Raw starch degrading alkaline α-amylase from Geobacillus kaustophilus TSCCA02: Production, characterization, and its potential for application as a detergent additive.

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.

Conversion of golden oyster mushroom, Pleurotus citrinopileatus to sugar syrup using enzymatic hydrolysis as a substrate for novel bacterial cellulose (Nata) fermentation.

Enzymatic hydrolysis of the golden oyster mushroom (Pleurotus citrinopileatus) generated a new bacterial cellulose (BC). The sugar syrup obtained from the hydrolysis of mushroom powder by commercial enzymes gave maximum total soluble solids (TSS) content at 8.83 ± 0.29°Brix, while 8.82 ± 0.06 mg GAE/g substrate of total phenolic content (TPC) was obtained when using initial substrate and enzyme concentrations at 125 g/L and 5.0%, respectively. Glutamic acid, aspartic acid, alanine and valine were determined as the main amino acids found in P. citrinopileatus hydrolysis at 524.74 ± 0.03, 247.09 ± 0.04, 176.82 ± 0.07 and 174.57 ± 0.01 mg/100 g sample, respectively. Thin-layer chromatography revealed that the obtained sugar syrup was glucose. The hydrolyzed mushroom fermented with Komagataeibacter xylinus AGR 60 at 30 ± 2 °C for 9 days produced optimal conditions at 4.0°Brix of the initial mushroom syrup and 12.0% (v/v) of the starter culture. Maximum BC thickness was 0.88 ± 0.03 cm with 7.90 ± 0.07 g dry weight, equivalent to 39.50 ± 0.35 g/L and 4.39 ± 0.04 g/L/day for BC production (P) and BC production rate (R p), respectively. The obtained BC was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, small-angle X-ray scattering and wide-angle X-ray diffraction. These showed the structure and functional properties as a natural source of fiber from the fermentation of a novel substrate.

Application of raw starch degrading enzyme from Laceyella sacchari LP175 for development of bacterial cellulose fermentation using colored rice as substrate.

Brown and black rice substrates were applied for sugar syrup production by the hydrolysis of raw starch degrading enzyme (RSDE) from Laceyella sacchari LP175 (300 U/mL) and commercial glucoamylase (GA, 2.0 U/mL) at 50 °C for 12 h using a simplex centroid mixture design. Results indicated that 300 g/L of substrates, consisting of 255 g/L Leum Pua glutinous rice and 45 g/L Black Jasmine rice, gave the highest sugar syrup production at 124.6 ± 2.52 g/L with 2.00 ± 0.05 mg GAE/mL of total phenolic content (TPC), equivalent to 0.42 ± 0.01 g/g rice sample and 6.67 ± 0.15 mg GAE/g rice sample, respectively. The obtained sugar syrup was used as the substrate for production of bacterial cellulose (Nata) by Komagataeibacter xylinus AGR 60 in a plastic tray at room temperature for 9 days. The fermentation medium containing 200 mL of rice syrup (25 g/L), 2.0 g of ammonium sulfate [(NH4)2SO4] and 0.4 mL glacial acetic acid yielded 1.1 ± 0.08 cm thickness with 8.15 ± 0.12 g of dry weight. The obtained bacterial cellulose from colored rice was characterized compared with bacterial cellulose from the conventional coconut juice by scanning electron microscope (SEM) and Fourier-transform infrared spectroscopy (FTIR) which demonstrated that the sugar syrup from colored rice could use as substrate for a novel bacterial cellulose as a healthy product in the future through microbial enzyme technological process. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02673-3.

Production and development of vinegar fermentation from broken Riceberry rice using raw starch-degrading enzyme hydrolysis.

