Disruption of the acid protease gene in Saccharomycopsis fibuligera A11 enhances amylolytic activity and stability as well as trehalose accumulation

The acid protease gene in Saccharomycopsis fibuligera A11 was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into ORF (Open Reading Frame) of the acid protease gene. The mutant 12 obtained could grow in the medium containing hygromycin. No clear zone formed by the mutant grown on the plate containing milk protein was observed whereas a big clear zone formed by the strain A11 was detected. The acid protease and amylases activities produced by the mutant within 3 days were 0.89 U/ml and 424.7 U/ml, respectively while those produced by the strain A11 were 13.5 U/ml and 259.9 U/ml, respectively. The amylases preparations produced by the mutant 12 and the strain A11 kept 88.8% and 45.5% of amylase activity, respectively after they were incubated at 37 °C for two days. Trehalose amount accumulated in the mutant cells was 28.3% (w/w) while that accumulated in the cells of S. fibuligera A11 was 23.6 (w/w).     

Trehalose accumulation from cassava starch and release by a highly thermosensitive and permeable mutant of <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis>

Highly thermosensitive and permeable mutants are the mutants from which intracellular contents can be released when they are incubated both in low osmolarity water and at non-permissive temperature (usually 37°C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named A11-b was obtained from Saccharomycopsis fibuligera A11-12, a trehalose overproducer in which the acid protease gene has been disrupted. Of the total trehalose, 73.8% was released from the mutant cells suspended in distilled water after they had been treated at 37°C overnight. However, only 10.0% of the total trehalose was released from the cells of S. fibuligera A11-12 treated under the same conditions. The cell volume of the mutant cells suspended in distilled water and treated at 37°C overnight was much bigger than that of S. fibuligera A11-12 treated under the same conditions. The cell growth and trehalose accumulation of the mutant were almost the same as those of S. fibuligera A11-12 during the cultivation at the flask level and in a 5-l fermentor. Both could accumulate around 28.0% (w/w) trehalose from cassava starch. After purification, the trehalose crystal from the aqueous extract of the mutant was obtained.     

Production of cellulosic ethanol in Saccharomyces cerevisiae heterologous expressing Clostridium thermocellum endoglucanase and Saccharomycopsis fibuligera β-glucosidase genes

Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and β-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an α-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and β-glucosidase was able to produce ethanol from β-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst.     

The heterologous expression potential of an acid-tolerant <Emphasis Type="Italic">Talaromyces pinophilus</Emphasis> β-glucosidase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A filamentous fungus displaying high cellulase activity was isolated from a compost heap with triticale (a wheat-rye hybrid) as the main constituent. It was preliminarily identified as a Talaromyces pinophilus species. A 2577 base pair β-glucosidase gene was cloned from complementary DNA and heterologously expressed in Saccharomyces cerevisiae. The recombinant β-glucosidase production profile was assessed and compared to that of the Saccharomycopsis fibuligera β-glucosidase which served as a benchmark. The enzyme was also characterised in terms of pH and temperature tolerance as well as response to inhibitors. Maximal extracellular β-glucosidase activity of 0.56 nkat/mg total protein was measured using p-nitrophenyl-β-D-glucopyranoside as substrate. The recombinant protein displayed a pH optimum of 4.0, and good thermostability as 70% of maximal enzyme activity was retained after 1 h at 60 °C. Activity of the recombinant β-glucosidase was adversely affected by the presence of glucose and ethanol at higher concentrations while xylose had no effect. The expression of the T. pinophilus β-glucosidase did not reach the same titres as for the benchmark; however, in the context of constructing a yeast strain for bioethanol production in a consolidated bioprocess, the enzyme may still show good potential.     

Direct fermentation of cassava starch to ethanol by mixed cultures of Endomycopsis fibuligera and Zymomonas mobilis: Synergism and limitations

Summary A mixed culture of Endomycopsis fibuligera NRRL 76 and Zymomonas mobilis ZM4 could directly and more efficiently ferment cassava starch (22.5% w/v) to ethanol (10.5% v/v) than the monocultures. The combination of culture filtrate of E.fibuligera containing amylases and Z.mobilis simultaneously saccharified and fermented the cassava starch to ethanol equally well. Glucoamylase (0.01%) added to the fermenting medium improved ethanol (13.2% v/v) production by the above mixed culture to almost the theoretical level (98%) indicating that this enzyme is a rate-limiting factor in E.fibuligera. Z. mobilis alone converted the enzymehydrolyzed starch only to almost theoretical level (98%).     

