Identification of thermostable glycogen branching enzyme from Geobacillus sp. Geo5 by genome mining

Glycogen branching enzyme (EC 2.4.1.18) has increasing demand from food and beverages processing industries. This enzyme, which catalyses the formation of α-1,6-glycosidic branch points in glycogen structure, is used to enhance nutritional value and quality of food and beverages. To be applicable in industries, enzymes that are stable and active at high temperature are much desired. A thermophilic bacterium, Geobacillus sp. Geo5, was isolated from Sungai Klah Hot Springs at97°C and therefore it was postulated that this bacterium species would produce thermostable glycogen branching enzyme that is active at high temperature. The objectives of this research are to identify the branching enzyme gene (glgB) of Geobacillus sp. Geo5, to produce the enzyme using Escherichia coli and to characterise the biochemical properties of the enzyme. Using genome data mining, the nucleotide sequence of glgB was fished out from Geobacillus sp. Geo5 genome sequence provided by Malaysia Genome Institute. The size of the gene is 2013 bp and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the three dimensional structure of the enzyme using an online software, I-TASSER. The percentage sequence identity of the template (Mycobacterium tuberculosis H37RV; PDB ID: 3K1D) in the threading aligned region with the Geobacillus sp. Geo5 sequence was only 45%. Subsequently, glgB from Geobacillus sp. Geo5 was isolated using polymerase chain reaction (PCR). To study the enzyme, the gene was cloned into pET102/D-TOPO® vector by PCR cloning and overexpressed in BL21 Star TM (DE3) E. coli. The expression of active enzyme was the highest when the expression was induced with 0.75 mM of IPTG, at 30°C for 8 hours. The recombinant protein was also expressed together with bacteriocin release protein to secrete the protein into E. coli culture medium. The study shown that induction with 5 ng/mL of mitomycin C for 8 hour was enough to secrete the recombinant protein to extracellular environment (34.1 U/mL) although not entirely since 43.0 U/mL of the activity was still in the cell. Therefore, the intracellular expression system was chosen for further studies on the enzyme. The recombinant protein from intracellular expression was then purified by affinity chromatography using HisTrap HP column with the recovery of 84%. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55°C and the half-life at 60°C and 70°C was 24 hours and 5 hours, respectively. The enzyme was stable at pH 5 to pH 9 and the optimum pH for enzyme activity was at pH 6. Metal ions, Mn2+, Zn2+, Cu2+, Fe2+ and Ca2+ seem to inhibit the activity of this enzyme. Mg2+ however does not affect the enzyme activity. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus sp. Geo5 by genome mining together with molecular biology technique. The stability of this enzyme would be very practical for industrial applications especially in carbohydrates processing such as nutraceutical, food and beverages industries

Spray-dried immobilized lipase from Geobacillus sp. strain ARM in sago

Sago starch is traditionally used as food especially in Southeast Asia. Generally, sago is safe for consumption, biodegradable, easily available and inexpensive. Therefore, this research was done to expand the potential of sago by using it as a support for enzyme immobilization. In this study, ARM lipase, which was isolated from Geobacillus sp. strain ARM, was overexpressed in Escherichia coli system and then purified using affinity chromatography. The specific activity of the pure enzyme was 650 U/mg, increased 7 folds from the cell lysate. The purified enzyme was immobilized in gelatinized sago and spray-dried by entrapment technique in order to enhance the enzyme operational stability for handling at high temperature and also for storage. The morphology of the gelatinized sago and immobilized enzyme was studied by scanning electron microscopy. The results showed that the spray-dried gelatinized sago was shrunken and became irregular in structure as compared to untreated sago powder. The surface areas and porosities of spray-dried gelatinized sago with and without the enzyme were analyzed using BET and BJH method and have shown an increase in surface area and decrease in pore size. The immobilized ARM lipase showed good performance at 60-80 °C, with a half-life of 4 h and in a pH range 6-9. The immobilized enzyme could be stored at 10 °C with the half-life for 9 months. Collectively, the spray-dried immobilized lipase shows promising capability for industrial uses, especially in food processing

Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05

The glycogen branching enzyme (EC 2.4.1.18), which catalyses the formation of α-1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene (glgB) was extracted from the Geobacillus mahadia Geo-05 genome sequence provided by the Malaysia Genome Institute. The size of the gene is 2013 bp, and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the thermostability, function and the three dimensional structure of the enzyme. The gene was cloned and overexpressed in E. coli to verify the predicted result experimentally. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55 °C, and the half-life at 60 °C and 70 °C was 24 h and 5 h, respectively. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus mahadia Geo-05 by genome mining together with molecular biology technique