Broken Riceberry rice was used as a substrate for sugar syrup production by the hydrolysis of raw starch-degrading enzyme as a low-temperature amylase (iKnowZyme® LTAA, Thailand). Response surface methodology (RSM) with a central composite design (CCD) showed that an optimized substrate concentration of 250 g/L yielded 13°Brix of total soluble solid (TSS) content when incubated at 50 °C for 12 h. The major product from the broken Riceberry rice hydrolysis was glucose with lesser amounts of maltose and maltotriose. Maximum alcohol content (16% w/v) for broken Riceberry rice wine was obtained after fermentation with two mixed strains of Saccharomyces cerevisiae for 10 days. Scanning electron micrographs showed that yeast strains could grow on the solid residue of broken Riceberry rice that supported yeast cell survival under stress conditions. Broken Riceberry rice wine was used as the substrate for vinegar fermentation by Acetobacter aceti TISTR 354. Maximum acetic acid concentration was achieved at 5.4% when incubated at room temperature for 6 days, containing 10.92 mg/L and 965.53 ± 7.74 mL sample/g DPPH of anthocyanin content and antioxidant assay, respectively. Our finding revealed the feasibility of broken Riceberry rice substrate for sugar syrup, wine and vinegar production by raw starch-degrading enzyme hydrolysis which increased the value of low-cost agricultural crops through biotechnological processes.

Development of biodegradation process for Poly(DL-lactic acid) degradation by crude enzyme produced by Actinomadura keratinilytica strain T16-1

Background: Plastic waste is a serious problem because it is difficult to degrade, thereby leading to global environment problems. Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester derived from renewable resources, and it can be degraded by various enzymes produced by microorganisms. This study focused on the scale-up and evaluated the bioprocess of PLA degradation by a crude microbial enzyme produced by Actinomadura keratinilytica strain T16-1 in a 5 L stirred tank bioreactor. Results: PLA degradation after 72 h in a 5 L bioreactor by using the enzyme of the strain T16-1 under controlled pH conditions resulted in lactic acid titers (mg/L) of 16,651 mg/L and a conversion efficiency of 89% at a controlled pH of 8.0. However, the PLA degradation process inadvertently produced lactic acid as a potential inhibitor, as shown in our experiments at various concentrations of lactic acid. Therefore, the dialysis method was performed to reduce the concentration of lactic acid. The experiment with a dialysis bag achieved PLA degradation by weight loss of 99.93%, whereas the one without dialysis achieved a degradation of less than approximately 14.75%. Therefore, the dialysis method was applied to degrade a commercial PLA material (tray) with a conversion efficiency of 32%, which was 6-fold more than that without dialysis. Conclusions: This is the first report demonstrating the scale-up of PLA degradation in a 5 L bioreactor and evaluating a potential method for enhancing PLA degradation efficiency.

Actinomycetospora endophytica sp. nov., isolated from wild orchid (Podochilus microphyllus Lindl.) in Thailand.

A novel endophytic actinomycete, designated strain A-T 8314T, was isolated from a wild orchid, Podochilus microphyllus Lindl., collected from Trat Province, Thailand. The taxonomic position of strain A-T 8314T was established using a combination of genotypic and phenotypic analyses. The isolate was a Gram-positive bacterium that developed bud-like spore chains. Strain A-T 8314T grew aerobically at an optimum temperature of 20-25 °C and an optimal pH 6.0. The cell wall contained meso-diaminopimelic acid, and the whole-cell sugars were ribose, arabinose and galactose. The predominant menaquinone was MK-8 (H4). The polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylmonomethylethanolamine, hydroxy-phosphatidylmonomethylethanolamine and hydroxyl phosphatidylethanolamine. The predominant cellular fatty acid was iso-C16 : 0. The DNA G+C content of the genomic DNA was 73.2±0.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A-T 8314T belonged to the genus Actinomycetospora, and was most closely related to Actinomycetospora chiangmaiensis YIM 0006T (98.8 %) and Actinomycetosporacorticicola 014-5T (98.6 %). The DNA-DNA relatedness values that distinguished A-T 8314T from its closest species were below 70 %. Following an evaluation of phenotypic, chemotaxonomic and genotypic studies, it was concluded that the new isolate represents as a novel species, for which the name Actinomycetospora endophytica sp. nov is proposed. The type strain is A-T 8314T (=TBRC 5722T=NBRC 113235T).

