJAR S. (2004). Glucose Isomerase of the Streptomyces sp. SK strain: Purification, Sequence
Analysis and Implication of Alanine 103 Residue on the Enzyme Thermostability and Acidotolerance.
Biochimie, 86: 561-568.
2.       MEZGHANI M., BORGI M.A., KAMMOUN R., AOUISSAOUI H. AND BEJAR S.(2005).
Construction of new stable strain over-expressing the glucose isomerase of the Streptomyces sp. SK
strain. Enzyme and Microbial Technology, 37: 735-738
3.       RHIMI M. AND BEJAR S.(2006). Cloning, purification and biochemical characterization of
metallic-ions independent and thermoactive L-arabinose isomerase from the Bacillus
stearothermophilus US100 strain” "Biochimica and Biophysica Acta (BBA) 1760: 191-199.
4.       RHIMI M., Ben MESSAOUD E., BORGI M.A., BEN KHADHRA K. and BEJAR S. (2006). Co-
expression of l-arabinoseisomerase and D-glucose isomerase in E. coli and development of an
efficient process producing simultaneously D-tagatose and D-fructose. Enzyme and Microbial
Technology (In press).
5.       BORGI M.A, RHIMI M. AND BEJAR S (2006) Involvement of Alanine 103 residue in the kinetic
properties of glucose isomérases from Streptomyces species Biotechnology journal (In press).
6.       RHIMI M., JUY M., AGHAJARI N., HASER R. AND BEJAR S. (2006). Probing the essential
calaytic residues and the substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-
arabinose isomerase by site-directed mutagenesis. In revision; Journal of Bacteriology.

*Patents

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1.      BELGUITH-SRIH K., ELLOUZ R. AND S. BEJAR (2002). Polypeptides having glucose
isomerase activity and acids encoding the same. US Patent No: 6, 372, 476
2.      RHIMI M., CHOUAYEKH H., BEN ALI M., NAILI B., JEMLI S. et BEJAR S. (2005) : Une L-
arabinose isomérase ayant une dépendance minime en ions métalliques pour son activité et pour sa
thermostabilité, codée par une séquence nucléotidique nouvellement isolée et caractérisée.
Application N° WO 2006/071203 A2 6 July 2006

*Main International Projects
- ICGEB: Collaborative project CRP-TUN-01: 91-93; Over-expression and secretion of glucose
isomerase from Streptomyces:
-        Collaborative Project with Novozymes Compagny 1999-2000: Amylolytics enzymes and
Glucose isomerase
-        ICGEB: Collaborative project CRP/TUN00-02, (2001-2003): Correlation between the aa
sequence and the Physico-chemicals Properties of three Enzymes involved in the Glucose and
fructose Production.
- Projet CMCU N° 04/0905: S.BEJAR :R.HASER : Criblage, clonage et étude de la structure-fonction
de quelques enzymes d'intérêt industriel : Grants for small and long period fellowship
- International Fondation for Sciences (IFS) :



III. Antifungal and bacteriocins activities for use as food additives from new isolated lactic acid and
actinomycete bacteria. (Main investigators: Dr. Lotfi MELLOULI, Dr. Hichem Chouayakeh and Prof.
Samir BEJAR)


