Extracellular Invertase Research Articles

Carbon and nitrogen sources consumption by ale and lager yeast strains: a comparative study during fermentation.

The rapid and efficient consumption of carbon and nitrogen sources by brewer's yeast is critical for the fermentation process in the brewing industry. The comparison of the growth characterizations of typical ale and lager yeast, as well as their consumption preference to carbon and nitrogen sources were investigated in this study. Results showed that the ale strain grew faster and had a more extended stationary phase than the lager strain. However, the lager strain was more tolerant to the stressful environment in the later stage of fermentation. Meanwhile, the ale and lager yeast strains possessed varying preferences for metabolizing the specific fermentable sugar or free amino acid involved in the wort medium. The lager strain had a strong capacity to synthesize the extracellular invertase required for hydrolyzing sucrose as well as a strong capability to metabolize glucose and fructose. Furthermore, the lager strain had an advantage in consuming Lys, Arg, Val, and Phe, whereas the ale strain had a higher assimilation rate in consuming Tyr. These findings provide valuable insights into selecting the appropriate brewer's yeast strain based on the wort components for the industrial fermentation process. KEY POINTS: • The lager strain is more tolerant to the stressful environment. • The lager strain has the great capability to synthesize the extracellular invertase. • The assimilation efficiency of free amino acid varies between ale and lager.

Improved Sugarcane-Based Fermentation Processes by an Industrial Fuel-Ethanol Yeast Strain.

In Brazil, sucrose-rich broths (cane juice and/or molasses) are used to produce billions of liters of both fuel ethanol and cachaça per year using selected Saccharomyces cerevisiae industrial strains. Considering the important role of feedstock (sugar) prices in the overall process economics, to improve sucrose fermentation the genetic characteristics of a group of eight fuel-ethanol and five cachaça industrial yeasts that tend to dominate the fermentors during the production season were determined by array comparative genomic hybridization. The widespread presence of genes encoding invertase at multiple telomeres has been shown to be a common feature of both baker's and distillers' yeast strains, and is postulated to be an adaptation to sucrose-rich broths. Our results show that only two strains (one fuel-ethanol and one cachaça yeast) have amplification of genes encoding invertase, with high specific activity. The other industrial yeast strains had a single locus (SUC2) in their genome, with different patterns of invertase activity. These results indicate that invertase activity probably does not limit sucrose fermentation during fuel-ethanol and cachaça production by these industrial strains. Using this knowledge, we changed the mode of sucrose metabolism of an industrial strain by avoiding extracellular invertase activity, overexpressing the intracellular invertase, and increasing its transport through the AGT1 permease. This approach allowed the direct consumption of the disaccharide by the cells, without releasing glucose or fructose into the medium, and a 11% higher ethanol production from sucrose by the modified industrial yeast, when compared to its parental strain.

The Influence of Yarrowia lipolytica Glycosylation on the Biochemical Properties and Oligomerization of Heterologous Invertase

Invertases are important enzymes used in the food industry. Despite many studies on the invertase-encoding SUC2 gene expression in the industrial yeast Yarrowia lipolytica, no biochemical characteristics of this enzyme expressed as heterologous protein have been provided. Here, two isoforms of extracellular invertase produced by Y. lipolytica were detected using ion-exchange chromatography. Specific activities of 226.45 and 432.66 U/mg for the first and second isoform, respectively, were determined. Basic characteristics of this enzyme were similar to the one isolated from Saccharomyces cerevisiae (optimum pH and temperature, metal ions inhibition, substrate specificity and fructooligosaccharides (FOS) biosynthesis). The apparent differences were higher KM for sucrose (67 mM) and lower molecular mass (66 kDa) resulting from lower N-glycosylation level (9.1% of mass). The N-glycan structures determined by MALDI-TOF and HPLC represented high mannose structures, though with much shorter chains than hypermannosylated glycans from S. cerevisiae. Furthermore, galactose was detected as the modifying sugar in the glycan structures of invertase expressed in Y. lipolytica. N-glycans did not affect invertase activity but were important for its oligomerization. The expressed enzyme aggregated into dimers, tetramers, hexamers, and octamers, as well as structures of higher molecular mass, which might be decamers, which have not been described so far in the literature.

