Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

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Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

 First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1 _P ) and terminator (PGK1 _T ) sequences on a multicopy episomal plasmid. The resulting construct PGK1 _P -abf B-PGK1 _T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively. Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996     

Molecular Cloning of the Phytase Gene from Citrobacter braakii and its Expression in Saccharomyces cerevisiae

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppA_Ec and rAppA_Sc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppA_Sc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppA_Ec.     

Cloning and Characterization of the nagA Gene that Encodes β-N-Acetylglucosaminidase from Aspergillus nidulans and Its Expression in Aspergillus oryzae

We isolated a β-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed β-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of β-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of β-N-acetylglucosaminidase in a wheat bran solid culture.     

Expression and Secretion of Bovine β-Lactoglobulin in Saccharomyces cerevisiae

A bovine β-lactoglobulin (β-LG) was expressed in Saccharomyces cerevisiae carrying bovine pre-β-LG cDNA and secreted into its growth medium. The expression plasmid was constructed by inserting the whole coding region of the cDNA encoding pre-β-LG between the promoter and terminator of the yeast glyceraldehyde 3-phosphate dehydrogenase gene of pYG100, a yeast expression vector. In the supernatant of the yeast growth medium, β-LG with a native conformation was detected by sandwich ELISA, and its amount was estimated to be 1.1 mg/l. A Western-immunoblotting analysis revealed that β-LG secrected in the growth medium had the same mobility as that of authentic bovine β-LG. The N-terminal sequence was also identical with that of authentic mature bovine β-LG.     

Expression and secretion of barley α-amylase and A.niger glucoamylase in Saccharomyces cerevisiae

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Characterization of Active Barley α-Amylase 1 Expressed and Secreted by Saccharomyces cerevisiae

Recombinant barley α-amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified α-amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k_cat/K_m was 2.7 × 10² mM^?1.s^?1, consistent with those of α-amylases from plants and other sources.     

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 and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum

The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.α-Galactosidase (αGal) (EC 3.2.1.22) is of particular interest in view of its biotechnological applications. αGal from coffee beans demonstrates a relatively broad substrate specificity, cleaving a variety of terminal α-galactosyl residues, including blood group B antigens on the erythrocyte surface. Treatment of type B erythrocytes with coffee bean αGal results in specific removal of the terminal α-galactosyl residues, thus generating serological type O erythrocytes (8). Cyamopsis tetragonoloba (guar) αGal effectively liberates the α-galactosyl residue of galactomannan. Removal of a quantitative proportion of galactose moieties from guar gum by αGal improves the gelling properties of the polysaccharide and makes them comparable to those of locust bean gum (18). In the sugar beet industry, αGal has been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar (28). Raffinose and stachyose in beans are known to cause flatulence. αGal has the potential to alleviate these symptoms, for instance, in the treatment of soybean milk (16).αGals are also known to occur widely in microorganisms, plants, and animals, and some of them have been purified and characterized (5). Dey et al. showed that αGals are classified into two groups based on their substrate specificity. One group is specific for low-M_r α-galactosides such as pNPGal (p-nitrophenyl-α-d-galactopyranoside), melibiose, and the raffinose family of oligosaccharides. The other group of αGals acts on galactomannans and also hydrolyzes low-M_r substrates to various extents (6).We have studied the substrate specificity of αGals by using galactomanno-oligosaccharides such as Gal³Man₃ (6³-mono-α-d-galactopyranosyl-β-1,4-mannotriose) and Gal³Man₄ (6³-mono-α-d-galactopyranosyl-β-1,4-mannotetraose). The structures of these galactomanno-oligosaccharides are shown in Fig. ​Fig.1.1. Mortierella vinacea αGal I (11) and yeast αGals (29) are specific for the Gal³Man₃ having an α-galactosyl residue (designated the terminal α-galactosyl residue) attached to the O-6 position of the nonreducing end mannose of β-1,4-mannotriose. On the other hand, Aspergillus niger 5-16 αGal (12) and Penicillium purpurogenum αGal (25) show a preference for the Gal³Man₄ having an α-galactosyl residue (designated the stubbed α-galactosyl residue) attached to the O-6 position of the third mannose from the reducing end of β-1,4-mannotetraose. The M. vinacea αGal II (26) acts on both substrates to almost equal extents. The difference in specificity may be ascribed to the tertiary structures of these enzymes. Open in a separate windowFIG. 1Structures of galactomanno-oligosaccharides.Genes encoding αGals have been cloned from various sources, including humans (3), plants (20, 32), yeasts (27), filamentous fungi (4, 17, 24, 26), and bacteria (1, 2, 15). αGals from eukaryotes show a considerable degree of similarity and are grouped into family 27 (10).Here we describe the cloning of P. purpurogenum αGal cDNA, its expression in Saccharomyces cerevisiae, and the purification and characterization of the recombinant enzyme.     

Molecular Cloning and Expression of Proteinaceous α-Amylase Inhibitor Gene from Streptomyces nitrosporeus in Escherichia coli

The proteinaceous α-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous α-amylase inhibitors.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.     

Chemical Modification of Amino Groups of Acid-stable α-Amylase and Acid-unstable α-Amylase from Aspergillus niger

Monochlorotrifluoro-p-benzoquinone (CFQ) was used for investigating the state of the amino groups of acid-stable α-amylase and acid-unstable α-amylase. About half of the total amino groups in both enzyme molecules were reacted with the reagent. The unreactive amino groups seemed to exist in a different state from the reactive ones. Both enzymes whose amino groups were modified by CFQ still maintained the α-phenylmaltosidase activity in spite of losing or decreasing the amylase activity. These facts suggest that the amino groups of both enzymes were not in the active site but the modification of them caused steric hindrance.

The pH-stability of the acid-unstable α-amylase whose one or two amino groups were modified with succinic anhydride or 2,4,6-trinitrobenzene-l-sulfonate (TNBS) increased on the acidic side and decreased on the alkaline side, but further modification of them led to decrease the stability on both sides.     

Cell Bound α-Amylase in Aspergillus oryzae

It was previously reported that α-amylase accumulation is caused within the mycelium grown in a phosphate deficient medium and the concentration of anions or pH in a surrounding medium is responsible for its liberation. As it was subsequently found that α-amylase liberation from the mycelium of Aspergillus oryzae is stimulated by peptone, an attempt was made on purification of effective substances from it. The present paper describes on purification and properties of phosphopeptides found as an effective substance for α-amylase liberation, and discusses on the stimulation effect, comparing with the effects on pH and concentration of anions which were previously observed.