Since centuries, foods have been preserved by heating, chilling, drying, salting, conserving,
acidification, oxygen-removal, fermenting, adding various synthetic preservatives, etc. Although these
methods were often applied in combinations, effective limits against pathogenic microbial safety
(bacteria and fungus) were established. Actually, consumers demand food products with fewer or
without synthetic additives but increased safety, quality and shelf-life. These demands have led to
increasing interest in the use of natural antimicrobials such bacteriocins and antifungal to preserve
foods. However, despite the wide range of potential antimicrobials, relatively few are suitable for use in
practice in particular food products and several works are actually oriented toward the research of new
natural antimicrobials compounds (bacteriocins and antifungal. Bacteriocins are inhibitory peptides or
proteins, produced by different groups of bacteria, which have bactericidal effects on micro-organisms
closely related to the producer. They have received increasing interest, especially those produced by
lactic acid bacteria (LAB), because of their potential use as food additives. Antifungal, are active
compounds that either destroy or inhibit the growth of fungi. The filamentous soil bacteria belonging to
the genus Streptomyces are rich sources of a high number of bioactive natural products, such as
antifungal, extensively used as pharmaceuticals and agrochemicals: human and animal therapy, food
industries and agriculture.
Among the research topics of the LEMP-CBC, one is interested to the metabolites having biological
activities: antibiotics, antifungal, antitumoral and bacteriocin which can be used in health, agriculture
and food. The aim of this present project is to search for new bacterial antifungal and bacteriocins
which can be used as food additives to increase safety, quality and shelf-life. For the screening of
interesting bacteriocins, we attempt to isolate new lactic acid bacteria from different origins such as
traditional fermented foods (yoghurt, milk cream, sour dough, milk, etc), fermented vegetables, fruits
and silages, etc. It should be noted that until now, we have isolated one hundred LAB strains from
different origins and ten of these bacteria were selected for their important antifungal and antibacterial
activities against several fungus and pathogenic bacteria. Two strains of these ten selected bacteria
have been identified (new Lactobacillus bacteria) and the physico-chemical characteristics of
corresponding bacteriocins have been studied. We possess also a collection of six hundreds
actinomyctes strains isolated from different uncommon water and grounds Tunisian habitats.
Biological test against several funguses and Gram+ and Gram- bacteria has permitted us to select fifty
strains for further studies. However, six bacteria from these selected actinomycete strains have been
already identified (new Streptomyces bacteria) and their corresponding active molecules have been
purified and characterized. These molecules, belonging to different families (macrolides,
diketopiperazine derivatives, isocoumarins, tyramines, rhamnopyranosides, etc), possess different
biological activities and can be used as pharmaceuticals, agriculture and food additives compounds.
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Some genes involved in two biosynthesis pathways of these characterized molecules have been
detected and analysed.
Therefore, during this proposed project and besides the already selected (lactic acid and actinomycete
bacteria), we plan to carry on the screening and selection of other bacteria producing interesting
activities (bacteriocins and antifungal), to purify and characterize the active compounds from some
already selected bacteria and from new ones which could be selected and to study the application of
these molecules as food additives. While our interest will be especially focused on the research of
interesting compounds for food additives to increase safety, quality and shelf-life, it should be noted
that in this project we also plan the detection and analysis of genes involved in the biosynthesis
pathways of the most interesting active compounds. This molecular study will permit subsequently the
production of new hybrid molecules with interesting activities.

Expertise and references in this filed

* Expertise
        Isolation of novel strains from original and uncommon Tunisian biotope (soil of hot spring,
desert, salt lake etc…) producing bioactive molecules.
        Amelioration and optimisation of the culture conditions of the selected strains for active
compounds production
        Purification and structure determination of the bioactive couponds
        Cloning and expression of genes involved in the biosynthesis pathways of the interesting
active compounds.

* Recent publications in this field
1.       Mellouli L*., Ben Ameur-Mehdi R., Sioud S, Salem M. and Bejar S. (2003). Isolation,
purification and partial characterization of antibacterial activities produced by a new isolated
Streptomyces sp. US24 strain. Research in Microbiology 154: 345-352.
2.       Mellouli L*., Karray-Rebai, I, Sioud, S, Naili, B and Bejar, S. (2004). Efficient Transformation
Procedure of a Newly Isolated Streptomyces sp. US58 strain Producing Antibacterial Activities.
Current Microbiology 49. V6: 400-406
3.       Ben Ameur-Mehdi R, Mellouli L*., Chabchoub, F., Fotso, S., and Bejar, S. (2004). Purification
and Structure Elucidation of two Biologically Active Molecules from a new Isolated Streptomyces sp.
US24 strain Chemistry of Natural Compounds: Vol. 40. No 5: 510-513.
4.       Lilia Fourati Ben Fguira, Serge Fotso, raoudha Ben Ameur Mehdi, Lotfi Mellouli*, Hartmut
Laatsch (2005). Purification and Structure Elucidation of Antifungal and Antibacterial Activities from a
Newly Isolated Streptomyces sp. US80 strain. Research in Microbiology. Vol. 156, 3: 341-347.
5.       Raoudha Ben Ameur Mehdi, Samiha Sioud, Lilia Fourati Ben Fguira, Samir Bejar and Lotfi
Mellouli*. (2006). Purification and structure determination of four bioactive molecules from a newly
isolated Streptomyces sp. TN97 strain. Process Biochemistry. 41, 1506-1513.
6.       S. Sioud, I. Karray-Rebai, H. Aouissaoui, B. Aigle, S. Bejar and L. Mellouli* . Targeted gene
disruption of the Cyclo (L-Phe, L-Pro) biosynthetic pathway in Streptomyces sp. US 24 strain
(Submitted)