Engineering proton-coupled hexose uptake in Saccharomyces cerevisiae for improved ethanol yield.

BackgroundIn the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S. cerevisiae was replaced by hexose–proton symport.ResultsIntroduction of heterologous glucose– or fructose–proton symporters in an hxt0 yeast background strain (derived from CEN.PK2-1C) restored growth on the corresponding sugar under aerobic conditions. After applying an evolutionary engineering strategy to enable anaerobic growth, the hexose–proton symporter-expressing strains were grown in anaerobic, hexose-limited chemostats on synthetic defined medium, which showed that the biomass yield of the resulting strains was decreased by 44.0-47.6%, whereas the ethanol yield had increased by up to 17.2% (from 1.51 to 1.77 mol mol hexose−1) compared to an isogenic strain expressing the hexose uniporter HXT5. To apply this strategy to increase the ethanol yield on sucrose, we constructed a platform strain in which all genes encoding hexose transporters, disaccharide transporters and disaccharide hydrolases were deleted, after which a combination of a glucose–proton symporter, fructose–proton symporter and extracellular invertase (SUC2) were introduced. After evolution, the resulting strain exhibited a 16.6% increased anaerobic ethanol yield (from 1.51 to 1.76 mol mol hexose equivalent−1) and 46.6% decreased biomass yield on sucrose.ConclusionsThis study provides a proof-of-concept for the replacement of the endogenous hexose transporters of S. cerevisiae by hexose-proton symport, and the concomitant decrease in ATP yield, to greatly improve the anaerobic yield of ethanol on sugar. Moreover, the sugar-negative platform strain constructed in this study acts as a valuable starting point for future studies on sugar transport or development of cell factories requiring specific sugar transport mechanisms.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-022-02145-7.

A transcriptional landscape underlying sugar import for grain set in maize.

Developing seed depends on sugar supply for its growth and yield formation. Maize (Zea mays L.) produces the largest grains among cereals. However, there is a lack of holistic understanding of the transcriptional landscape of genes controlling sucrose transport to, and utilization within, maize grains. By performing in-depth data mining of spatio-temporal transcriptomes coupled with histological and heterologous functional analyses, we identified transporter genes specifically expressed in the maternal-filial interface, including (i) ZmSWEET11/13b in the placento-chalazal zone, where sucrose is exported into the apoplasmic space, and (ii) ZmSTP3, ZmSWEET3a/4c (monosaccharide transporters), ZmSUT1, and ZmSWEET11/13a (sucrose transporters) in the basal endosperm transfer cells for retrieval of apoplasmic sucrose or hexoses after hydrolysis by extracellular invertase. In the embryo and its surrounding regions, an embryo-localized ZmSUT4 and a cohort of ZmSWEETs were specifically expressed. Interestingly, drought repressed those ZmSWEETs likely exporting sucrose but enhanced the expression of most transporter genes for uptake of apoplasmic sugars. Importantly, this drought-induced fluctuation in gene expression was largely attenuated by an increased C supply via controlled pollination, indicating that the altered gene expression is conditioned by C availability. Based on the analyses above, we proposed a holistic model on the spatio-temporal expression of genes that likely govern sugar transport and utilization across maize maternal and endosperm and embryo tissues during the critical stage of grain set. Collectively, the findings represent an advancement towards a holistic understanding of the transcriptional landscape underlying post-phloem sugar transport in maize grain and indicate that the drought-induced changes in gene expression are attributable to low C status.