Poly(DL-lactide)-degrading enzyme production by immobilized Actinomadura keratinilytica strain T16-1 in a 5-L fermenter under various fermentation processes

Background: Poly(DL-lactic acid), or PDLLA, is a biodegradable polymer that can be hydrolyzed by various types of enzymes. The protease produced by Actinomadura keratinilytica strain T16-1 was previously reported to have PDLLA depolymerase activity. However, few studies have reported on PDLLA-degrading enzyme production by bacteria. Therefore, the aims of this study were to determine a suitable immobilization material for PDLLA-degrading enzyme production and optimize PDLLA-degrading enzyme production by using immobilized A. keratinilytica strain T16-1 under various fermentation process conditions in a stirrer fermenter. Results: Among the tested immobilization materials, a scrub pad was the best immobilizer, giving an enzyme activity of 30.03 U/mL in a shake-flask scale. The maximum enzyme activity was obtained at aeration 0.25 vvm, agitation 170 rpm, 45°C, and 48 h of cultivation time. Under these conditions, a PDLLA-degrading enzyme production of 766.33 U/mL with 15.97 U/mL·h productivity was observed using batch fermentation in a 5-L stirrer fermenter. Increased enzyme activity and productivity were observed in repeated-batch (942.67 U/mL and 19.64 U/mL·h) and continuous fermentation (796.43 U/mL and 16.58 U/mL·h) at a dilution rate of 0.013/h. Scaled-up production of the enzyme in a 10-L stirrer bioreactor using the optimized conditions showed a maximum enzyme activity of 578.67 U/mL and a productivity of 12.06 U/mL·h. Conclusions: This research successfully scaled-up the enzyme production to 5 and 10 L in a stirrer fermenter and is helpful for many applications of poly(lactic acid).

Polycladomyces subterraneus sp. nov., isolated from soil in Thailand.

A thermophilic poly(L-lactide)-degrading Gram-stain-positive filamentous bacterial strain that develops single spores on the aerial mycelium was isolated from forest soil at Srinagarind Dam, Kanchanaburi Province, Thailand. The results of a polyphasic taxonomic study showed that our isolate had characteristics typical of members of the genus Polycladomyces. The isolate grew aerobically at an optimum temperature of 50-55 °C and optimal pH 6-7. Meso-diaminopimelic acid was present as the diagnostic diamino acid in the peptidoglycan but no characteristic sugars are detected. The predominant menaquinone was MK-7. The diagnostic phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine diphosphatidylglycerol, phosphatidylglycerol and phosphatidylserine. The predominant cellular fatty acid was iso-C15 : 0. The DNA G+C content of strain KSR 13T was 53.4 mol%. The 16S rRNA gene sequence analysis also indicated that strain KSR 13T belonged to the genus Polycladomyces, being most closely related to Polycladomyces abyssicola JIR-001T (99.2 %). The DNA-DNA relatedness values that distinguished KSR 13T from P. abyssicola JIR-001T were 17.8-32.1 %, which were significantly below the 70 % cutoff value recommended for species delineation. Following an evaluation of phenotypic, chemotaxonomic and genotypic studies, the new isolate is proposed as a novel species and named Polycladomyces subterraneus sp. nov. The type strain is KSR 13T (=BCC 50740T=NBRC 109332T).

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production.

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8 g:10 g:2 g yielded the highest enzyme production of 201.6 U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5 × 106 spores/mL inoculum, which gave the highest enzyme activity of 389.5 U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2 g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300 g raw cassava chips/L with cane molasses.

Antagonistic activity of endo-ß-1,3-glucanase from a novel isolate, Streptomyces sp. 9X166, against black rot in orchids.

A total of 123 actinomycetes was isolated from 12 varieties of wild orchids and screened for potential antagonistic activity against Phytophthora, which causes black rot disease in orchids. In vitro and in vivo experimental results revealed that Streptomyces sp. strain 9X166 showed the highest antagonistic activity; its ß-1,3-glucanase production ability was a key mechanism for growth inhibition of the pathogen. PCR amplification and DNA sequencing of the 16S ribosomal RNA gene allowed the identification of this strain, with high similarity (99.93%) to the novel species Streptomyces similaensis. The glucanase enzyme, purified to homogeneity by anion exchange and gel filtration chromatography, showed a specific activity of 58 U mg(-1) (a 3.9-fold increase) and yield of 6.4%. The molecular weight, as determined by SDS-PAGE and gel filtration, was approximately 99 and 80 kDa, respectively, suggesting that the enzyme was a monomer. The purified enzyme showed the highest substrate specificity to laminarin, indicating that it was ß-1,3-glucanase. The hydrolyzed products of cello-oligosaccharides suggested that this enzyme was endo-type ß-1,3-glucanase. Streptomyces sp. 9X166 culture filtrate, possessing ß-1,3-glucanase activity, could degrade both freeze-dried and living mycelium. This is the first report on a ß-1,3-glucanase-producing Streptomyces sp. that could be an effective biocontrol agent for black rot disease in orchids.

