Mandelate dehydrogenases from Rhodotorula graminis

Rhodotorula graminis is a yeast with the ability to utilise mandelate as a source of carbon and energy. Mandelate exists in two enantiomeric forms; D-mandelate and L-mandelate. R. graminis possesses both D(-)-mandelate dehydrogenase and L(+)-mandelate dehydrogenases which enable this organism to use both forms of mandelate. The L(+)- mandelate dehydrogenase (LMDH) is a soluble component of the mitochondrial intermembrane space and is similar to L(+)-lactate dehydrogenase (LLDH) from S. cerevisiae and H. anomala which is a flavocytochrome b2. The D(-)-mandelate dehydrogenase is an unrelated enzyme which is a soluble NAD-dependent enzyme.In order to study these two proteins the genes encoding LMDH and DMDH were isolated, sequenced and expressed. An 81 bp fragment was amplified by RT-PCR and used to probe the gene encoding the LMDH from R. graminis.The genomic DNA was isolated from a genomic library and then the cDNA was amplified by RT- PCR. Comparison of the genomic DNA and the cDNA sequences reveals the presence of eleven introns in the genomic DNA encoding LMDH. The conserved intron sequences of LMDH in R. graminis are similar to the intron sequences from the phenylalanine- ammonia lyase (PAL) gene in Rhodosporidium toruloides and Rhodotorula rubra. The LMDH from R. graminis is predicted to contain a 74 amino acid extension at the N- terminus. This presequence is highly basic and contains a long stretch of non -polar amino acids, typical of a subclass of mitochondrial targeting sequences. The 1479 bp cDNA specifies a mature LMDH of 492 amino acids including the incorporated N- terminal methionine. The calculated Mr is 54,604. Computer search with other proteins in the database shows similarity with other FMN- dependent 2- hydroxyacid oxidising enzymes. LMDH is very closely related to flavocytochrome b2 from S. cerevisiae and H. anomala. Based on the crystal structure and the amino acid sequence comparison with L(+)-lactate dehydrogenase from S. cerevisiae, LMDH from R. graminis could be divided into two domains, the haem binding domain and the flavin binding domain. The amino acid sequence of the predicted cytochrome domain shows high similarity with microsomal b5. Comparison of the amino acid sequence of LMDH and LLDH from S. cerevisiae shows that all the residues important in catalysis and substrate binding of the enzyme are highly conserved. Amino acid sequence comparison also reveals that Leu230 in LLDH from S. cerevisiae, which determins the substrate specificities of the enzyme, is replaced by a smaller glycine in LMDH from R. graminis.The genomic DNA and the cDNA of D(-)-mandelate dehydrogenase have been isolated and amplified respectively. Comparison of the genomic DNA and the cDNA revealed the presence of three introns. All the introns start with GT, have the sequence of CAG at the 3' end and have an internal consensus sequence of CTGAC. The entire 1053 bp sequence of the amplified cDNA specifies a protein (DMDH) of 351 amino acids with the calculated Mr of 38,591. A computer search with other proteins in the database shows that DMDH from R. graminis belongs to the D-isomer-2 hydroxyacid dehydrogenase family. Based on the crystal structure and amino acid sequence from D-glycerate dehydrogenase from Hyphomicrobium methylovorum it is predicted that the polypeptide chain of DMDH is divided into two domains, the catalytic domain which is formed by approximately residues 1 to 111 and 308 to 351 and the second coenzyme-binding domain which is located in the middle of the polypeptide chain approximately between residue 111 to 307. Arg259 in DMDH is believed to be important in substrate binding as in the other D- isomer specific dehydrogenases. His304 acts as an acid/base catalyst. The highly conserved sequence G-X-G-X-X-G-17X-D which is common to the NAD-binding domain is conserved in DMDH except that the third glycine is replaced by glutamine. The function of glutamine here is not clear and can only be determined when the DMDH crystal structure becomes available.Both LMDH and DMDH were succesfully expressed by using the expression vector pRC23 which is thermoinducible. The calculated kcal and Km of the partially purified recombinant LMDH is 350 s⁻¹ and 0.35 mM respectively. The specific activity for DMDH is 0.0528 units /mg

Protein sequences classification based on weighting scheme

We present a new technique to recognize remote protein homologies that rely on combining probabilistic modeling and supervised learning in high-dimensional feature spaces. The main novelty of our technique is the method of constructing feature vectors using Hidden Markov Model and the combination of this representation with a classifier capable of learning in very sparse high-dimensional spaces. Each feature vector records the sensitivity of each protein domain to a previously learned set of sub-sequences (strings). Unlike other previous methods, our method takes in consideration the conserved and non-conserved regions. The system subsequently utilizes Support Vector Machines (SVM) classifiers to learn the boundaries between structural protein classes. Experiments show that this method, which we call the String Weighting Scheme-SVM (SWS-SVM) method, significantly improves on previous methods for the classification of protein domains based on remote homologies. Our method is then compared to five existing homology detection methods