Extraction and characterization of β-fructofuranosidases produced by Kluyveromyces marxianus CCMB 322

Invertase (β-fructofuranosidase, EC 3.2.1.26) catalyzes sucrose hydrolysis into glucose and fructose and it is one of the simplest carbohydrases. These enzymes occur widely in nature and their presence has been reported in microorganisms and plants. Since yeasts are the main industrial source, most researches concerning this enzyme have focused on invertase extracted from such source. This study extracted and characterized inverted intracellular (Inv-I) and extracellular (Inv-E) of Kluyveromyces marxianus CCMB 322 isolated in the baiano semi-arid region. Kluyveromyces marxianus CCMB 322 produces intracellular and extracellular invertase with different characteristics. The optimum activity was achieved at approximately pH 3.9 and 45ºC, in Inv-I and Inv-E. The invertases produced by K. marxianus CCMB 322 showed thermal stability similar to that found in other studies. The Km and Vmax values of the Inv-I enzyme were 61.12mM and 5.56 µmol/mL.min-1, but the Km and Vmax values of the Inv-E enzyme were 76.5mM and 0.364 µmol/mL.min-1. Inverted from K. marxianus is a higher affinity for sucrose compared to enzymes from other sources.

Effect of Temperature on Production Intracellular and Extracellular Invertase by Potential Indigenous Strain Kluyveromyces Marxianus Using Sugarcane Molasses

This study is focused on enzymolgy regarding enzyme purification technique and application of thermotolerant specie Kluyveromyces marxianus yeast in bio reactions for the research and optimization of fermentation temperature for intracellular and extracellular enzyme production. Fermentation studies were carried out in shake flask level to optimize intracellular and extracellular enzyme production by changing process conditions like temperature range from (30 to 55 0 C), the pH (5.5), speed (350). The substrate type sugar cane molasses 15% added as a carbon and ammonium sulphate (0.75%) as nitrogen source. The optimized fermentation temperature was found 45 o C, at pH 5.5, and rpm was 350. The production of intracellular invertase was (890µmoles/min/g) while the extracellular (120µmoles/min/g) Kluyveromyces marxianus was greater efficiency as compared to other specie because of its metabolic activity, which express more heat stability and Invertase activty upto 65 0 C. Keywords: Yeast, Fermentation, K.marxianus DOI : 10.7176/JNSR/9-9-02 Publication date :May 31 st 2019

Genome-Wide Survey of Invertase Encoding Genes and Functional Characterization of an Extracellular Fungal Pathogen-Responsive Invertase in Glycine max.

Invertases are essential enzymes that irreversibly catalyze the cleavage of sucrose into glucose and fructose. Cell wall invertase (CWI) and vacuolar invertase (VI) are glycosylated proteins and exert fundamental roles in plant growth as well as in response to environmental cues. As yet, comprehensive insight into invertase encoding genes are lacking in Glycine max. In the present study, the systematic survey of gene structures, coding regions, regulatory elements, conserved motifs, and phylogenies resulted in the identification of thirty–two putative invertase genes in soybean genome. Concomitantly, impacts on gene expression, enzyme activities, proteins, and soluble sugar accumulation were explored in specific tissues upon stress perturbation. In combination with the observation of subcellular compartmentation of the fluorescent fusion protein that indeed exported to apoplast, heterologous expression, and purification in using Pichia pastoris system revealed that GmCWI4 was a typical extracellular invertase. We postulated that GmCWI4 may play regulatory roles and be involved in pathogenic fungi defense. The experimental evaluation of physiological significance via phenotypic analysis of mutants under stress exposure has been initiated. Moreover, our paper provides theoretical basis for elucidating molecular mechanisms of invertase in association with inhibitors underlying the stress regime, and will contribute to the improvement of plant performance to a diverse range of stressors.

