Glucoamylase, a saccharifying enzyme, is applied in the brewing industry to hydrolyse the dextrins of malted barley into simple sugars which can then be fermented by brewer's yeast. In order to establish the potential of glucoamylase from Lactobacillus amylovorus for application in the brewing industry, the main objectives of this study were: (1) to determine the cultural conditions for growth and glucoamylase production, (2) to purify the enzyme to homogeneity using chromatography and electrophoretic techniques, (3) to study biochemical properties of the purified enzyme, and (4) to clone the gene coding for glucoamylase, and characterize the recombinant glucoamylase. / The actively amylolytic Lactobacillus amylovorus ATCC 33621 produced an intracellular glucoamylase activity. Conditions for growth and glucoamylase production were maximized by using dextrose free MRS medium supplemented with 1% dextrin, at pH 5.5 and 37$ sp circ$C. Enzyme production was maximal during the late logarithmic phase of growth from 16-18 h. Crude cell extract showed optimal activity at pH 6.0 and 55$ sp circ$C. / Native and SDS-PAGE of the purified enzyme showed a monomeric protein of 47 kD. Glucoamylase activity was confirmed by activity staining using a starch/polyacrylamide gel where a zone of clearing was visible on a blue/black background stained with Kl/I$ sb2.$ Optimal pH, pl and temperature of purified glucoamylase were 4.5, 4.39 and 45$ sp circ$C, respectively. The enzyme was rapidly inactivated by temperatures above 55$ sp circ$C and was inhibited by heavy metals, e.g. Pb$ sp{2+}$ and Cu$ sp{2+}$ at 1.0 mM. EDTA did not inhibit the enzyme activity at a final concentration of 10 mM. Enzyme inhibition by 1 mM of p-chloromercuribenzoic acid (pCMB) and iodoacetate suggested that a sulfhydryl group was present in the enzyme active site. Kinetic studies of glucoamylase confirmed that the enzyme reacted preferentially with polysaccharides. HPLC analyses of the end products of enzyme action showed that glucose was the major end product of enzyme action and this glucose was responsible for end product inhibition. / The gene coding for glucoamylase was cloned into Escherichia coli using the STA2 glucoamylase gene of Saccharomyces diastaticus as a probe. Three glucoamylase producing transformants were identified as the insert sizes of about 5.2 Kb, 6.4 Kb and 5.9 Kb, respectively. When the characteristics of both recombinant and purified wild type glucoamylases were compared, both enzymes showed a similar pH range of 3.0-8.0, and temperature optimum of 45$ sp circ$C. The recombinant enzyme pH profiles were broader than that of the wild type and an optimum pH of 6.0 was obtained. This study has shown that glucoamylase from Lb. amylovorus is less heat stable than other bacterial glucoamylases and thus may be suitable for application in the brewing industry. Successful cloning of this gene coding for glucoamylase in brewer's yeast, Saccharomyces cerevisiae, would reap the advantageous properties of the enzyme while eliminating the costs of adding commercial enzymes.

No description available.

To improve the existing methods based on phenotype and/or genotype a new genotyping method was investigated to identify Lactobacillus species. / The method used the polymerase chain reaction (PCR) to amplify specific sequences of aminopeptidase (pepN) genes. The primers for the PCR reactions derived from a pepN sequence of Lactobacillus rhamnosus S93. PepN amplification products of 387 bp were obtained from forty three Lactobacillus strains and from some strains of Lactococcus (3), Streptococcus (2) and Bifidobactertium (5). / Restriction fragment length polymorphisms (RFLPs) and single-strand conformation polymorphisms (SSCP) methods were used to detect polymorphisms among amplified aminopeptidase DNA fragments from the different Lactobacillus strains. / The results of RFLPs after digestions with Sau3A I, Rsa I and Tru9 I confirmed that the PCR products were specific. According to the fingerprints generated, Lactobacillus species tested could be grouped in four. / SSCP allowed a good discrimination between different pepN PCR products of the same size. Some Lactobacillus strains, Lb. plantarum and Lb. rhamnosus showed the different ssDNA patterns. Though for many strains of Lactobacillus the SSCP patterns were similar, no general comparison can be made because all the samples were not loaded on the same SSCP polyacrylamide gel. The SSCP, PCR-based method can be easily modified to increase the rate of polymorphism detection. / This new genetic identification method is different from others because it uses specific pepN DNA sequences for each strain tested and it uses SSCP to detect the presence of polymorphisms. The method is also applicable to other genera of lactic acid bacteria.