Effect of cross-linked enzyme aggregates in hierarchically mesocellular mesoporous magnetic silica preparation conditions towards enzyme activity retention

This work aimed to optimize ten preparation factors that might influence the cellulase and xylanase activity retention of cross-linked enzyme aggregates in hierarchically mesocellular mesoporous magnetic silica. The factors were optimized using the fractional factorial design (210-5). The optimized output was occurred at 2 mL of enzyme amount, magnet-to-enzyme ratio of 1:0.15, enzyme adsorption at 26°C and 162 rpm in 40 min, enzyme-to-precipitant ratio of 1:11, 0.05% (v/v) of glutaraldehyde concentration, and cross-linking process at 37°C and 300 rpm in 2 h. The factors were examined to observe the effect of every factor towards cellulase and xylanase acrivity retention

Cloning and expression of pullulanase gene from locally isolated bacillus SP

Bacterial pullulanase represents one of th e starch-degrading enzymes that are widely used in the starch processing indu stry along with amylases. Amylases hydrolyze a -(1,4 )-glycosidic linkage in starch to produce a mixture of glucose , maltooligo sacchari de and limited a-dextrin. All the remaining a -(1,6)-glycosid ic branches in the products are hydrolyzed by p ullulanase. This is an advantage t o improve glucose production by coupling pullulanase and amylase in the p rocess. As such, many pullulana e enzyme has been isolated and one has been showing optimum pH of 10-10.5 which is suitable for use in dishwasher detergent additive in removal of star ch stain. We have recently iso lated a few bacterias that have shown potentially pullulanase producers by the holo-zone in pullulan-plate assay. One of them, we named Bacillus –1 sho ws a bigger holo-zone among others, Bacillus- 1 is highly active in pH more than 7. The enzyme also shows a mo derate activity to wards starch that may be indicates be side hydrolyzes a -(1,6)-glycosidic linkage in starch, it also hydrolyzes a -(1,4)- glycosidi c simi lar to a -amylase. Unfortunately the enzyme from wild-type bacteria is in lower yield an d in this studies, we intend to clone and sequence the pullulanase gene and also expressed the gene in a high expression system to be able to produce in a high yield before characterizing expressed protein

Optimization and characterization of exo-polygalacturonase by Aspergillus niger cultured via solid state fermentation

Polygalacturonases represent an important member of pectinases group of enzymes with immense industrial applications. The activity of exo-polygalacturonase produced by Aspergillus niger was studied in solid state fermentation (SSF) using Nephrolepis biserrata leaves as substrate. Central composite design (CCD) was used to optimize four significant variables resulted from the screening process that has been initially analyzed for the production of exo-polygalacturonase which are incubation time, temperature, concentration of pectin and moisture content. The optimum exo-polygalacturonase production obtained was 54.64 U/g at 120 hours of incubation time, temperature at 340C, 5.0 g/L of pectin concentration and 75.26% of moisture content. For partial characterization of exo-polygalacturonase, the optimum temperature and pH were obtained at 50°C and pH 4.0, respectively. SDS-PAGE analysis showed that molecular weight of exo-polygalacturonase were 35 and 71 kDa. This study has revealed a significant production of exo-polygalacturonase by A. niger under SSF using cheap and easily available substrate and thus could found immense potential application in industrial sectors and biotechnology

Effect of agitation and aeration rates on chitinase production using Trichoderma virens UKM1 in 2 L stirred tank reactor

Shrimps have been a popular raw material for the burgeoning marine and food industry contributing to increasing marine waste. Shrimp waste, which is rich in organic compounds is an abundant source of chitin, a natural polymer of N-acetyl-D-glucosamine (GluNac), a reducing sugar. For this respect, chitinase-producing fungi have been extensively studied as biocontrol agents. Locally isolated Trichoderma virens UKM1 was used in this study. The effect of agitation and aeration rates using colloidal chitin as control substrate in a 2-l stirred tank reactor gave the best agitation and aeration rates at 200 rpm and 0.33 vvm with 4.1 U/l per hour and 5.97 U/l per hour of maximum volumetric chitinase activity obtained, respectively. Microscopic observations showed shear sensitivity at higher agitation rate of the above system. The oxygen uptake rate during the highest chitinase productivity obtained using sun-dried ground shrimp waste of 1.74 mg of dissolved oxygen per gram of fungal biomass per hour at the kappaL a of 8.34 per hour

Optimization of medium for the production of β-cyclodextrin glucanotransferase using Central Composite Design (CCD)

