Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11.
Dissertation (MSc (Microbiology))--University of Pretoria, 2006.
Saved in:
| Other Authors: | |
|---|---|
| Format: | Thesis |
| Published: |
University of Pretoria
2013
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613500214345728 |
|---|---|
| access_status_str | Open Access |
| author2 | Theron, Jacques |
| author_browse | Theron, Jacques |
| author_facet | Theron, Jacques |
| collection | Thesis |
| dc_rights_str_mv | © 2004, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSc (Microbiology))--University of Pretoria, 2006. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/24613 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:37:08.061Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/24613 Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11. Theron, Jacques upetd@up.ac.za Louw, Maureen E. Mnisi, Stephens Mkhevu Escherichia coli genetics Enzymes synthesis Esterases biotechnology Bacillus bacteria UCTD Dissertation (MSc (Microbiology))--University of Pretoria, 2006. Over the past few years, the use of enzymes as catalysts for the preparation of novel organic molecules has received a steadily increasing amount of attention. Lipolytic enzymes are widely distributed in nature and attract great attention because of their biotechnological potential, as they catalyse the enantio- and regioselective hydrolysis and synthesis of a broad range of natural and non-natural esters. Bacteria produce different lipolytic enzymes, such as esterases (EC 3.1.1.1), which hydrolyse ester-containing molecules at least partly soluble in water, and lipases (EC 3.1.1.3), which hydrolyse water-insoluble long-chain triglycerides. In this study, a bacterial isolate, B. coagulans strain 81-11, isolated from popcorn seeds, was characterized with the specific aim of isolating and characterizing genes encoding novel lipolytic enzymes. A genomic library of B. coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4-kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723-bp open reading frame (ORF), designated estCl, encoding a protein of 240 amino acids with an estimated molecular mass of 27 528 Da and a pI of 9.15. The deduced amino acid sequence of the estCl gene exhibited significant amino acid sequence identity with carboxyl esterases and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrate confirmed the anticipated esterase activity. EstCl exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50°C, although the enzyme was active in the pH range 7-9 and displayed activity at temperatures up to 55°C. Since bacterial esterases are potentially important for a variety of biotechnological applications, there is a considerable industrial interest to produce these enzymes at a larger scale. Among the many systems that are available for heterologous protein production, attempts were made to over express the newly identified B. coagulans estCI esterase¬encoding gene in different Gram-positive bacteria, as they are well known for their important contribution to food biotechnology and as production organisms for industrial enzymes. A recombinant expression vector was thus constructed (pMG36-EstCl) and introduced in Lactococcus lactis, Lactobacillus plantarum and Bacillus subtilis strains 154 and lA297. Of these different bacterial hosts, high levels of intracellular esterase activity were detected in B. subtilis lA297 only. In an attempt to increase extracellular expression of the B. coagulans EstCl esterase, a recombinant secretion plasmid (pNW-EstClaps) was constructed that contained an alkaline protease promoter and signal sequence from a Bacillus species. Following introduction of the construct in B. subtilis lA297, the derived recombinant strain displayed 2.3-fold higher extracellular esterase-activity levels than the parent B. coagulans strain, and the extracellular esterase activity represented 82% of the total esterase activity. Microbiology and Plant Pathology unrestricted 2013-09-06T18:03:54Z 2005-05-20 2013-09-06T18:03:54Z 2004-09-01 2006-05-20 2005-05-13 Dissertation Mnisi, SM 2004, Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 81-11, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/24613 > H776/ag http://hdl.handle.net/2263/24613 http://upetd.up.ac.za/thesis/available/etd-05132005-132458/ © 2004, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | Escherichia coli genetics Enzymes synthesis Esterases biotechnology Bacillus bacteria UCTD Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11. |
| title | Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11. |
| title_full | Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11. |
| title_fullStr | Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11. |
| title_full_unstemmed | Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11. |
| title_short | Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11. |
| title_sort | cloning properties and expression of a novel esterase from bacillus coagulans strain 18 11 |
| topic | Escherichia coli genetics Enzymes synthesis Esterases biotechnology Bacillus bacteria UCTD |
| url | http://hdl.handle.net/2263/24613 http://upetd.up.ac.za/thesis/available/etd-05132005-132458/ |