RESEARCH OF INVERTASE ACTIVITY WHEN CHANGING THE PARAMETERS OF THE FERMENTATION PROCESS SUGAR-MINERAL MEDIUM AND HYDROLYSATE OF STARCH BY THE MICROMYCETE Aspergillus niger

The studies found that strains of micromycete Aspergillus niger L-4 and B-3, producers of citric acid, can synthesize hydrolytic enzymes with invertase activity with a deep cultivation method on a sugar-mineral medium and medium based on starch hydrolysate. The most preferred regimes for conducting the biotechnological process are: the age of the inoculated mycelium is 24 h, the fermentation temperature for the sugar-mineral medium is32 °C and for starch hydrolysate is29 °C. For the Aspergillus niger strain L-4, the extracellular invertase activity for 120 h of the biotechnological process as a result of the fermentation of the sugar-mineral medium was (0,847 ± 0,068) u/cm 3 of the native solution, and for strain B-3 — (0,966 ± 0,077) u/cm 3 of the native solution, respectively. During fermentation of the medium, based on starch hydrolysate, extracellular invertase activity for strain L-4 was (1,379 ± 0,097) u/cm 3 of native solution, and for strain B-3 — (1,597 ± 0,144) u/cm3 of native solution, respectively. The strains of the fungus Aspergillus niger L-4 and B-3 when cultivated on a sugar-mineral medium and medium, based on starch hydrolysate, have the ability to synthesize enzymes with invertase activity, the invertase activity of strain B-3 at the end of the process higher. The obtained data can be applied in further researches to develop a technology for the production of citric acid and invertase in one biotechnological process.

Metabolic Control of Tobacco Pollination by Sugars and Invertases.

Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrates.

Invertase production and molasses decolourization by cold-adapted filamentous fungus Cladosporium herbarum ER-25 in non-sterile molasses medium

This study was undertaken to remove the coloring compounds of molasses as well as produce extracellular (exo) invertase in sterile and non-sterile molasses medium by using cold-adapted filamentous fungus Cladosporium herbarum ER-25. It was determined that a combination of low culture pH (5.5), temperature (20°C) and high molasses concentration (6%) could completely prevent undesired bacterial contamination during the cultivation of C. herbarum ER-25. Under the optimized non-sterile culture conditions, the maximum invertase activity (36.1U/mL) was attained after 72h. On the other hand, the fungus could remove toxical dark brown pigments (melanoidins) in non-sterilized molasses medium through biodegradation and bioadsorption mechanisms. A color removal rate of 64.8% in non-sterile medium could be achieved at the end of 144-h cultivation period. It was found that laccase and manganese peroxidase were responsible for biodegradation. No ligninase activity was detected for the fungus during the cultivation. Maximum laccase (4.6U/mL) and manganese peroxidase (3.5U/mL) activities could be reached after 120h. Higher invertase activity and color removal rate were achieved in non-sterilized medium compared to sterilized one. This is the first report on invertase production from cold-adapted microorganisms under non-sterile culture conditions. As an additional contribution, use of cold-adapted fungi for molasses decolourization was investigated for the first time in the present study.

In Silico Analysis of the Structure of Fungal Fructooligosaccharides-Synthesizing Enzymes

Fructooligosaccharides (FOS) are prebiotics commonly manufactured using fungal fructosyltransferases (FTases) or β-fructofuranosidases. Several reports have attempted to optimize FOS production by changing operational conditions. Nevertheless, there is a lack of information related to the molecular enzyme-substrate interaction. In this study, we present an in silico evaluation of the interactions between substrates (i.e., glucose, sucrose, GF2, GF3, and GF4) and native FOS-synthesizing enzymes from fungi, with reported FOS production yield. In addition, a molecular dynamic simulation was conducted to assess the stability of these interactions. Six fungal enzymes with reported data of FOS production were selected: sucrose-sucrose 1-fructosyltransferase from A. foetidus (GenBank No. CAA04131); intracellular invertase from A. niger (GenBank No. ABB59679); extracellular invertase from A. niger (GenBank No. ABB59678); β-fructofuranidase from A. japonicus ATCC 20611 (GenBank No. BAB67771); fructosyltransferase from A. oryzae N74 (GenBank No. ACZ48670); and fructosyltransferase from A. japonicus (PDB ID 3LF7). These enzymes shared an identity between 15 and 96%, but have a highly conserved folding, and the characteristic FTases domains. Docking results showed that these enzymes also share a similar protein-ligand interaction profile. It was observed that the production yield of total FOS correlated with the sum of affinity energies for GF2, GF3, and GF4. Finally, we present the first molecular dynamic simulation for FOS and fungal FOS-synthesizing enzymes, showing that the protein-ligand interaction does not induce significant changes on the enzyme stability. Overall, these results represent valuable information to continue understanding the FOS synthesis process by fungal FOS-synthesizing enzymes, and they can have a significant impact toward the improvement in their catalytic properties and the synthesis of specific FOS.

Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter.

Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform.

L-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei.

BackgroundTrichoderma reesei is the main producer of lignocellulolytic enzymes that are required for plant biomass hydrolysis in the biorefinery industry. Although the molecular toolbox for T. reesei is already well developed, repressible promoters for strain engineering and functional genomics studies are still lacking. One such promoter that is widely employed for yeasts is that of the l-methionine repressible MET3 gene, encoding ATP sulphurylase.ResultsWe show that the MET3 system can only be applied for T. reesei when the cellulase inducing carbon source lactose is used but not when wheat straw, a relevant lignocellulosic substrate for enzyme production, is employed. We therefore performed a transcriptomic screen for genes that are l-methionine repressible in a wheat straw culture. This analysis retrieved 50 differentially regulated genes of which 33 were downregulated. Among these, genes encoding transport proteins as well as iron containing DszA like monooxygenases and TauD like dioxygenases were strongly overrepresented. We show that the promoter region of one of these dioxygenases can be used for the strongly repressible expression of the Aspergillus niger sucA encoded extracellular invertase in T. reesei wheat straw cultures. This system is also portable to other carbon sources including d-glucose and glycerol as demonstrated by the repressible expression of the Escherichia coli lacZ encoded ß-galactosidase in T. reesei.ConclusionWe describe a novel, versatile set of promoters for T. reesei that can be used to drive recombinant gene expression in wheat straw cultures at different expression strengths and in an l-methionine repressible manner. The dioxygenase promoter that we studied in detail is furthermore compatible with different carbon sources and therefore applicable for manipulating protein production as well as functional genomics with T. reesei.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0308-3) contains supplementary material, which is available to authorized users.

Production and Characterization of a Crude Thermostable Invertase from Cryptococcus laurentii S23

A yeast strain isolated from soil and identified as Cryptococcus laurentii was found capable of producing thermostable extracellular invertase. The extracellular enzyme was thermostable, and the optimum temperature, pH and time for enzyme-substrate reaction were found to be 60°C, 5.5 and 5 min, respectively. Mn2+ stimulated the activity of enzyme significantly while Co2+, Ca2+, Cu2+ and Fe3+ showed little stimulatory effects. Cetyltrimethylammonium bromide showed stimulatory and ethylenediaminetetraacetic acid showed inhibitory effects on enzyme activity. Enzyme was found to be thermostable up to 60°C at pH 5.5. The maximum invertase production (35.32 IU ml−1) was achieved at 30°C, 120 rpm and pH 4.5 on 48 h using 1.0% cell suspension (24 h old) as inoculum in 50 ml medium in 250 ml Erlenmeyer flask. The superior carbon and nitrogen sources for the production of invertase were found to be sucrose (2.0%), peptone (nitrogen level 1 mg/ml). The Km and Vmax were 2.9 mg ml−1 and 39.7 mg min−1 ml−1, respectively. The maximum enzyme activity obtained was 44.72.

