Removal of carboxymethyl cellulase activity from the culture filtrate of Termitomyces clypeatus producing xylanase

Termitomyces clypeatus produced 450 IU xylanase ml^–1 in a medium containing starch-free wheat bran powder as the carbon source. Carboxymethyl cellulase (CMCase) activity in the culture filtrate was removed by keeping the filtrate at pH 10 for 60 min followed by a change to pH 6. Treatment of Kraft pulp (bamboo) with the filtrate at pH 7 decreased the kappa number from 10.5 to 5 with release of reducing groups equivalent to 0.15 mg glucose g^–1 pulp.     

Cellobiase from Termitomyces clypeatus: activity and secretion in presence of glycosylation inhibitors

In presence of the glycosylation inhibitors, 2-deoxy-d-glucose (1 mg/ml), tunicamycin (30 μg/ml), 1-deoxynojirimycin (30 μg/ml) and d-glucono-δ-lactone (1 mg/ml), total cellobiase activity, in the extracellular, intracellular and cell bound fractions, of the fungus Termitomyces clypeatus grown in 20 ml cellobiose medium (1%, w/v) increased by 50-, 1.8-, 2.4-, 1.3-fold, respectively, with respect to control medium (16.3 U). The inhibitors also stimulated secretion of 95% of the total protein in culture medium, except d-glucono-δ-lactone which released 60% of the total protein. 2-Deoxy-d-glucose (1 mg/ml) led to production of extracellular cellobiase up to 40 U/ml, whereas in absence of the inhibitors only 0.59 U/ml enzyme was detected.     

Characterization of a novel low molecular weight sucrase from filamentous fungus Termitomyces clypeatus

An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%, w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties, molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) medium in the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 °C and pH 5.0. K_m and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity.     

Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli

An amylase gene was identified in a Streptococcus bovis 033 gtWESB genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.     

Properties of an extracellular amylase purified from a Bacillus species

A strain of Bacillus produced an amylase with properties characteristically different from known bacterial amylases. The purified 80 kDa protein of pI 5.1 dextrinized starch, glycogen and pullulan. The temperature and pH optima of the enzyme were 60 °C and 6.6 respectively. In the presence of 0.05 M CaCl₂, the enzyme retained stability for 15 min at 80 °C. Antibodies raised to the amylase protein showed no reaction with -amylases of Bacillus sp. and B. licheniformis. In culture, proteolytic degradation of the enzyme was observed.     

Raw starch digesting amylase from Thermoactinomyces thalpophilus F13

Thermoactinomyces thalpophilus produced a raw-starch digesting amylase (RSDA) when grown on both cereal and tuber starches. Glucose, maltose and glycerol repressed enzyme production. Highest activity was recorded on rice starch (39 U ml^-1). Considerable variability existed in the effectiveness of nitrogenous nutrients to stimulate expression of RSDA. Multiple pH optima for RSDA production and activity suggests enzyme heterogeneity.     

Expression and characterization of amylase encoded by a gene cloned from Cellulomonas sp. NCIM 2353

A Cellulomonas genomic fragment encoding extracellular amylase activity was isolated as a clone (ACs2) in Escherichia coli DH10B. The amylase was expressed in the absence of IPTG and in the presence of starch or maltose. This enzyme corresponded to the low mobility activity of Cellulomonas amylases as demonstrated on gel electrophoresis. Maltose, as well as lactose, xylose and xylan cross-induced the amylase of clone ACs2. Maltose-induced amylase was purified to homogeneity. ACs2-coded amylase is a 70kDa acidic protein, with a pH optimum of 7.0 at 45°C. This enzyme exhibited an endo mode of action, similar to the corresponding Cellulomonas enzyme.     

Production of amylase by Arthrobacter psychrolactophilus

Arthrobacter psychrolactophilus ATCC 700733 grew with a doubling time of 1.5–2.3 h (22°C) and produced up to 0.2 units/mL (soluble starch assay) of extracellular amylase in tryptic soy broth without dextrose (TSBWD) containing 0.5% or 1.0% (w/v) soluble starch or maltose as the fermentable substrate. Time-course experiments in media containing soluble starch as substrate showed that amylolytic activity appeared in cultures at 24 h (after exponential growth had ceased), reached peak levels in 72–96 h, and declined rapidly after reaching peak levels. Peak levels were highest in TSBWD containing 1.0% soluble starch. Proteolytic activity appeared at about the same time as amylolytic activity and increased during the period of amylase production. Significant amylase production was not observed in cultures in TSBWD with 0.5% glucose or in cultures grown at 28°C, but low levels of amylase were observed in TSBWD cultures grown at 19–23°C which contained no added carbohydrate. A single band of activity was observed after electrophoresis of supernatant fractions in non-denaturing gels, followed by in situ staining for amylolytic activity. The amylase possessed a raw starch-binding domain and bound to uncooked corn, wheat or potato starch granules. It was active in the Phadebas assay for -amylase. Activity was maximum on soluble starch at a temperature between 40°C and 50°C. The amylase after purification by affinity chromatography on raw starch granules exhibited two starch-binding protein bands on SDS gels of 105 kDa and 26 kDa.     