*International Projects

1.       Réseau Formation Franco-Tunisien : "1993-1996", Grant for short and long period
2.       Project PICS Franco-Tunisian (DGRST-CNRS) In collaboration with the laboratory of Biology
and Molecular Genetic of Orsay - French "1999-2001": Caractérisation de gènes de biosynthèse
d'antibiotiques macrolides chez Streptomyces ambofaciens et chez des Streptomyces nouvellement
isolés
3.       Project Tunisian-Morocco "Research Programme CMPTM "2000-2001": Isolement de
bactéries Actinomycétales productrices d'activités antibactériennes et/ou antifongiques:
caractérisation biochimique des activités et étude moléculaire des gènes impliqués dans les voies de
biosynthèses
4.       Projet TWAS N°. 02-173 RG/BIO/AF/AC "2003". Isolation of new actinomycetes strains having
antibiotic activities: identification of strains, extraction and purification of actives molecules. Detection,
cloning and analysis of genes involved in biosynthesis pathways.
5.       Project TWAS N° 04-287 RG/BIO/AF/AC “2005”. Antibacterial activities: Isolation, selection
and identification of producer strains. Extraction and purification of active molecules. Detection,
cloning and analysis of genes involved in biosynthesis pathways

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 7.     Project CMCU (2006): N° 06/S 0901 MELLOULI / AIGLE: Three and Three Tunisian Partners:
 Research and Study of new active antifungal activities for agricultural applications
 8.     Project Tuniso-Egyptian (2007-2008) MELLOULI / SHAABAN: Novel Bioactive Secondary
 Metabolites from Actinomycetes Bacteria.



Keywords describing          Sustainable production and management of biological resources from
the project idea             land, forest and aquatic environments:
                                Biological resources                   Novel feeds
                                Biodiversity                           Novel plants
                                Genomics/Proteomics/Metabolomics       Plant Health
                                Bioinformatics                         Animal Production and Welfare
                                Agriculture                            Animal husbandry
                                Forestry                               Vaccines and Diagnostics
                                Fisheries                              Organic production methods
                                Aquaculture                            Dairy Production
                                Horticulture                           Tracking and tracing
                             Fork to Farm: Food, health and well being
                                Consumer behaviour                  Potable/Safe Drinking Water
                                Functional Food                     Animal Feed
                                Nutrition Science                   Chemical Food Safety
                                Physiology                          Microbiological Food Safety
                                Food Technology                     New detection methods
                                Food Processing                     Risk Assessment
                                Packaging                           Food Chain Analysis/Management
                                Food safety                         Pesticide/BioActive
                                                                  Additives/Substances Control
                             Life sciences and biotechnology for sustainable non-food products and
                             processes
                                 Biomass production                      Wood-production
                                 Bio-products                            Pollution
                                 Bio-refinery                            Ecology
                                 Bio-processes                           Waste Processing
                                 Fibres (Wool, cotton, novel-bio-
                             fibres)


PROFILE OF PARTNER SOUGHT


Type

  Research Organisation           University       SME              Other, please specify:


Role to cover in the project

  technology development                   research                                 training

  dissemination                            demonstration                            other



Country / Region         Tunisia- Mediterranean Partner countries region


Start of collaboration
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 start-up phase                     mid-term                       end-phase



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