Characterization of thermostable α-glucosidases from newly isolated <Emphasis Type="Italic">Geobacillus</Emphasis> sp. A333 and thermophilic bacterium A343

We have partially purified and characterized two new thermostable exo-α-1,4-glucosidases (E.C.3.2.1.20) isolated from Geobacillus sp. A333 and thermophilic bacterium A343 strains. A333 α-glucosidase showed optimum activity at 60°C, pH 6.8 and had a value of 1.38 K _m for the pNPG substrate, whereas these results were found to be 65°C, 7.0 and 0.85, respectively for A343 enzyme. Specificity for 20 different substrates and thin layer chromatography studies demonstrated that the A333 enzyme had high transglycosylation activity, and A343 had wide substrate specificity. The substrate specificity of A333 α-glucosidase was determined as maltose, dextrin, turanose, maltotriose, maltopentaose, meltotetraose, maltohexaose and phenyl-α-d-glycopyranoside. On the other hand, the A343 α-glucosidase mostly hydrolyzed dextrin, turanose, maltose, phenyl-α-d-glucopyranoside, maltotriose, maltotetraose, maltopentaose, isomaltose, saccharose and kojibiose by acting α-1,2, α-1,3, α-1,4 and α-1,6 bonds of these substrates. The relative activites of A333 and A343 enzymes were determined to be 83 and 92% when incubated at 60°C for 5 h whereas, the pH of 50% inactivation at 60°C for 15 h were determined to be pH 4.5/10.0 and pH 5.0/10.0, respectively. In addition, the results not only showed that both of the α-glucosidases were stable in a wide range of pH and temperatures, but were also found to be resistant to most of the denaturing agents, inhibitors and metal ions tested. With this study, thermostable exo-α-1,4-glucosidases produced by two new thermophilic strains were characterized as having biotechnological potential in transglycosylation reactions and starch hydrolysis processes.     

Expression and characterization of an <Emphasis Type="Italic">α</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> JW200 with potential for industrial application

In this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K _m and V _max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).     

Cloning,expression, and characterization of an insoluble glucan-producing glucansucrase from <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> NRRL B-1118

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K _M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching.     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Separation and properties of α-mannosidase and mannanase from the basidiomycetePhellinus abietis

Proteins of a crude enzyme preparation obtained from the cultivation medium of the basidiomycetePhellinus abietis were separated by gel filtration and ion-exchange chromatography. The preparation contained a minimum of three enzymes capable of splitting α-d-mannosidic bonds: α-mannosidase, exomannanase, and endomannanase, which were separated. Some properties of the mannanase complex of the crude enzyme preparation, and of a partially purified α-mannosidase were examined. The mannanase complex exhibited two pH optima, its temperature optimum being at 46 °C The pH optimum of purified α-mannosidase was at pH 5.0, the temperature optimum was at 60 °C; the enzyme had a relatively high heat stability. The K_m of α-mannosidase forp-nitrophenyl α-d-mannopyranoside was 1.5 x 10⁻⁵ M. Pure α-mannosidase did not split mannan.     

Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the <Emphasis Type="Italic">Anoxybacillus</Emphasis> species that produce high levels of maltose

Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD–Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6–10. At 60°C in the absence of Ca²⁺, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca²⁺ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.     

Purification and characterization of a highly thermostable α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Geobacillus caldoxylolyticus</Emphasis> TK4

The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C), and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity with p-nitrophenyl-α-l-arabinofuranoside, with apparent K _m and V _max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K _m and V _max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.     

Cloning, purification, and characterization of a thermostable alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari

The gene, AbfAC26Sari, encoding an α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a V _max and K _m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu²⁺ and Hg²⁺. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.     

Characterization of thermostable α-glucosidase from Clostridium thermohydrosulfuricum 39E

