Kluyveromyces marxianus MTCC 1389 Augments Multi-stress Tolerance After Adaptation to Ethanol Stress.

During fermentation, yeast cells undergo various stresses that inhibit cell growth and ethanol production. Therefore, the ability to tolerate multiple stresses during fermentation is one of the important characteristics for yeast cells that can be used for commercial ethanol production. In the present study, we evaluated the multi-stress tolerance of parent and ethanol adapted Kluyveromyces marxianus MTCC1389 and their relative gene expression analysis. Multi-stress tolerance was confirmed by determining its cell viability, growth, and spot assay under oxidative, osmotic, thermal, and ethanol stress. During oxidative (0.8% H2O2) and osmotic stress (2 M NaCl), there was significant cell viability of 90% and 50%, respectively, by adapted strain. On the other hand, under 45 °C of thermal stress, the adapted strain was 80% viable while the parent strain was 60%. In gene expression analysis, the ethanol stress responsive gene ETP1 was significantly upregulated by 3.5 folds, the osmotic stress gene SLN1 was expressed by 3 folds, and the thermal stress responsive gene MSN2 was expressed by 7 folds. This study shows adaptive evolution for ethanol stress can develop other stress tolerances by changing relative gene expression of osmotic, oxidative, and thermal stress responsive genes.

Heterologous expression in Pichia pastoris and characterization of a novel GH11 xylanase from saline-alkali soil with excellent tolerance to high pH, high salt concentrations and ethanol.

A GH11 xylanase gene (xyn11-1) cloned from saline-alkali soil was successfully expressed in Pichia pastoris GS115. The purified recombinant Xyn11-1 showed its maximal activity at pH 6.0, and retained more than 60.4% of activity at pH 10.0, with good pH stability. Its optimal temperature was 50 °C and it was stable after incubation for 1 h at 30 °C. Furthermore, Xyn11-1 was highly salt-tolerant, retaining more than 77.4% of activity in the presence of 0.25-4 M NaCl and displaying more than 47.2% relative activity after being incubated in the presence of 5 M NaCl at 37 °C for 10 min. In addition, 5 mM ß-Mercaptoethanol, Cu2+, Co2+, and Mn2+ increased the xylanase activity by 22.3%, 8.8%, 7.1%, and 4.4%, respectively. Significantly, 93.4% and 59.8% of the optimal activity was retained in the presence of 2% and 10% (v/v) ethanol, respectively. Under optimal conditions, the Km,Vmax, and Kcat value of Xyn11-1 for beechwood xylan were 3.7 mg ml-1, 101.0 µmol min-1 mg-1 and 42.1 s-1, respectively. Xyn11-1 is a strict endo-ß-1,4-xylanase, its main enzymatic products being xylotetraose and xylopentaose. Xyn11-1 is the first reported GH11 xylanase isolated from saline-alkali soil, and has excellent tolerance of high pH, high salt concentrations and ethanol, which indicates its great potential for basic research and industrial applications.

Characterization of an ethanol-tolerant 1,4-ß-xylosidase produced by Pichia membranifaciens.

AIMS: The purification and biochemical properties of the 1,4-ß-xylosidase of an oenological yeast were investigated. METHODS AND RESULTS: An ethanol-tolerant 1,4-ß-xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G-100. The relative molecular mass of the enzyme was determined to be 50kDa by SDS-PAGE. The activity of 1,4-ß-xylosidase was optimum at pH 6·0 and at 35°C. The activity had a Km of 0·48±0·06mmol l(-1) and a Vmax of 7·4±0·1µmol min(-1)mg(-1) protein for p-nitrophenyl-ß-d-xylopyranoside. CONCLUSIONS: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may be useful for assessing the ability of the 1,4-ß-xylosidase from P. membranifaciens to be used in the bioethanol production process.

Ethanol Production Potential of Ethanol-Tolerant Saccharomyces and Non-Saccharomyces Yeasts.

Four ethanologenic ethanol-tolerant yeast strains, Saccharomyces cerevisiae (ATKU132), Saccharomycodes ludwigii (ATKU47), and Issatchenkia orientalis (ATKU5-60 and ATKU5-70), were isolated by an enrichment technique in yeast extract peptone dextrose (YPD) medium supplemented with 10% (v/v) ethanol at 30°C. Among non-Saccharomyces yeasts, Sd. ludwigii ATKU47 exhibited the highest ethanol-tolerance and ethanol production, which was similar to S. cerevisiae ATKU132. The maximum range of ethanol concentrations produced at 37°C by S. cerevisiae ATKU132 and Sd. ludwigii ATKU47 from an initial D-glucose concentration of 20% (w/v) and 28% (w/v) sugarcane molasses were 9.46-9.82% (w/v) and 8.07-8.32% (w/v), respectively.