Grimelysin, a novel metalloprotease from Serratia grimesii, is similar to ECP32

Limited actin proteolysis is the hallmark of bacterial metalloprotease ECP32. While ECP32 has long been considered an Escherichia coli protein, the N-terminal amino acid sequence of the active enzyme described previously, could not been retrieved in the E. coli genome. We cloned, sequenced and characterized Serratia grimesii protease grimelysin and show that grimelysin is similar to the previously described protease ECP32.     

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (μ_max) could reach 0.03 and 0.05 h⁻¹ under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobiose L⁻¹ and produced 2.3 g ethanol L⁻¹ in 48 h, while S. cerevisiae secreting β-glucosidase into culture broth used 3.6 g cellobiose L⁻¹ and produced 1.5 g ethanol L⁻¹ over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11 h⁻¹) on cellobiose.     

Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalose purification

In this study, corn starch was used as the substrate for cell growth and trehalose accumulation by Saccharomycopsis fibuligera A11. Effect of different aeration rates, agitation speeds, and concentrations of corn starch on direct conversion of corn starch to trehalose by S. fibuligera A11 were examined using a Biostat B2 2-l fermentor. We found that the optimal conditions for direct conversion of corn starch to trehalose by this yeast strain were that agitation speed was 200 rpm, aeration rate was 4.0 l/min, concentration of corn starch was 2.0% (w/v), initial pH was 5.5, fermentation temperature was 30°C. Under these conditions, over 22.9 g of trehalose per 100 g of cell dry weight was accumulated in the yeast cells, cell mass was 15.2 g/l of the fermentation medium, 0.12% (w/v) of reducing sugar, and 0.21% (w/v) of total sugar were left in the fermented medium within 48 h of the fermentation. It was found that trehalose in the yeast cells could be efficiently extracted by the hot distilled water (80°C). After isolation and purification, the crystal trehalose was obtained from the extract of the cells.     

The nucleotide sequence of the β-glucosidase gene from Cellvibrio gilvus

A gene encoding β-glucosidase from Cellvibrio gilvus, a cellobiose-producing bacterium, was cloned into Escherichia coli and sequenced. The structural gene consisted of 2565 bp encoding 854 amino acid residues with a characteristic signal peptide. A typical promoter sequence and SD region were located upstream of the initiation ATG codon. A sequence (180 amino acids) having high homology with those of β-glucosidases from several microorganisms was found in the deduced amino acid sequence of C. gilvus β-glucosidase. This sequence contains the aspartic acid residue which was found to be an active site residue in Aspergillus wentii β-glucosidase A3. The β-glucosidase gene of C. gilvus contains a high amount (69.4%) of G+C. These bases are localized not in the 3rd position of the codon, as is usually observed in G+C-rich genes, but rather in the 1st position. This result in a peptide which contains an extremely high amount (48%) of four amino acids (Pro, Ala, Arg, Gly) coded by CCN, GCN, CGN, and GGN.     

Molecular and biochemical characterization of a cyanogenic β-glucosidase in the inner bark tissues of rubber tree (Hevea brasiliensis Muell. Arg.)

Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67 kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67 kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67 kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584 bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of β-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both β-glucosidase and linamarase and was thus characterized as a cyanogenic β-glucosidase. Proteins immuno-related to the 67 kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67 kDa protein is a vacuole-localized cyanogenic β-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.     