Summary Clostridium thermohydrosulfuricum 39E produced a cell-bound -glucosidase. It was partially purified 140-fold by solubilizing with Triton X-100, ammonium sulfate treatment, DEAE-Sepharose CL-6B, octyl-Sepharose and acarbose-Sepharose affinity chromatography. The optimum temperature for the action of the enzyme was at 75°C. It had a half-life of 35 min at 75°C, 110 min at 70°C and 46 h at 60°C. The enzyme was stable at pH 5.0–6.0 and had an optimum pH at 5.0–5.5. It hydrolyzed the -1,4-linkages in maltose, maltotriose, maltotetraose and maltohexaose, the rate decreasing in order of higher-sized oligosaccharides. The enzyme preparation also hydrolyzed the -1,6 linkages in isomaltose and isomaltotriose. It rapidly hydrolyzed p-nitrophenyl -d-glucoside (pNPG). The K _m values for maltose, isomaltose, panose, maltotriose, and pNPG were 1.85, 2.95, 1.72, 0.58, and 0.31 mm, respectively, at pH 5.5 and 60°C. The enzyme produced glucose from all these substrates. The enzyme preparation did not require any metal ion for activity. The -glucosidase activity was inhibited by acarbose. Offprint requests to: B. C. Saha     

Expression,purification and characterization of recombinant α-glucosidase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An expression plasmid containing the agdA gene encoding Aspergillus oryzae ZL-1 α-glucosidase was constructed and expressed in Pichia pastoris X-33. The molar mass of the purified protein was estimated by SDS-PAGE. HPLC analysis showed that the purified enzyme has a transglucosylating activity with maltose as substrate. The main component of the enzyme products was panose, while amounts of isomaltose and isomaltotriose were very low or absent. pH 5.2 and temperature of 37 °C were optimum for enzyme activity.     

Zhihengliuella salsuginis sp. nov., a moderately halophilic actinobacterium from a subterranean brine

A novel moderately halophilic, alkaliphilic, non-motile, non-sporulating, catalase-positive, oxidase-negative, aerobic, coccus-shaped, Gram-positive bacterium, designated strain JSM 071043^T, was isolated from a subterranean brine sample collected from a salt mine in Hunan Province, China. Growth occurred with 0.5–20% (w/v) NaCl (optimum 5–10%) at pH 6.5–10.5 (optimum pH 8.5) and at 10–40°C (optimum 25–30°C). Good growth also occurred in the presence of 0.5–20% (w/v) KCl (optimum 5–8%) or 0.5–25% (w/v) MgCl₂·6H₂O (optimum 5–10%). The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu) and major cell-wall sugars were tyvelose and mannose. The major cellular fatty acids were anteiso-C_15:0, iso-C_16:0 and anteiso-C_17:0. Strain JSM 071043^T contained MK-9 and MK-8 as the predominant menaquinones and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The DNA G + C content was 67.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JSM 071043^T was a member of the suborder Micrococcineae, and was most closely related to Zhihengliuella halotolerans YIM 70185^T (sequence similarity 98.9%) and Zhihengliuella alba YIM 90734^T (98.2%), and the three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis, DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 071043^T represents a novel species of the genus Zhihengliuella, for which the name Z. salsuginis sp. nov. is proposed. The type strain is JSM 071043^T (= DSM 21149^T = KCTC 19466^T).     

A thermostable α-galactosidase from Lenzites elegans (Spreng.) ex Pat. MB445947: purification and properties

An α-galactosidase was isolated from a culture filtrate of Lenzites elegans (Spreng.) ex Pat. MB445947 grown on citric pectin as carbon source. It was purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration chromatography and anion-exchange chromatography. The relative molecular mass of the native purified enzyme was 158 kDa determined by gel filtration and it is a homodimer (Mr subunits = 61 kDa). The optimal temperature for enzyme activity was in the range 60–80 °C. This α-galactosidase showed a high thermostability, retaining 94 % of its activity after preincubation at 60 °C for 2 h. The optimal pH for the enzyme was 4.5 and it was stable from pH 3 to 7.5 when the preincubation took place at 60 °C for 2 h. It was active against several α-galactosides such as p-nitrophenyl-α-d-galactopyranoside, α-d-melibiose, raffinose and stachyose. The α-galactosidase is a glycoprotein with 26 % of structural sugars. Galactose was a non-competitive inhibitor with a Ki = 22 mM versus p-nitrophenyl-α-d-galactoside and 12 mM versus α-d-melibiose as substrates. Glucose was a simple competitive inhibitor with a Ki = 10 mM. Cations such as Hg²⁺ and p-chloromercuribenzoate were also inhibitors of this activity, suggesting the presence of –SH groups in the active site of the enzyme. On the basis of the sequence of the N-terminus (SPDTIVLDGTNFALN) the studied α-galactosidase would be a member of glycosyl hydrolase family 36 (GH 36). Given the high optimum temperature and heat stability of L. elegans α-galactosidase, this fungus may become a useful source of α-galactosidase production for multiple applications.     