A novel ß-glucosidase from a hot-spring metagenome shows elevated thermal stability and tolerance to glucose and ethanol.

ß-glucosidase causes hydrolysis of ß-1,4-glycosidic bond in glycosides and oligosaccharides. It is an industrially important enzyme owing to its potential in biomass processing applications. In this study, computational screening of an extreme temperature aquatic habitat metagenomic resource was done, leading to the identification of a novel gene, bglM, encoding a ß-glucosidase. The comparative protein sequence and homology structure analyses designated it as a GH1 family ß-glucosidase. The bglM gene was expressed in a heterologous host, Escherichia coli. The purified protein, BglM, was biochemically characterized for ß-glucosidase activity. BglM exhibited noteworthy hydrolytic potential towards cellobiose and lactose. BglM, showed substantial catalytic activity in the pH range of 5.0-7.0 and at the temperature 40 °C-70 °C. The enzyme was found quite stable at 50 °C with a loss of hardly 20% after 40 h of heat exposure. Furthermore, any drastically negative effect was not observed on the enzyme's activity in the presence of metal ions, non-ionic surfactants, metal chelating, and denaturing agents. A significantly high glucose tolerance, retaining 80% relative activity at 1 M, and 40% at 5 M glucose, and ethanol tolerance, exhibiting 80% relative activity in 10% ethanol, enrolled BglM as a promising enzyme for cellulose saccharification. Furthermore, its ability to catalyze the hydrolysis of daidzin and polydatin ascertained it as an admirably suited biocatalyst for enhancement of nutritional values in soya and wine industries.

Purification and biochemical characterization of an extracellular ß-d-fructofuranosidase from Aspergillus sp.

This study focused on the purification and characterization of an extracellular ß-d-fructofuranosidase or invertase from Aspergillus sojae JU12. The protein was purified by size exclusion chromatography with 5.41 fold and 10.87% recovery. The apparent molecular mass of the enzyme was estimated to be ~ 35 kDa using SDS-PAGE and confirmed by deconvoluted mass spectrometry. The fungal ß-d-fructofuranosidase was suggested to be a monomer by native PAGE and zymography, and was found to be a glycoprotein possessing 68.92% carbohydrate content. The products of enzyme hydrolysis were detected by thin layer chromatography and revealed the monosaccharide units, d-glucose and d-fructose. ß-d-fructofuranosidase showed enhanced activity at broad pH 4.0-9.0 and activity at a temperature range from 30 to 70 °C, while the enzyme was stable at pH 8.0 and 40 °C, respectively. The ß-d-fructofuranosidase activity was lowered by metal ion inhibitors Ag2+ and Hg2+ whereas elevated by SDS and ß-ME. The fungal ß-d-fructofuranosidase was capable of hydrolyzing d-sucrose and the kinetics were determined by Lineweaver-Burk plot with Km of 10.17 mM and Vmax of 0.7801 µmol min-1. Additionally, the extracellular ß-d-fructofuranosidase demonstrated tolerance to high ethanol concentrations indicating its applicability in the production of alcoholic fermentation processes.

Thermotolerant genes essential for survival at a critical high temperature in thermotolerant ethanologenic Zymomonas mobilis TISTR 548.

BACKGROUND: High-temperature fermentation (HTF) technology is expected to reduce the cost of bioconversion of biomass to fuels or chemicals. For stable HTF, the development of a thermotolerant microbe is indispensable. Elucidation of the molecular mechanism of thermotolerance would enable the thermal stability of microbes to be improved. RESULTS: Thermotolerant genes that are essential for survival at a critical high temperature (CHT) were identified via transposon mutagenesis in ethanologenic, thermotolerant Zymomonas mobilis TISTR 548. Surprisingly, no genes for general heat shock proteins except for degP were included. Cells with transposon insertion in these genes showed a defect in growth at around 39 °C but grew normally at 30 °C. Of those, more than 60% were found to be sensitive to ethanol at 30 °C, indicating that the mechanism of thermotolerance partially overlaps with that of ethanol tolerance in the organism. Products of these genes were classified into nine categories of metabolism, membrane stabilization, transporter, DNA repair, tRNA modification, protein quality control, translation control, cell division, and transcriptional regulation. CONCLUSIONS: The thermotolerant genes of Escherichia coli and Acetobacter tropicalis that had been identified can be functionally classified into 9 categories according to the classification of those of Z. mobilis, and the ratio of thermotolerant genes to total genomic genes in Z. mobilis is nearly the same as that in E. coli, though the ratio in A. tropicalis is relatively low. There are 7 conserved thermotolerant genes that are shared by these three or two microbes. These findings suggest that Z. mobilis possesses molecular mechanisms for its survival at a CHT that are similar to those in E. coli and A. tropicalis. The mechanisms may mainly contribute to membrane stabilization, protection and repair of damage of macromolecules and maintenance of cellular metabolism at a CHT. Notably, the contribution of heat shock proteins to such survival seems to be very low.