Simultaneous production of l-lactic acid with high optical activity and a soil amendment with food waste that demonstrates plant growth promoting activity.

A unique method to produce highly optically-active l-lactic acid and soil amendments that promote plant growth from food waste was proposed. Three Bacillus strains Bacillus subtilis KBKU21, B. subtilis N3-9 and Bacillus coagulans T27, were used. Strain KBKU21 accumulated 36.9 g/L l-lactic acid with 95.7% optical activity and 98.2% l-lactic acid selectivity when fermented at 43°C for 84 h in a model kitchen refuse (MKR) medium. Residual precipitate fraction (anaerobically-fermented MKR (AFM) compost) analysis revealed 4.60%, 0.70% and 0.75% of nitrogen (as N), phosphorous (as P2O5), and potassium (as K2O), respectively. Additionally, the carbon to nitrogen ratio decreased from 13.3 to 10.6. AFM compost with KBKU21 promoted plant growth parameters, including leaf length, plant height and fresh weight of Brassica rapa (Komatsuna), than that by chemical fertilizers or commercial compost. The concept provides an incentive for the complete recycling of food waste, contributing towards a sustainable production system.

Kineococcus mangrovi sp. nov., isolated from mangrove sediment.

An aerobic, Gram-stain-positive, motile, coccus-shaped actinobacterium, designated strain L2-1-L1T, was isolated from mangrove sediment in Thailand. The organism was deep orange, oxidase-negative and catalase-positive. Growth occurred at temperatures between 17 and 32 °C and with NaCl concentrations up to 10 %. Chemotaxonomic characteristics of strain L2-1-L1T were typical of the genus Kineococcus. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Whole-cell hydrolysates contained arabinose, galactose, glucose, mannose and ribose. The menaquinone was MK-9(H2). Mycolic acids were not detected. Anteiso-C15 : 0 and iso-C14 : 0 were detected as the major cellular fatty acids. The major polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol and an unidentified phosphoglycolipid. The G+C content of the DNA was 74.7 mol%. A phylogenetic tree, based on 16S rRNA gene sequences, revealed that strain L2-1-L1T represents a novel member of the genus Kineococcus. The most closely related species were Kineococcus endophytica KLBMP 1274T, Kineococcus aurantiacus NBRC 15268T and Kineococcus rhizosphaerae RP-B16T, with 98.9, 98.6 and 98.5 % 16S rRNA gene sequence similarity, respectively. On the basis of phylogenetic analysis, DNA-DNA relatedness data, phenotypic characteristics and chemotaxonomic data, a novel species of the genus Kineococcus is proposed: Kineococcus magrovi sp. nov. The type strain is strain L2-1-L1T ( = BCC 75409T = NBRC 110933T).

Purification and characterization of three ß-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28.

Production and utilization of cellulosic ethanol has been limited, partly due to the difficulty in degradation of cellulosic feedstock. ß-Glucosidases convert cellobiose to glucose in the final step of cellulose degradation, but they are inhibited by high concentrations of glucose. Thus, in this study, we have screened, isolated, and characterized three ß-glycosidases exhibiting highly glucose-tolerant property from Aspergillus niger ASKU28, namely ß-xylosidase (P1.1), ß-glucosidase (P1.2), and glucan 1,3-ß-glucosidase (P2). Results from kinetic analysis, inhibition study, and hydrolysis of oligosaccharide substrates supported the identification of these enzymes by both LC/MS/MS analysis and nucleotide sequences. Moreover, the highly efficient P1.2 performed better than the commercial ß-glucosidase preparation in cellulose saccharification, suggesting its potential applications in the cellulosic ethanol industry. These results shed light on the nature of highly glucose-tolerant ß-glucosidase activities in A. niger, whose kinetic properties and identities have not been completely determined in any prior investigations.