Purification and characterization of an extracellular alpha-L-arabinosidase from a novel isolate Bacillus pumilus ARA and its over-expression in Escherichia coli

The α-l-arabinosidase, AraB, was induced when Bacillus pumilus ARA was grown at 50°C in a minimal medium containing xylan. A 56-kDa protein with α-l-arabinosidase activity was purified from culture supernatant to gel electrophoretic homogeneity. The optimal activity was at pH 6.4 and 60°C over a 10-min assay. The purified enzyme was stable over a pH range of 5.2–7.6 and had a 1-h half life at 70°C. The enzyme released arabinose from oat spelt xylan. Kinetic experiments at 60°C with p-nitrophenyl α-l-arabinofuranoside as substrate gave a K _m, and V _max of 1.05 mM and 240 U per mg of protein. The NH₂-terminal amino acid sequence of the enzyme was determined, and its gene araB was subsequently cloned, sequenced, and over-expressed in Escherichia coli. The open reading frame of araB consists of a 1,479-bp fragment encoding a protein of 472 amino acids, which belonged to family 51 of the glycoside hydrolases with an identity of 67% to the protein encoded by abfB of Bacillus subtilis 168.     

Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine

A gram-negative, rod-shaped bacterium capable of utilizing l-asparagine as its sole source of carbon and nitrogen was isolated from soil and identified as Enterobacter cloacae. An intracellularly expressed l-asparaginase was detected and it deaminated l-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction mixture indicated that 2.8 mM l-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase activity was found in cells cultured on l-fructose, d-galactose, saccharose, or maltose, and in cells cultured on l-asparagine as the sole nitrogen source. The pH and temperature optimum of l-asparaginase was 8.5 and 37–42 °C, respectively. The half-life of the enzyme at 30 °C and 37 °C was 10 and 8 h, respectively. Received: 19 February 1998 / Received last revision: 4 June 1998 / Accepted: 10 July 1998     

Amylases of the thermophilic fungusThermomyces lanuginosus: Their purification,properties, action on starch and response to heat

A thermophilic fungus Thermomyces lanuginous strain IISc 91, secreted one form each of α-amylase and glucoamylase during growth. Both enzymes were purified to homogeneity by ion-exchange and gel-filtration chromatography and obtained in mg quantities. α-Amylase was considered to be a dimeric protein of ∼ 42 kDa and contained 5% (by mass) carbohydrate. It was maximally active at pH 5.6 and at 65°C. It had an activation energy of 44 kJ mol^-1. The apparent K_m for soluble starch was 2.5 mg ml^-1. The enzyme produced exceptionally high levels of maltose from raw potato starch. At 50°C, the enzyme was stable for > 7h. At 65°C, α-amylase was nearly 8-times more stable in the presence of calcium. Addition of calcium increaed the melting temperature of α-amylase from 66°C to 73°C. Upon incubation at 94°C, α-amylase was progressively and irreversibly inactivated, and converted into an inactive 72 kDa trimeric species. Glucoamylase was a monomeric glycoprotein of ∼ 45 kDa with a carbohydrate content of 11% (by mass). It effected up to 76% conversion of starch in 24 h producing glucose as the sole product. Its apparent K_m for soluble starch was 0.04 mg ml^-1 and V_max was 660 Mmol glucose min^-1 mg protein^-1. It also hydrolyzed maltose. Its activity on maltooligosaccharides increased with the chain length of the substrates. Glucoamylase was stable at 60°C for over 7h. Its activation energy was 61 kJ mol^-1 Glucoamylase did not show synergistic effect with α-amylase. The properties of α-amylase and glucoamylase of Thermomyces lanuginosus strain IISc 91 suggest their usefulness in the commercial production of maltose and glucose syrups.     

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 °C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 °C, with 87% inactivation after 21 h at 30 °C, 45% inactivation after 40 min at 40 °C and 90% inactivation after 10 min at 50 °C. Proteases known to be present in crude preparations had a temperature optimum of 50 °C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 °C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 °C, and the amylase required Ca⁺² for activity. The optimal pH for activity was 5.0–9.0 on soluble starch (30 °C), and the amylase exhibited a K _m with 4-nitrophenyl-α-D-maltoheptaoside-4,6-O-ethylidene of 120 μM at 22 °C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 °C at about the same rate as an equal number of units of barley α-amylase, but lost most of its activity after only a few hours.     