Purification and characterization of an extracellular ß-xylosidase from Pseudozyma hubeiensis NCIM 3574 (PhXyl), an unexplored yeast.

This paper reports on the production of ß-xylosidase from an unexplored yeast, Pseudozyma hubeinsis. The expression of this enzyme could be induced by beech wood xylan when the yeast was grown at 27 °C. The enzyme was purified to homogeneity as a glycoprotein with 23 % glycosylation. The purification protocol involved ammonium sulphate precipitation, QAE-Sephadex A50 ion exchange chromatography and sephacryl-200 column chromatography which resulted in 8.3-fold purification with 53.12 % final recovery. The purified enzyme showed prominent single band on SDS-PAGE. It is a monomeric protein of 110 kDa molecular weight confirmed by SDS-PAGE followed by MALDI-TOF mass spectrometry (112.3 kDa). The enzyme was optimally active at 60 °C and pH 4.5 and stable at pH range (4-9) and at 50 °C for 4 h. Chemical modification studies revealed that active site of the purified enzyme comprised of carboxyl, tyrosine and tryptophan residues. The carboxyl residue is involved in catalysis and tryptophan residue is solely involved in substrate binding. The best match from the search of the NCBInr database was with gi|808364558 glycoside hydrolase of Pseudozyma hubeiensis SY62 with 26 % sequence coverage confirming that it is a glycoside hydrolase/beta-glucosidase. From the search of customized SWISSPROT database, it was revealed that SWISSPROT does not contain any entries that are similar to the purified enzyme.

Effect of ultraviolet radiation on physiological and biochemical properties of yeast Saccharomyces cerevisiae during fermentation of ultradispersed starch raw material

Background: Study of correlation between pretreatment of yeast with ultraviolet radiation and efficiency of further fermentation of wort made of ultrafine grain particles to ethanol. Results: We investigated three races of industrial yeast Saccharomyces cerevisiae (native and irradiated by ultraviolet). Physiological properties during fermentation of starchy wort were tested in all variants. It was shown that activation of the yeast by ultraviolet radiation allows to further increase the ethanol yield by 25% on average compared with the native yeast races when using thin (up to micro- and nano-sized particles) or standard grain grinding. Conclusions: Using mechanical two-stage grinding of starchy raw materials and ultraviolet pretreatment of yeast, the efficiency of saccharification of starch and fermentation of wort to ethanol was increased.

Improvement of ethanol production from sweet sorghum juice under batch and fed-batch fermentations: effects of sugar levels, nitrogen supplementation, and feeding regimes

Background: Fermentation process development has been very important for efficient ethanol production. Improvement of ethanol production efficiency from sweet sorghum juice (SSJ) under normal gravity (NG, 160 g/L of sugar), high gravity (HG, 200 and 240 g/L of sugar) and very high gravity (VHG, 280 and 320 g/L of sugar) conditions by nutrient supplementation and alternative feeding regimes (batch and fed-batch systems) was investigated using a highly ethanol-tolerant strain, Saccharomyces cerevisiae NP01. Results: In the batch fermentations without yeast extract, HG fermentation at 200 g/L of sugar showed the highest ethanol concentration (PE, 90.0 g/L) and ethanol productivity (QE, 1.25 g/L·h). With yeast extract supplementation (9 g/L), the ethanol production efficiency increased at all sugar concentrations. The highest PE (112.5 g/L) and QE (1.56 g/L·h) were observed with the VHG fermentation at 280 g/L of sugar. In the fed-batch fermentations, two feeding regimes, i.e., stepwise and continuous feedings, were studied at sugar concentrations of 280 g/L. Continuous feeding gave better results with the highest PE and QE of 112.9 g/L and 2.35 g/L·h, respectively, at a feeding time of 9 h and feeding rate of 40 g sugar/h. Conclusions: In the batch fermentation, nitrogen supplementation resulted in 4 to 32 g/L increases in ethanol production, depending on the initial sugar level in the SSJ. Under the VHG condition, with sufficient nitrogen, the fed-batch fermentation with continuous feeding resulted in a similar PE and increased QP by 51% compared to those in the batch fermentation.