Industrial Applications of Enzymes Cyclodextrin - DOC by neg11394

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                                  Date 2006     February           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

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

PPOJECT IDEA

 Title      Enzymes used for the production of sweeteners useful for       Acronym
            diabetic and obese persons as well as those used as
            additive in the bakery or in starch bioprocessing

 Project type: Specific Targeted Research Project

 CALL REFERENCE

 Call identification code:      KBBE-2007-3-2-03
 Topic addressed:        Designer Enzymes-Improved biocatalysts for biprocesses

 Short description of the project idea:
 Enzymes have extensive applications in a range of industrial processes. They are applied in all agro-
 food processes and in many other domains as the pharmaceutical, laundering and animal feed
 industries. The LEMP could be implicated in the screening study and improvement of some enzymes
 of industrial interest. This includes enzymes used for the production of sweeteners useful for diabetic
 and obese persons as well as those used as additive in the bakery or in starch bioprocessing.

 Some isomerases used for the production of sweeteners as low caloric substrates and/or useful for
 diabetic and obese persons.

 Marketing new lower caloric products will undoubtedly find success especially for diabetes and low
 caloric diet persons. In this goal the LEMP studied some isomerases used in the production of this
 kind of products. This includes L-arabinose isomerase (L-AI)(EC 5.3.1.4) which catalyses the
 conversion of L-arabinose to L-ribulose in biological systems. It is also referred to as D-galactose
 isomerase due to its ability, in vitro, to isomerize the D-galactose into D-tagatose. This later sweetner
 is the subject of recent interests in food industry, when one considers its privileged position within
 sweeteners. It is a rare natural ketohexose having a taste and physical properties similar to sucrose
 and it is an anti-hyperglycemiant factor, efficient anti-biofilm with a very low calorie carbohydrate and
 bulking agent. Nowadays, the compound D-tagatose attracted commercial attention and has been
 considered a Generally Recognized as Safe (GRAS) substance in the United States. D-tagatose is
 approved for use in foods and beverages in Korea, Australia, New Zeland and the USA.
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The second type of enzyme concern Xylose isomerase (D-xylose ketol isomerase EC 5.3.1.5) able to
catalyse the reversible isomerization of D-xylose into D-xylulose. It is also referred as a glucose
isomerase (GI) because of its ability to convert D-glucose to D-fructose. This property is widely
exploited industrially for the production of high-fructose syrup from starch. D-fructose is also an anti-
hyperglycemiant sugar having a largest market in the food industry.
 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 GISK was also largely demonstrated. We planed to achieve this
study by crystallization and the determination of the structure of this enzyme. In addition we plan to
study the best potentiality and conditions for using this enzyme (alone or in combination with other
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 distinguishable 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. Furthermore, 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.

Amylases used on starch processing, baking, textile and detergent:

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 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 applications.
The genes encoding for these enzymes have been cloned, sequenced and analyzed and their
structure-function relationship has been studied.
Hence we plan to screen new enzymes which are able to be used in bread baking as antirassissant
agents, on textile or detergents and for the production of specific maltooligosachharides or
cyclodxtrines.

In the same time we plan to use the enzyme engineering tools to enhance and adapt some of the
already studied enzymes for a best use in some industrial applications. In this context we intend to
enhance the thermostability and the resistance to oxidant and bleach-agents of the Amylase of B.
Stearothermophilus US100 strain (AmyUS100) in order to its better compatibility and performance in
detergent or in textile.. We also aim to ameliorate the properties of the CGtase of Paenibacillus pabuli
US132 strain in order to be more suitable for its application as an antirassissant agent or to be more
efficient on the production of specific cyclodextrines. The amelioration of performance of amylase of
B.subtilis US116 (AmyUS116) for the production of specific oligosaccharides namely maltoheptaose
and mlatohexaose is also among our objectives.

Proteases
We are in progress on the studying of a Novel oxidant- surfactant- and bleach-stable serine alkaline
protease from a newly isolated Bacillus pumilus CBS strain. This enzyme sound to be very interesting
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for its application in detergent. In addition of characterisation, molecular cloning and analysis of the
corresponding gene enzyme, we program to study its structure-function relationship in order to
enhance more and more its performance in detergent application.

Expertise and references in the filed of biocatalyst

Our research group possesses a good experience in:
         * Isolation of microorganisms from original Tunisian biotope in particular (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 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 proteins.
         * Scale-up and application of enzyme technology in collaboration with national and
international industrial partners.


Main Publications from 2004 in this field

1.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.
2.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.
3.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
4.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
5.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” for publication:"Biochimica and Biophysica Acta (BBA) 1760: 191-199.
6.ZOUARI-AYEDI D., CHOUAYEKH H., MHIRI S, ZERRIA K, DAHMANI M. F. AND BEJAR S (2006).
Expression and efficient secretion from Streptomyces lividans of recombinant α integrin CD11bA-
domain. Journal of Biomedecine and Biotechnology (In press).
7.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).
8.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 dans Biochemical Engineering Journal (In press).
9.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).
10.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.
11.KAMMOUN R., NAILI B. AND BEJAR S. Application of a statistical design to the optimization of
parameters -amylase production by Aspergillus oryzea CBS 819.72 and culture medium for grown
on gruel (wheat grinding by-product). Submitted to Bioresources Technology.
12.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

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 closely identical to that from Bacillus amyloliquefaciens but distinct in thermal stability. Submitted to
 Biochemical Engineering Journal.


 *Main International Projects
 - ICGEB: Collaborative project CRP-TUN-01: 91-93; Over-expression and secretion of glucose
 isomerase from Streptomyces:
 - European Community: Contract CII*-CT94-021; with IGM, Orsay-FRANCE, 96-99: New amylolytics
 activities: Grants for small and long period fellowship
 -PICS (DGRST-CNRS) Franco-Tunisien, IGM, Orsay-FRANCE: Antibiotics: 1999-2001, Grants for
 small and long period fellowship
 -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.
 -Collaborative Project with Lesaffre-Développement
 - International Fondation for Sciences (IFS N) E4126-1) : Expression of L-arabinose isomerase
 activities in the yoghurt lactic acid 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)


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Role to cover in the project

  technology development                  research                                training

  dissemination                           demonstration                           other



Country / Region         Tunisia- Mediterranean Partener countries region


Start of collaboration

  start-up phase                          mid-term                                end-phase



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