Production of cyclodextrin glucanotransferase (CGTase) from Bacillus G 1, a new bacterial isolate was optimized in shake-flask cultures using statistical design approach. An efficient fermentation medium producing CGTase with high activity (54.9U/ml) was determined: A 2(5) Central Composite Design (CCD; half fraction) has been chosen to elucidate the combined effect of five process variables; tapioca starch, peptone, yeast extract, magnesium sulphate and potassium phosphate concentration. Concentrations of 4% tapioca starch, 2% peptone, 0.04% magnesium sulphate and 1 % sodium carbonate have been shown to be optimum for the production of CGTase

Enhancement of isobutanol and 3-methyl-1-butanol production yields in saccharomyces cerevisiae without genetic modification

Bio-based fuel produced from the renewable resources is efficiently overcome the shortcomings of fossil fuels. Several factors such as the increasing awareness on environmental problems, fossil fuel prices and the sustainability of energy has encouraged the initiative in finding another source of transportation fuels. Higher alcohols have proved to be a better candidate to replace gasoline as vehicle fuel due to characteristics of higher energy content, low solubility in water, lower vapor pressure and higher blending ability with gasoline. Biologically, isobutanol and 3-methyl-1-butanol are produced through the fermentation of renewable feedstock with microorganism. Saccharomyces cerevisiae is known to be able to produce isobutanol and 3-methyl-1-butanol titers naturally without heterologous pathways. However, the production of these alcohols by Saccharomyces cerevisiae is only in a small quantity, thus several efforts in enhancing the isobutanol and 3-methyl-1-butanol yields have been conducted. In this study, the amino acids (valine and leucine) and amino acid precursor (2-ketoisovalerate) were added into the fermentation medium prior to the fermentation. The results obtained show that the supplementation of 2-ketoisovalerate and leucine individually into the fermentation broth leads to the increased in isobutanol and 3-methyl-1-butanol titers by 3.3 folds and 1.9 folds, respectively. The combination of 2-ketoisovalerate and valine increased the isobutanol yield by 4.3 folds while the 3-methyl-1-butanol was increased by 2.5 folds when supplemented with 2-ketoisovalerate and leucine. These results portray that the isobutanol and 3-methyl-1-butanol titers can be improved by manipulating several factors which is important for future production of higher alcohols

Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris

A cellobiohydrolase gene from the thermophilic fungus Humicola insolens ATCC 16454 was expressed in the methylotrophic yeast Pichia pastoris X-33, and the biochemical properties of the recombinant protein were characterised. The full-length cDNA of the cellobiohydrolase gene avi2 was cloned into the P. pastoris expression vector pPICZαC and expressed extracellularly as a recombinant cellobiohydrolase protein with a molecular weight of approximately 52.3 kDa. The purified recombinant Avi2 enzyme displayed an optimal activity at 50°C and was found stable between temperatures of 30°C and 60°C. The optimal pH of the enzyme was pH 5.0. More than 80% of the enzyme activity was retained at pH values ranging from pH3.0 to pH9.0. Recombinant Avi2 enzyme showed its highest activity towards the substrates Avicel (0.075 U mg-1) and Sigmacell-cellulose (0.018 U mg-1). Very low or undetectable hydrolysis was observed with cellobiose and filter paper. Metal ions, such as Mn2+, Co2+, and Ba2+, increased the activity of the recombinant enzyme. Manganese ions caused the highest increase in activity of approximately 1.38-fold compared to the control assay. Other ions such as Pd2+, Cu2+, Zn2+, Fe2+, and SDS, however, inhibited Avi2 enzyme activity. Interestingly, this recombinant enzyme showed high pH stability when it was incubated in either acidic or basic solutions

In silico structural characterization of L. lactis subsp. cremoris MG1363 Ffh-Ftsy complex in protein targeting interaction

In bacteria, gene conservation and experimental data show that Lactococcus lactis has the simplest version of protein secretion system compared to Escherichia coli and Bacillus subtilis whose systems are more complex. L. lactis only possess the signal recognition particle (SRP) pathway, where the specific interaction of Ffh and FtsY is known to be essential for the efficiency and fidelity of its protein targeting. Therefore, modelling and structural characterization study of Ffh and FtsY will give an idea of its crucial region and amino acids that are critical in Ffh-FtsY interaction during protein targeting. This work is the first attempt to model L. lactis Ffh-FtsY complex, which was derived by computational docking, where a blind dock was applied. Results showed that the complex interface was predominantly stabilized by four hydrophobic interactions and 17 hydrogen bonds, where these putative binding interfaces are mostly confined at the motifs II and III in each G domain of Ffh and FtsY. Several residues were expected to play important roles in initiating or regulating guanosine triphosphate hydrolysis, including residue R142. This structural information will allow for the rational design of L. lactis Ffh-FtsY association in the future