Short communication: Purification and properties of a glucose-forming amylase of Lactobacillus brevis

An extracellular glucose-forming amylase was produced by Lactobacillus brevis isolated from Kagasok tea. The enzyme was purified 70-fold and had optimal activity at 55°C and pH 6.5. Its K _m value for starch was 0.27 mg ml^-1 and its M _r was approx. 75,900 Da. The activity of the enzyme was enhanced by Ca²⁺, Mg²⁺, Na⁺ or K⁺ and inhibited by EDTA, KCN, citric acid and l-cysteine.     

Caldimonas taiwanensis sp. nov., a amylase producing bacterium isolated from a hot spring

During screening for amylase-producing bacteria, a strain designated OnlT was isolated from a hot spring located in Pingtung area, which is near the very southern part of Taiwan. Cells of this organism were Gram-negative rods motile by means of a single polar flagellum. Optimum conditions for growth were 55 degrees C and pH 7. Strain On1(T) grew well in minimal medium containing starch as the sole carbon source, and its extracellular products expressed amylase activity. The 16S rRNA gene sequence analysis indicates that strain On1(T) is a member of beta-Proteobacteria. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA-DNA similarity data, physiological and biochemical characteristics, as well as fatty acid compositions, the organism belonged to the genus Caldimonas and represented a novel species within this genus. The predominant cellular fatty acids of strain On1(T) were 16:0 (about 30%), 18:1 omega 7c (about 20%) and summed feature 3 (16:1 omega 7c or 15:0 iso 2OH or both [about 31%]). Its DNA base ratio was 65.9 mol% G + C. We propose to classify strain On1(T) (= BCRC 17405T = LMG 22827T) as Caldimonas taiwanensis sp. nov.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

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.     

Production and stabilization of amylase preparations from rice bran solid medium

A low-cost amylase preparation of dried fermented bran was developed from rice bran solid cultures of Aspergillus oryzae supplemented with soya bean flour (SBF) and cassava starch (3:1) and dried at 50 °C for 4 h. Storage stability of preparations at 4 °C or 30 °C was significantly enhanced (P 0.05) by adding SBF or partially hydrolyzed starch (PHS). While amylase preparations without stabilizer retained 59 and 48% of their activity after 12 weeks storage at 4 and 30 °C respectively, the same preparations fortified with SBF (5% w/v) retained 95 and 94% stability respectively, during the same period. PHS at 5% (w/v) also gave a maximum stability of 94 and 91.8% at 4 and 30 °C, respectively. The unstabilized preparation retained only 42% of its activity compared to the stabilized forms, which retained 82–90% activity after 15 min incubation at 100 °C.     

Changes in relative mobility of pancreatic amylase variants in isoelectric focusing

Using isoelectric focusing with one ampholytic solution, double- and single-banded amylase phenotypes were found in a sample of rhesus monkeys,Macaca mulatta. When applying different ampholytic solutions, these variants were shown to change their position relative to each other. Single-banded phenotypes showed either a position corresponding to one of the bands of the double-banded phenotype or to an intermediate one. Family studies, however, suggested that the differences between the observed patterns were not caused by genetic differences. This discloses a problem with respect to the interpretation of electrophoretic data, i.e. bands with different positions produced by isoelectric focusing may not necessarily represent genetic differences.     

Mycelial amylase activities of thermophilic species ofRhizomucor,Humicola andPapulaspora

Amylase activities in mycelia ofRhizomucor pusillus, Humicola grisea var.thermoidea, Humicola lanuginosa andPapulaspora thermophilia do not correspond directly with previously-measured extracellular values, and appear to decline within the time period corresponding to reduction in mycelial dry weight. The results are compared with previously-reported data on extracellular amylase.     