Sinosporangium siamense sp. nov., isolated from soil and emended description of the genus Sinosporangium.

An actinomycetes strain A-T 1946(T) that developed spherical sporangia containing non-motile spores on aerial mycelia was isolated from dry deciduous forest soil in Thailand. 16S rRNA gene sequence analysis indicated that strain A-T 1946(T) belongs to the genus Sinosporangium, being closely related to Sinosporangium album 6014(T) (98.8% sequence similarity). The DNA-DNA relatedness values were 43.7-50.9%, which were significantly below 70% and differentiated strain A-T 1946(T) from the closest species. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars contained rhamnose, ribose, madurose and glucose. The predominant menaquinone was MK-9(H2). The diagnostic phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, lysophosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol-mannoside, N-acetylglucosamine-containing phospholipids, two unknown phosphoglycolipids and two unknown phospholipids. The predominant cellular fatty acids were unsaturated C(16 : 1) and C(17 : 1), and saturated C(16 : 0) and 10-methyl-C(17 : 0). Following an evaluation of phenotypic, chemotaxonomic and genotypic characteristics, the isolate is proposed to represent a novel species of genus Sinosporangium to be named Sinosporangium siamense sp. nov. The type strain is A-T 1946(T) ( = BCC 29081(T) = NBRC 109515(T)). An emended description of the genus Sinosporangium is also provided.

Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175.

Eleven strains of poly(L-lactide) (PLLA)-degrading thermophilic bacteria were isolated from forest soils and selected based on clear zone formation on an emulsified PLLA agar plate at 50°C. Among the isolates, strain LP175 showed the highest PLLA-degrading ability. It was closely related to Laceyella sacchari, with 99.9% similarity based on the 16S rRNA gene sequence. The PLLA-degrading enzyme produced by the strain was purified to homogeneity by 48.1% yield and specific activity of 328 U·mg-protein-1 with a 15.3-fold purity increase. The purified enzyme was strongly active against specific substrates such as casein and gelatin and weakly active against Suc-(Ala)3-pNA. Optimum enzyme activity was exhibited at a temperature of 60°C with thermal stability up to 50°C and a pH of 9.0 with pH stability in a range of 8.5-10.5. Molecular weight of the enzyme was approximately 28.0 kDa, as determined by gel filtration and SDS-PAGE. The inhibitors phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetate (EDTA), and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) strongly inhibited enzyme activity, but the activity was not inhibited by 1 mM 1,10-phenanthroline (1,10-phen). The N-terminal amino acid sequences had 100% homology with thermostable serine protease (thermitase) from Thermoactinomyces vulgaris. The results obtained suggest that the PLLA-degrading enzyme produced by L. sacchari strain LP175 is serine protease.

Poly(L-lactide)-degrading enzyme production by Actinomadura keratinilytica T16-1 in 3 L airlift bioreactor and its degradation ability for biological recycle.

The optimal physical factors affecting enzyme production in an airlift fermenter have not been studied so far. Therefore, the physical parameters such as aeration rate, pH, and temperature affecting PLA-degrading enzyme production by Actinomadura keratinilytica strain T16-1 in a 3 l airlift fermenter were investigated. The response surface methodology (RSM) was used to optimize PLA-degrading enzyme production by implementing the central composite design. The optimal conditions for higher production of PLA-degrading enzyme were aeration rate of 0.43 vvm, pH of 6.85, and temperature at 46° C. Under these conditions, the model predicted a PLA-degrading activity of 254 U/ml. Verification of the optimization showed that PLA-degrading enzyme production of 257 U/ml was observed after 3 days cultivation under the optimal conditions in a 3 l airlift fermenter. The production under the optimized condition in the airlift fermenter was higher than un-optimized condition by 1.7 folds and 12 folds with un-optimized medium or condition in shake flasks. This is the first report on the optimization of environmental conditions for improvement of PLA-degrading enzyme production in a 3 l airlift fermenter by using a statistical analysis method. Moreover, the crude PLA-degrading enzyme could be adsorbed to the substrate and degraded PLA powder to produce lactic acid as degradation products. Therefore, this incident indicates that PLA-degrading enzyme produced by Actinomadura keratinilytica NBRC 104111 strain T16-1 has a potential to degrade PLA to lactic acid as a monomer and can be used for the recycle of PLA polymer.