Efficient maltotriose fermentation through hydrolysis mediated by the intracellular invertase of Saccharomyces cerevisiae

Background It is well known that in the yeast S. cerevisiae the sugars sucrose and maltose/maltotriose are metabolized by different pathways: sucrose is hydrolyzed by extracellular invertase (encoded by SUC genes), while maltose and maltotriose are actively transported into the cell and hydrolyzed by intracellular a-glucosidases (both proteins encoded by the MAL genes). Nevertheless, several reports have shown that some SUC genes can be located proximal to MAL genes at the telomeres of different chromosomes. Furthermore, the SUC genes also allow the synthesis of an intracellular form of invertase, an enzyme with no obvious function in yeasts [1]. We have already shown that sucrose can be metabolized by yeast cells through MAL-encoded transporters and a-glucosidases. Methods, results and conclusions Now, our results will show that maltotriose can be efficiently fermented by S. cerevisiae cells through its active transport mediated by the AGT1 permease, a MAL transporter required for maltotriose utilization [2,3], and its intracellular hydrolysis mediated by the cytoplasmic invertase. The Brazilian industrial fuel-ethanol strain CAT-1 cannot ferment maltotriose efficiently due to a defective promoter of the AGT1 gene [4]. To increase maltotriose fermentation by this strain, we placed a strong promoter (PGPD )i n theAGT1 gene of strain CAT-1, generating strain GMY05. While the AGT1 gene was indeed overexpressed in this strain (measured by real-time PCR and a specific transport assay), maltotriose was still not fermented efficiently. However, when we over-expressed the intracellular form of invertase, by replacing the signal sequence of the SUC2 gene with the strong PPGK promoter, the resulting iSUC2 strain GMY08 fermented maltotriose efficiently. Using conditions were the MAL-encoded a-glucosidases would not be expressed, we could show that the intracellular form of invertase hydrolyzes maltotriose efficiently (but not maltose or p-nitrophenyl-a-glucoside), specially at the cytoplasmic pH of 7.0. Under the same conditions we purified the intracellular invertase by ion-exchange chromatography, and the identity of the enzyme confirmed by mass spectrophotometry. With the purified enzyme we performed enzymatic tests that corroborated our previous analysis, showing that intracellular invertase hydrolyzes maltotriose. Thus, our results indicate an unexpected overlap in sucrose-maltotriose metabolism by yeast cells, showing that the intracellular invertase allows efficient maltotriose hydrolysis, and offers new approaches that can be applied to optimize several industrial fermentation processes that use starch hydrolysates, including production of distilled beverages, brewing and backing.

Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization.

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml(-1)) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5-6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5-6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l(-1) sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l(-1) sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μmax from 0.035 to 0.070 h(-1) and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.

Season and Nitrogen Effects on Activities of Three Hydrolytic Enzymes in Soils of the Gurbantunggut Desert, Northwest China

The activities of three extracellular hydrolytic enzymes, soil invertase, urease, and alkaline phosphatase (AlP), were measured across seasons and with the experimental addition of nitrogen (N) in the soil of the Gurbantunggut Desert, Northwest China. Seasonal fluctuations in hydrolytic enzyme activities were not correlated with seasonal variations in soil temperature, water content, pH, conductance, and organic carbon. Invertase and AlP activities increased with low rates of N addition, peaked at a N addition rate of 3.0 g N m−2 y−1, and then decreased at higher N addition rates. Urease activity decreased with increasing N addition. Higher organic matter content in the upper depths of soil resulted in higher hydrolytic enzyme activity at depths of 0–5 cm in soil samples and hydrolytic enzyme activity at that depth was more sensitive to N addition and seasonal environmental factors than that at depths of 5–10 cm in soil samples.

Molecular and Functional Characterization of an Invertase Secreted by Ashbya gossypii

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-μm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.

Kinetics and Thermodynamics of Ethanol Production by Saccharomyces cerevisiae MLD10 Using Molasses

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48 ± 0.02 g g(-1)), theoretical yield (93 ± 3%), and extracellular invertase productivity (1,430 ± 50 IU l(-1) h(-1)), respectively, when fermenting 180 g sugars l(-1) in molasses medium at 43 °C in 300 m(3) working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M(-1)) and entropy (ΔS*) (-202.88 J M(-1) K(-1)) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l(-1) h(-1)) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.