<Emphasis Type="Italic">Yaniella soli</Emphasis> sp. nov., a new actinobacterium isolated from non-saline forest soil in China

A novel Gram-stain-positive, slightly halophilic, facultatively alkaliphilic, non-motile, non-sporulating, catalase-positive, oxidase-negative, aerobic bacterium, designated strain JSM 070026^T, was isolated from non-saline forest soil in China. Growth occurred with 0–20% (w/v) NaCl (optimum, 2–4%) and at pH 6.0–10.5 (optimum, pH 8.0) and 5–40°C (optimum, 30°C). Good growth also occurred in the presence of 0–28% (w/v) KCl (optimum, 2–5%) or 0–25% (w/v) MgCl₂·6H₂O (optimum, 1–4%). The peptidoglycan type was A4α (l-Lys–Gly–l-Glu). Cell-wall sugars contained mannose and xylose. The major cellular fatty acids were anteiso-C15:0 and iso-C15:0. Strain JSM 070026^T contained menaquinone 8 as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The DNA G + C content of strain JSM 070026^T was 56.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JSM 070026^T was a member of the suborder Micrococcineae and most closely related to Yaniella flava YIM 70178^T (sequence similarity 99.4%) and Yaniella halotolerans YIM 70085^T (97.9%). The three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis, DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 070026^T represents a novel species of the genus Yaniella, for which the name Yaniella soli sp. nov. is proposed. The type strain is JSM 070026^T (=DSM 22211^T = KCTC 13527^T).     

Inter organ comparison of amylases and starch content in mungbean seedlings

During seedling growth of mungbean in dark, depletion of cotyledonary starch is reflected by an increase in starch content of root and shoot. With progress of seedling growth, amylolytic activity increases in all organs i.e. cotyledons, shoots and roots. A rapid turnover of starch in shoots and roots has been proposed. Amylase activity of seedlings was in the order of cotyledons>shoots>roots. Five days after germination (DAG) α-amylase from cotyledons of mungbean seedlings was purified using ammonium sulphate precipitation, DEAE cellulose and sephadex G-150 column chromatography. Phytic acid was a stronger inhibitor of α-amylase than EDTA. Phytic acid, Hg²⁺, Zn²⁺ and Mn²⁺ were non-competitive inhibitors and the corresponding Ki values were 5.0–5.7, 0.36–0.38, 2.6–3.8 and 0.7–0.8 mol·M⁻³. Elution patterns of α-amylases of cotyledons, shoots and roots on sephadex G-100 column showed that cotyledonary α-amylase had a higher molecular mass than that of shoot and root α-amylases which had identical molecular masses. All α-amylases showed the same optimum pH 5.0 whereas optimum temperature was 55 °C for cotyledonary and 45 °C for shoot and root α-amylases. In all these tissues α-amylases were stable to 30 min heat treatment at 50 °C however unlike cereal α-amylases they lost activity at 70 °C. Km for α-amylases from cotyledons, shoots and roots with starch was 1.9, 4.3 and 6.6 mg per cm³, respectively. α-amylase of cotyledons and roots showed activity in reactions with various substrates in the order of starch>amylose>dextrin-I=dextrin-IV>α-cyclodextrin=β-cyclodextrin>amylopectin>pullulan. The shoot α-amylase showed high activity with amylopectin, which was comparable with that obtained with amylose, and the activity with α and β-cyclodextrin was higher in comparison with dextrin-I and IV. The α-amylases from these tissues liberated maltose, maltotriose and higher oligosaccharides from starch. It could be concluded that amylases from different organs of a seedling could have different physical and kinetic properties.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.     

Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone—1-butanol—Ethanol solvent byClostridium acetobutylicum

A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch byClostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent,i.e. 1.5–2 times more than with pure maltose or glucose used as carbon sources. Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch. The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation. The pH optima for α-amylase and glucoamylase were found to be at 5.3 and 4.0–4.4, respectively. α-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0–8.0 after a 1-d incubation at 37°C. SinceC. acetobutylicum α-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch. However, theC. acetobutylicum glucoamylase was only stable at pH 4–5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37°C.