An Alkaline Active Cyclodextrin Glycosyltransferase from an Alkaliphilic Bacillus agaradhaerens

Rita Martins

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Soda lakes represent the most stable naturally-occurring alkaline environments on Earth and harbour a diverse population of alkaliphiles. Alkaliphilic microorganisms have attracted a great interest due to their ability to produce extracellular enzymes that are active and stable at high pH values. Screening for alkaliphilic starch-hydrolyzing bacteria from samples of soda lakes at the Ethiopian Rift Valley (Lake Shalla, Lake Abijata, and Lake Aranguadi) resulted in the isolation of strains from the alkaliphilic species Bacillus pseudofirmus, B. cohnii, B. vedderi, and B. agaradhaerens, and one isolate closely related to the halophilic species Nesterenkonia halobia. A cyclodextrin glycosyltransferase (CGTase) from one of the B. agaradhaerens isolates (strain LS-3C) was purified and characterized with respect to catalysis, cyclodextrins (CDs) production, and stability. CDs are cyclic sugar molecules with a vast application in pharmaceutical, cosmetic and food industries, as well as in bioconversions and separation processes. The enzyme is a monomer with molecular weight of about 88 kDa and optimally active at pH 9.0 and 55 C. Although not so thermostable (only stable up to 40 C), this CGTase showed to have the widest pH span of stability (5.0-11.4) among other CGTases. Using maltodextrin as substrate, B. agaradhaerens LS-3C CGTase produced 89 % of beta-CD with only alpha-CD as secondary cyclic product. Product specificity could be further improved by addition of up to 14 mM CaCl2. Interestingly, compared to other CGTases, B. agaradhaerens LS-3C enzyme displayed significant hydrolysis activity in relation to its cyclization activity. CD ring-opening activity was minor, and coupling and disproportionation activities were also significantly lower than those of other reported CGTases. The gene encoding the enzyme was cloned and sequenced in order to investigate such differential characteristics at the molecular level. The deduced primary sequence of B. agaradhaerens CGTase notably revealed the presence of some residue replacements at the usually conserved positions in the sequence of these enzymes. The relation between such unusual replacements and the hydrolytic activity features of B. agaradhaerens LS-3C CGTase was analyzed. Few of the important amino acid replacements are indeed similar to those in the maltooligosaccharide-producing amylases, the so-called “intermediary” enzymes, which provides incipient evidence that B. agaradhaerens CGTase form a link between CGTases and alpha-amylases. Finally, B. agaradhaerens LS-3C cells were entrapped into Poly(vinyl alcohol) cryogel (PVA cryogel) beads and used as a source of immobilized CGTase. The immobilized biocatalyst was characterized and its application in a system with continuous CD removal by integrated adsorption was tested for beta-CD production.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Biotechnology
Supervisors/Advisors
  • [unknown], [unknown], Supervisor, External person
Award date2003 Jun 13
Publisher
ISBN (Print)91-89627-16-4
Publication statusPublished - 2003

Bibliographical note

Defence details

Date: 2003-06-13
Time: 10:30
Place: Lecture Hall B Center of Chemistry and Chemical Engineering, Sölvegatan 39, Lund Institute of Technology

External reviewer(s)

Name: Larsen, Kim
Title: Dr
Affiliation: Department of Life Science, University of Aalborg, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark

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Article: Martins R, Davids W, Al-Soud W A, Levander F, Rådström P and Hatti-Kaul R. Starch-hydrolysing Bacteria from Alkaline Ethiopian Soda Lakes. Extremophiles (2001) 5:135-144.

Article: Martins R and Hatti-Kaul R. A New Cyclodextrin Glycosyltransferase from an Alkaliphilic Bacillus agaradhaerens Isolate: Purification and Characterization. Enz. Microb. Technol. (2002) 30:116-124.

Article: Martins R and Hatti-Kaul R. Bacillus agaradhaerens LS-3C Cyclodextrin Glycosyltransferase: Activity and Stability Features. Enz. Microb. Technol. (submitted)

Article: Martins R, Delgado O and Hatti-Kaul R. Sequence Analysis of Cyclodextrin Glycosyltransferase from the Alkaliphilic Bacillus agaradhaerens strain LS-3C. Biotechnol. Lett. (submitted)

Article: Martins R, Plieva F M, Santos A, and Hatti-Kaul R. Integrated immobilized cell reactor-adsorption system for beta-cyclodextrin production: a model study using PVA-cryogel entrapped Bacillus agaradhaerens cells. Biotechnol. Lett. (submitted)

Subject classification (UKÄ)

  • Industrial Biotechnology

Keywords

  • cloning
  • characterization
  • purification
  • 16S rRNA gene analysis
  • screening
  • alkaliphiles
  • soda lakes
  • Bacillus agaradhaerens
  • cyclodextrins
  • cyclodextrin glycosyltransferase
  • alpha-amylase family
  • sequencing
  • immobilization
  • Biotechnology
  • Bioteknik

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