Cloning, properties and expression of a novel esterase from Bacillus coagulans strain 18-11.
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...
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2013
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| Online Access: | http://hdl.handle.net/2263/24613 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 > http://upetd.up.ac.za/thesis/available/etd-05132005-132458/ |
| Summary: | 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. === Dissertation (MSc (Microbiology))--University of Pretoria, 2006. === Microbiology and Plant Pathology === unrestricted |
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