No description available.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Lactobacillus plantarum 285, isolated from sorghum beer, produces bacteriocin 285, which displays activity against several food spoilage organisms. For future application of bacteriocin 285 in the food industry, it was important to characterize the peptide and identify the genes encoding its production. The effect of bacteriocin 285 on sensitive cells was determined through the use of an indicator (sensitive) organism, Lactobacillus sakei DSM 20017. The indicator strain was genetically modified to express GFP (green fluorescent protein), with the aim of quantifying the antibacterial activity of bacteriocin 285 as a function of GFP fluorescence. Bacteriocin 285 proved to be identical to plantaricin 423 produced by L. plantarum 423. Plantaricin 423 is a class lIa bacteriocin and displays antimicrobial activity towards a broad spectrum of bacteria, including several food spoilage organisms. The sensitivity of L. sakei DSM 20017 towards antibacterial peptides produced by Lactobacillus curvatus DF38, L. plantarum 285, Lactobacillus casei LHS and Lactobacillus salivarius 241 is not limited to the growth stage of the organism. Cells remained sensitive to all four of these bacteriocins, from lag phase to late exponential growth. To inhibit growth of up to 90% of the cells of L. sakei DSM 20017, 1 AU/ml bacteriocin 285 (7 ng/ml) of partially purified bacteriocin 285 was required. However, to kill all viable cells of L. sakei DSM 20017, 16 AU/ml (110 ng/ml) of partially purified bacteriocin 285 was required. The gfpuv gene, encoding GFPuv, was cloned downstream of the Idh promoter and successfully expressed in L. sakei DSM 20017. However, GFPuv fluorescence could not be used as a direct method to quantify the antimicrobial activity of bacteriocin 285, since cells of strain DSM 20017 remained fluorescent for prolonged periods after treatment with lethal concentrations of the bacteriocin. The non-viability of the cells was confirmed with epifluorescence microscopy and a L1VE/DEAD® Baclight™ Bacterial Viability Probe. Cells that were stained with the viability probe indicated that the majority of untreated L. sakei DSM 20017 cells were viable. However, treatment of strain DSM 20017 with 16 AU/ml bacteriocin 285 rendered all visible cells non-viable. / AFRIKAANSE OPSOMMING: Lactobacillus plantarum 285 wat uit sorgumbier geïsoleer is, produseer bakteriosien 285. Die bakteriosien toon aktiwiteit teen verskeie organismes wat voedselbederi veroorsaak. Vir toekomstige aanwending van bakteriosien 285 in die voedselindustrie was dit belangrik om die peptied te karakteriseer en die gene wat vir die produksie daarvan kodeer, te identifiseer. Die effek van bakteriosien 285 op sensitiewe selle is bepaal deur die gebruik van 'n indikator (sensitiewe)-organisme, Lactobacillus sakei DSM 20017. Die indikator-organisme is geneties verander om die GFP (groen fluoreserende proteïen) uit te druk. Die doel was om die antibakteriese aktiwiteit van bakteriosien 285 te kwantifiseer as 'n funksie van GFP fluorisensie. Bakteriosien 285 is identies aan plantarisien 423 wat deur L. plantarum 423 produseer word. Plantarisien 423 is 'n klas Iia bakteriosien en vertoon antimikrobiese aktiwiteit teenoor 'n wye verskeidenheid bakterieë, insluitende verskeie organismes wat voedsel bederf. Die sensitiwiteit van L. sakei DSM 20017 teenoor antibakteriese peptiede wat deur Lactobacillus cutveius DF38, L. plantarum 285, Lactobacillus casei LHS en Lactobacillus salivarius 241 geproduseer word, word nie beïnvloed deur die groeifase van die organisme nie. Selle het sensitief gebly teenoor al vier die bakteriosiene van sloer- tot laat eksponensiële groei. Om groei van tot 90% van L. sakei DSM 20017 selle te inhibeer, word 1 AU/ml (7 ng/ml) gedeeltelik gesuiwerde bakteriosien 285 benodig. Om alle lewensvatbare L. sakei DSM 20017 selle te dood, word 16 AU/ml (110 ng/ml) gedeeltelik gesuiwerde bakteriosien 285 benodig. Die gfpuv-geen, wat GFPuv kodeer is stroomaf van die Idh-promoter gekloneer en suksesvol in L. sakei DSM 20017 uitgedruk. GFPuv fluoresensie kon nie as direkte metode gebruik word om die antimikrobiese aktiwiteit van bakteriosien 285 te bepaal nie, aangesien die selle van L. sakei DSM 20017 fluoreserend gebly het lank na behandeling met dodelike konsentrasies van die bakteriosien. Die lewensvatbaarheid van die selle is bevestig deur epifluoresensiemikroskopie en 'n LlVE/DEAD® Bac/ight™ bakteriese lewensvatbaarheidspeiler. Selle van L. sakei DSM 20017 wat deur die peiler gekleur is, het gewys dat die meeste selle wat nie deur bakteriosien 285 behandel was nie, lewensvatbaar was. Behandeling van L. sakei DSM 20017 met 16 AU/ml bakteriosien 285 het al die sigbare selle gedood.