Integral kinetics of the cleavage of maltose catalysed by α-glucosidase fromSaccharomyces carlsbergensis

Summary A new, sensitive and continuous assay for -glucosidase is described exploiting the different angles of rotation for the substrate maltose and the product glucose. Kinetic experiments revealed a very pronounced product inhibition of -glucosidase fromSaccharomyces carlsbergensis with a K_i of 4.85·10^–3 M for glucose.The K_M of maltose was found to be 37.8·10^–3 M. Taking these values, an integral kinetic curve for the enzymatic hydrolysis of maltose was calculated, which is shown to fit the experimental data.Symbols used k₁ (min^–1) pseudo first-order rate constant (for enzymatic cleavage) - k₂ (min^–1) rate constant (for mutarotation reaction) - I, P (mol/1) inhibitor (product) concentration - k_i (mmol/1) inhibitor constant - K_M (mmol/l) Michaelis constant - [M] ₅₈₉ ³⁰ (degree/m · l/mol) molecular rotation at 30°C and 589 nm - s (mmol/l) substrate concentration - R (mmol/mg · min) reaction rate - V_max (mmol/mg · min) maximal rate - U (mol/min) activity unit (here at 30°C and pH=6.8) Indices O initial value - max maximal value     

Molecular Analysis of the α-Galactosidase MEL Genes in Yeast Saccharomyces sensu stricto

To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the -galactosidase MEL gene family, two new genes were cloned and sequenced from S. bayanus var. bayanus and S. pastorianus. Nucleotide sequence homology of the MEL genes of S. bayanus var. bayanus (MELb), S. pastorianus (MELpt), S. bayanus var. uvarum (MELu), and S. carlsbergensis (MELx) was rather high (94.1–99.3%), comparable with interspecific homology (94.8–100%) of S. cerevisiae MEL1-MEL11. Homology of the MEL genes of sibling species S. cerevisiae (MEL1), S. bayanus (MELb), S. paradoxus (MELp), and S. mikatae(MELj) was 76.2–81.7%, suggesting certain species specificity. On this evidence, the -galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae.     

L-Arabinose is not a gratuitous inducer of α-galactosidase from Saccharomyces carlsbergensis

L-Arabinose has been described as a gratuitous inducer of the yeast -galactosidase. This has been found to be an artefact resulting from galactose contamination of commercial samples of L-arabinose. The inactivation produced on UDP-glucose 4-epimerase by the pentose does not amplify the inducer activity of contaminating D-galactose.     

Selection for the α-Thalassemia Genes

Extremely high incidences of single and double deletions of α-globin genes are known among Asian populations. To study possible mechanisms for the maintenance of such deletions, mathematical analyses have been conducted. It has been shown that a stable polymorphism can be achieved easily through heterozygote advantage using deterministic models. The results strongly suggest that high incidences of single and double deletion of α-globin genes among Asian populations are maintained by some type of heterozygote advantage.     

Mapping of the Homothallic Genes, HMα and HMa, in Saccharomyces Yeasts

Two of the three homothallic genes, HM alpha and HMa, showed direct linkage to the mating-type locus at approximately 73 and 98 strans (57 and 65 centimorgans [cM], respectively, whereas, the other, HO, showed no linkage to 25 standard markers distributed over 17 chromosomes including the mating-type locus. To determine whether the HM alpha and HMa loci located on the left or right side of the mating-type locus, equations for three factor analysis of three linked genes were derived. Tetrad data were collected and were compared with expected values by chi 2 statistics. Calculations indicated that the HM alpha gene is probably located on the right arm at 95 strans (65 cM) from the centromere and the HMa locus at approximately 90 strans (64 cM) on the left arm of chromosome III.     

Dark-recovery of Gamma- and UV-irradiated Saccharomyces carlsbergensis at 4° C

Experiments were carried out to find the effect of dark-holding in distilled water at 4° C on recovery of Gamma- and UV-irradiated cells of a haploid strain ofSaccharomyces carlsbergensis. It was found that there was an appreciable increase in survival of the irradiated cells following 4 to 24 hours' holding while no increase in the number of control cells was observed following similar treatment. It is suggested that some common type of damage induced by both Gamma- and UV-radiations inS. carlsbergensis may be repaired efficiently under metabolic conditions associated with growth at 4° C in distilled water.     

Fast and reliable method for simultaneous zymographic detection of glucoamylase and α-amylase in fungal fermentation

Detection of α-amylase and glucoamylase in crude fermentation extracts using a single native electrophoresis gel and zymogram is described in this article. Proteins were printed on substrate gel and simultaneously onto a membrane in a three-sandwich gel. α-Amylase was detected on the substrate gel with copolymerized β-limit dextrins and iodine reagent. Glucoamylases were detected on the membrane using a coupled assay for glucose detection. Both amylases were detected in native gel using starch and iodine reagent. The described technique can be a helpful tool for monitoring and control of fermentation processes because fungal amylase producers almost always synthesize both amylases.     