Properties of trehalose-6-phosphate synthase fromSaccharomycopsis fibuligera

In this paper, we investigated the properties of trehalose-6- phosphate synthase (SfTps1) inSaccharomycopsis fibuligera sdu a high-trehalose-accumulating strain. The purified SfTps1 showed a band on Native-PAGE and SDS-PAGE of about 66 kDa. The optimal pH and temperature of the purified enzyme were 6.6 and 37 °C, respectively. The enzyme was activated by Ca²⁺, K⁺ and Mg²⁺, inhibited by Mn²⁺, Cu²⁺, Fe³⁺, Hg²⁺ and Co²⁺. Iodoacetic acid, EDTA and PMSF had inhibitory effect on the enzyme activity. K_m values of the enzyme for glucose-6-phosphate and UDP-glucose were 38.6 mM and 9.3 mM, respectively. The effects of various stress conditions on SfTps1 activity and trehalose content in this strain were also studied. Neither the activation of SfTps1 nor the change in trehalose content was observed under stress exposure ofSaccharomycopsis fibuligera cells. Our results indicate that the SfTps1 protein and trehalose metabolism in response to stress conditions inSaccharomycopsis fibuligera clearly differ from that ofSaccharomyces cerevisiae and most of other eukaryotes.     

Surface display of acid protease on the cells of Yarrowia lipolytica for milk clotting

The acid protease structural gene was amplified from the genomic DNA of Saccharomycopsis fibuligera A11. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in the cells of Yarrowia lipolytica, the cells displaying the acid protease could form clear zone on the plate-containing milk indicating that they had extracellular acid protease activity. The cells displaying the acid protease can be used to effectively clot skimmed milk. The highest clotting milk activity (1,142.9 U/ml) was observed under the conditions of pH 3.0, 40 °C, 20 mM of CaCl₂, and 10% skimmed milk powder. We found that the acid protease displayed on the cells of Y. lipolytica which has generally regarded as safe status could be easily isolated and concentrated compared to the free acid protease. Therefore, the displayed acid protease may have many potential applications in food and cheese industries. This is the first report that the yeast cells displaying the acid protease were used to clot milk.     

Subcellular fractionation of midgut cells of the sunn pest Eurygaster integriceps (Hemiptera: Scutelleridae): Enzyme markers of microvillar and perimicrovillar membranes

The subcellular distributions of six digestive and non-digestive enzymes (α-glucosidase, β-glucosidase, alkaline phosphatase, acid phosphatase, aminopeptidase and lactate dehydrogenase) of Eurygaster integriceps have been studied. The subcellular distributions of acid phosphatase and α-glucosidase are similar and the gradient ultracentrifugation profiles of these two enzymes overlap. Two partially membrane-bound enzymes, alkaline phosphatase and β-glucosidase have similar distributions in differential centrifugation fractions, which are different from that of α-glucosidase. Sucrose gradient ultracentrifugation of membranes from luminal contents showed that β-glucosidase carrying membranes are heavier. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the profile of proteins extracted from β-glucosidase carrying membranes is different from that of α-glucosidase carrying membranes. We conclude that β-glucosidase and aminopeptidase are markers of microvillar membrane (MM) and perimicrovillar space, respectively, while α-glucosidase and acid phosphatase are perimicrovillar markers. In E. integriceps V1 luminal content is a rich source of PMM and MM and that is used to resolve these membranes.     

A novel high molecular weight thermo-acidoactive β-glucosidase from <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

An extracellular high molecular weight β-glucosidase was secreted by a local strain P1 of Beauveria bassiana. The enzyme was produced in the presence of various carbon sources, namely glucose, maltose, lactose, glycerol, starch, wheat bran and gruel. The highest level of β-glucosidase activity was produced with wheat bran at the concentration of 3%. Glucose caused a repressor effect on the β-glucosidase expression in a dose-dependent manner. The highest enzyme production level was obtained at initial pH of 6.0 and 7.0 in the culture medium. The zymography analysis revealed that B. bassiana secreted a β-glucosidase with high molecular weight between 400 and 600 kDa. The enzymatic preparation was characterized and showed temperature and pH optima of 55°C and 5.0, respectively. The enzyme was stable at 40 and 50°C but its stability declined at 60°C. Interestingly, this β-glucosidase had high stability at acid and basic pH saving its initial activity after 24 h incubation at pH from 3.0 to 11.0. It was stable also in presence of monovalent Na⁺ and K⁺ ions saving 60% of its initial activity at 2 M salts. Bivalent metal ions preserved totally or partially the enzymatic activity; in addition, Ba²⁺ was revealed as an activator. This is the first report that focuses on the production and the biochemical characterization of a β-glucosidase from the entomopathogenic fungus, B. bassiana.     