Molecular cloning,characterization and expression of a gene encoding phosphoketolase from Termitomyces clypeatus

A phosphoketolase (pk) gene from the fungus Termitomyces clypeatus (TC) was cloned and partially characterized. Oligonucleotide primers specific for the phosphoketolase gene (pk) were designed from the regions of homologies found in the primary structure of the enzyme from other fungal sources related to TC, using multiple sequence alignment technique. The cDNA of partial lengths were amplified, cloned and sequenced in three parts by 3′ and 5′ RACE and RT-PCR using these oligonucleotide primers. The full length ds cDNA was constructed next by joining these three partial cDNA sequences having appropriate overlapping regions using Overlap Extension PCR technique. The constructed full length cDNA exhibited an open reading frame of 2487 bases and 5′ and 3′ UTRs. The deduced amino acid sequence, which is of 828 amino acids, when analyzed with NCBI BLAST, showed high similarities with the phosphoketolase enzyme (Pk) superfamily with expected domains. The part of the TC genomic DNA comprising of the pk gene was also amplified, cloned and sequenced and was found to contain two introns of 68 and 74 bases that interrupt the pk reading frame. The coding region of pk cDNA was subcloned in pKM260 expression vector in correct frame and the protein was expressed in Escherichia coli BL21 (DE3) transformed with this recombinant expression plasmid. The recombinant protein purified by His-tag affinity chromatography indicated the presence of a protein of the expected size. In vivo expression studies of the gene in presence of different carbon sources indicated synthesis of Pk specific mRNA, as expected. Phylogenetic studies revealed a common ancestry of the fungal and bacterial Pk. The TC is known to secrete several industrially important enzymes involved in carbohydrate metabolism. However, the presence of Pk, a key enzyme in pentose metabolism, has not been demonstrated conclusively in this organism. Cloning, sequencing and expression study of this gene establishes the functioning of this gene in T. clypeatus. The Pk from TC is a new source for commercial exploitation.     

Enzyme formation by a yeast cell wall lytic Arthrobacter species: formation of amylase

The kinetics of amylolytic enzyme formation by a yeast cell wall lytic Arthrobacter species were studied. Cultivation on autoclaved cells of baker's yeast showed that amylase formation was closely related to trehalose and glycogen dissimilation. Growth on yeast glycogen (0.5%) proceeded quite rapidly ( = 0.31 h^–1) with extensive amylase formation during exponential cell multiplication and a further low increase in activity during the stationary phase. Beside amylolytic activity [450 units (U) l^–1] the formation of a relatively high level of -glucosidase (90 U l^–1) was detected, the latter almost exclusively bound to bacterial cells. Growth on 0.5% trehalose occurred at a reduced rate ( = 0.22 h^–1) with post-logarithmic enzyme synthesis in the stationary phase. Amylase activity attained a level of 1200 U l^–1, whereas -glucosidase was very low at 7.7 U l^–1. Continuous culture experiments in the chemostat showed maximal volumetric productivity of amylase (105 U l^–1 h^–1) at a dilution rate of 0.15 h^–1. Growth on various carbohydrates revealed low levels of amylolytic activity (<100 U l^–1), which were increased by a -1,4-glucans and oligosaccharides such as starch, dextrin, maltotriose and maltose. On 0.5% maltose, growth-associated enzyme synthesis (230 U l^–1) was detected at a reduced growth rate ( = 0.14 h^–1). Amylolytic enzyme preparations from the culture fluid showed an unusual cleavage pattern; acting on starch, the polymer was almost completely hydrolysed to maltotriose and maltose in a molar ratio of 3:1.Correspondence to: W. A. Hampel     

Starch deposition and amylase accumulation during somatic embryogenesis in bamboo (Dendrocalamus hamiltonii)

In monocots, the zygotic embryo is protected and nourished by an endosperm. In the present study starch deposition and amylase accumulation was noticed during somatic embryogenesis in stem callus of a bamboo, Dendrocalamus hamiltonii. SEM studies revealed that starch grains were clearly visible in the scutellum during the maturation stage of the somatic embryo. As the somatic embryo developed further, the scutellum got reduced with corresponding increase in amylase. The amylase activity was tested periodically at different developmental stages of embryos. The role of scutellum in somatic embryos for starch deposition and amylase accumulation is discussed.     

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0–8.5 and active at pH 6.0–7.5. The optimum temperature at pH 7.0 was 35°C in the presence of 5 mM CaCl₂. The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.     

Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica)

A new thermophilic spore-forming strain MR3CT was isolated from geothermal soil located on Mount Rittmann in Antarctica. Strain MR3CT was Gram-positive, rod-shaped, occurring in pairs or filamentous. Growth was observed between 45 and 65 degrees C (optimum 61 degrees C) and at pH 5.0-6.5 (optimum pH 5.6). It was capable of utilizing galactose, trehalose, maltose and sucrose. The microorganism produced an exopolysaccharide and synthesized an extracellular constitutive amylolytic activity. The G + C content of DNA was 43.5 mol%. On the basis of 16S rRNA gene sequence similarity, strain MR3CT was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone-menaquinone-7; major fatty acid-iso-C15:0 and iso-C17:0) supported the affiliation of strain MR3C1T to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MR3CT from the validly published Anoxybacillus species. MR3CT therefore represents a new species, for which the name Anoxybacillus amylolyticus sp. nov., is proposed, with the type strain MR3CT (= ATCC BAA-872T = DSM 15939T = CIP 108338T).