Feeding strategies for the enhanced production of α-arbutin in the fed-batch fermentation of Xanthomonas maltophilia BT-112

To develop a cost-effective method for the enhanced production of α-arbutin using Xanthomonas maltophilia BT-112 as a biocatalyst, different fed-batch strategies such as constant feed rate fed-batch, constant hydroquinone (HQ) concentration fed-batch, exponential fed-batch and DO-control pulse fed-batch (DPFB) on α-arbutin production were investigated. The research results indicated that DPFB was an effective method for α-arbutin production. When fermentation with DO-control pulse feeding strategy to feed HQ and yeast extract was applied, the maximum concentrations of α-arbutin and cell dry weight were 61.7 and 4.21 g/L, respectively. The α-arbutin production was 394 % higher than that of the control (batch culture) and the molar conversion yield of α-arbutin reached 94.5 % based on the amount of HQ supplied (240 mM). Therefore, the results in this work provide an efficient and easily controlled method for industrial-scale production of α-arbutin.     

Coexpression of α-l-arabinofuranosidase and β-glucosidase in Saccharomyces cerevisiae

Monoterpenes are important aroma compounds in grape varieties such as Muscat, Gewürztraminer and Riesling, and are present as either odourless, glycosidically bound complexes or free aromatic monoterpenes. Commercial enzymes can be used to release the monoterpenes, but they commonly consist of crude extracts that often have unwanted and unpredictable side-effects on wine aroma. This project aims to address these problems by the expression and secretion of the Aspergillus awamoriα-l-arabinofuranosidase in combination with either the β-glucosidases from Saccharomycopsis fibuligera or from Aspergillus kawachii in the industrial yeast Saccharomyces cerevisiae VIN13. The concentration of five monoterpenes was monitored throughout alcoholic fermentation of Gewürztraminer grapes. The recombinant yeast strains that caused an early boost in the geraniol concentration led to a reduction in the final geraniol levels due to the downregulation of the sterol biosynthetic pathway. Monoterpene concentrations were also analysed 9 and 38 days after racking and the performance of the VB2 and VAB2 recombinant strains was similar, and in many cases, better than that of a commercial enzyme used in the same experiment. The results were backed by sensorial analysis, with the panel preferring the aroma of the wines produced by the VAB2 strain.     

Cloning of the α-Amylase cDNA of Aspergillus shirousamii and Its Expression in Saccharomyces cerevisiae

α-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3? non-coding regions, respectively, so far determined. The α-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active α-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.     

Phosphate effects in the fermentation of α-amylase by Bacillus amyloliquefaciens

Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding.     

Separation and purification of α-ketoglutarate and pyruvate from the fermentation broth of Yarrowia lipolytica

Bioprocess and Biosystems Engineering - A strategy to achieve the efficient co-production of α-ketoglutarate (KGA) and pyruvate (PYR) via Yarrowia lipolytica fermentation was established in...     

Nuclear overhauser effects for methyl β-maltoside and the conformational states of maltose in aqueous solution

A nuclear Overhauser enhancement in methyl β-maltoside, resulting from pre-irradiation of H-1′ of the non-reducing glucose residue, has been measured and calculated theoretically. Comparison of these data reveals a complicated conformational equilibrium in aqueous solutions of maltose derivatives.     

Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.     

Structural Analysis of Free N-Glycans in α-Glucosidase Mutants of Saccharomyces cerevisiae: Lack of the Evidence for the Occurrence of Catabolic α-Glucosidase Acting on the N-Glycans

Saccharomyces cerevisiae produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from N-glycans (Glc₃Man₉GlcNAc₂) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated N-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in S. cerevisiae. To this end, the precise structures of cytosolic free N-glycans (FNGs), mainly derived from the peptide:N-glycanase (Png1) mediated deglycosylation of N-glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on N-glycans are produced by this yeast.     

Molecular Cloning and Evolutionary Analysis of Hemoglobin α-Chain Genes in Bats

Bats are the only mammals with the capacity for powered flight. When flying, they need abundant energy and oxygen. According to previous works, the hemoglobin (Hb) oxygen loading function of bats is insensitive to variations in body temperature, although different bat species have different heat sensitivity. We cloned Hb α-chain sequences from eight bat species to investigate whether they have different characteristics. We found that Hb in the bat lineages is under purifying selection, which accords with the importance of its function in bats. Three turn regions in bat Hb, however, have distinct evolutionary rates compared with those of other mammals, and the codons in these regions have an accelerated rate of evolution. These codons are under divergent selection in bats. These changes in Hb may have occurred in response to the physiological requirements of the species concerned, as adaptations to different lifestyles.     

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode

Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals. Here, for the first time a Saccharomyces cerevisiae strain capable of producing high levels of α-santalene, the precursor of a commercially interesting compound, was constructed through a rationally designed metabolic engineering approach. Optimal sesquiterpene production was obtained by modulating the expression of one of the key metabolic steps of the mevalonate (MVA) pathway, squalene synthase (Erg9). To couple ERG9 expression to glucose concentration its promoter was replaced by the HXT1 promoter. In a second approach, the HXT2 promoter was used to express an ERG9 antisense construct. Using the HXT1 promoter to control ERG9 expression, it was possible to divert the carbon flux from sterol synthesis towards α-santalene improving the productivity by 3.4 fold. Combining this approach together with the overexpression of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) and deletion of lipid phosphate phosphatase encoded by LPP1 led to a strain with a productivity of 0.18mg/gDCWh. The titer was further increased by deleting DPP1 encoding a second FPP consuming pyrophosphate phosphatase yielding a final productivity and titer, respectively, of 0.21mg/gDCWh and 92mg/l of α-santalene.