Cellulase genes of Cellulomonas uda CB4. I. Cloning and expression of β-glucosidase genes in Escherichia coli

Two β-glucosidase genes in Cellulomonas uda CB4 were cloned in Escherichia coli with pAT325 constructed from pAT153 and pBR325. Plasmids pCC1 and pCG1 were isolated from the transformants producing β-glucosidase, and the β-glucosidase genes cloned were in 6.1 and 8.1 kb BamHI fragments, respectively. The amount of β-glucosidase expressed in E. coli harboring pCCl and pCGI was 1.2 and 4.0 times that in the present strain. E. coli harboring pCCl grew efficiently on cellobiose.     

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5–6.0 for E. fibuligera and 5.5–7.0 for S. castellii.     

Fermentation of cellobiose to ethanol by industrial Saccharomyces strains carrying the β-glucosidase gene (BGL1) from Saccharomycopsis fibuligera

Constructs carrying the Saccharomycopsis fibuligera β-glucosidase gene (BGL1) under the control of a constitutive actin or a galactose-inducible promoter were introduced into eleven Saccharomyces strains. In ten of these recombinant strains, BGL1 expression driven by the actin promoter was between 1.6- and 18-fold higher than that obtained with the galactose-inducible promoter. Strains carrying the actin promoter yielded ethanol concentrations from cellobiose of between 0.5% and 14%, depending on their ability to accumulate Bgl1 (between 30 and 250 mU/mL) but also on their genetic background. Comparative analysis of a S. cerevisiae strain and its corresponding petite version showed similar ethanol yields, despite a 3-fold lower β-glucosidase production of the latter, suggesting that respiratory activity could be one of the factors influencing ethanol production when using carbon sources other than glucose. This study provides a selection of strains that may be good candidates as hosts for ethanol biosynthesis from cellulosic substrates.     

Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases

Cloning of cDNA encoding an α-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative α-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.52. The deduced amino acid sequence shows a high degree of identity (70%) with two yeast glucoamylases, namely, the extracellular glucoamylase Gam from Schwanniomyces occidentalis and the cell surface glucoamylase Gca from Candida albicans. The recombinant product, synthesized in Saccharomyces cerevisiae, is localized on the cell surface and hydrolyses maltooligosaccharides exclusively without the ability to digest soluble starch, which is consistent with the specificity characteristic of α-glucosidase, EC. 3.2.1.20.     

Purification and biochemical characterization of a novel α-glucosidase from Aspergillus niveus

An extracellular α-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS–PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical α-glucosidase activity, hydrolyzing p-nitrophenyl α-d-glucopyranoside and presented an optimum temperature and pH of 65°C and 6.0, respectively. In the absence of substrate the purified α-glucosidase was stable for 60 min at 60°C, presenting t ₅₀ of 90 min at 65°C. Hydrolysis of polysaccharide substrates by α-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and β-ciclodextrin were poor substrates, and sucrose and α-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an α-helical content of 31% and a β-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.     

Overexpression of acid protease of <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and characterization of the recombinant acid protease for skimmed milk clotting

The gene encoding an acid protease natively produced by Saccharomycopsis fibuligera was cloned and overexpressed in Yarrowia lipolytica and the resultant recombinant acid protease was purified and characterized. The molecular mass of the purified enzyme was estimated as 94.8 kDa by gel filtration chromatography. The optimal pH and temperature of the purified acid protease were 3.5 and 33°C, respectively, and the enzyme was very stable over a pH range of 1.0 ∼ 3.0. The recombinant acid protease was activated by Zn²⁺, but was inhibited by Hg²⁺, Fe²⁺, Fe³⁺, and Mg²⁺, EDTA, EGTA, iodoacetic acid, and pepstatin. The purified recombinant acid protease from the positive transformant 71 had high milk clotting activity, suggesting that it may be used as a rennet substitute in the cheese industry.