Purification and characterization of a thermostable xylanase from Saccharopolyspora pathumthaniensis S582 isolated from the gut of a termite.

An extracellular thermostable xylanase produced by Saccharopolyspora pathumthaniensis S582 was purified 167-fold to homogeneity with a recovery yield of 12%. The purified xylanase appeared as a single protein band on SDS-PAGE, with a molecular mass of 36 kDa. The optimal temperature and pH of the xylanase were 70 °C and 6.5. The enzyme was stable within a pH range of 5.5-10.0. It retained its activity after incubation at 50 °C for 2 h. Its half lives at temperatures of 60 and 70 °C were 180 and 120 min respectively. Hydrolysis of beechwood xylan by the xylanase yielded xylobiose and xylose as major products. The enzyme acted specifically on xylan as an endo-type xylanase, and exhibited a K(m) value of 3.92 mg/mL and a V(max) value of 256 µmol/min/mg. Enzyme activity was completely inhibited by Hg(2+), and was stimulated by Rb(+) and Cs(+). The xylanase gene was cloned from genomic DNA of Saccharopolyspora pathumthaniensis S582 and sequenced. The ORF consisted of 1,107 bp and encoded 368 amino acid residues containing a putative signal peptide of 23 residues. This xylanase is a new member of family (GH) 10 that shows highest identity, of 63.4%, with a putative xylanase from Nocardiopsis dassonvillei subsp. dassonvillei.

Saccharopolyspora pathumthaniensis sp. nov., a novel actinomycetes isolated from termite guts (Speculitermes sp.).

Morphological and chemotaxonomic characterization of actinomycete strain S582 isolated from the gut of a termite (Speculitermes sp.) in Pathum Thani Province, Thailand, clearly demonstrated that this strain is a member of the genus Saccharopolyspora. 16S rDNA sequence analysis for the strain supported the assignment of the strain to the genus Saccharopolyspora. The similarity value of sequences between this strain and the closely related species Saccharopolyspora endophytica was 99.5%. The DNA G+C content was 70.2 mol%. DNA-DNA hybridization results (53.3%) and some physiological and biochemical properties indicated that strain S582(T) was distinguished from the phylogenetically closest relatives. Based on these genotypic and phenotypic data, strain S582(T) should be a new species in the genus Saccharopolyspora and the name Saccharopolyspora pathumthaniensis sp. nov. is proposed for the strain. The type strain is S582(T) (=NBRC 104112(T) =BCC 28624(T)).

Sphaerisporangium krabiense sp. nov., isolated from soil.

A Gram-staining-positive, filamentous bacterial strain, designated A-T 0308(T), was isolated from soil of a tropical mangrove forest in Thailand. Strain A-T 0308(T) developed spherical sporangia containing non-motile spores on aerial mycelium. The novel strain contained meso-diaminopimelic acid, N-acetyl-type peptidoglycan and madurose, mannose, ribose, galactose and glucose as whole-cell sugars. The predominant menaquinones were MK-9(H(4)) and MK-9(H(6)); a small amount of MK-9(H(2)) and MK-9 was also detected. Mycolic acids were not detected. The diagnostic phospholipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and phosphoglycolipid. The predominant cellular fatty acids were iso-C(16:0) and 10-methylated C(17:0). The G+C content of the DNA was 72 mol%. Phenotypic and chemotaxonomic analyses showed that the novel isolate had characteristics typical of members of the genus Sphaerisporangium. 16S rRNA gene sequence analysis also indicated that the strain belongs to the genus Sphaerisporangium and that it represents a clade distinct from other members of the genus with sequence similarities ranging from 96.3 to 97.8% between the novel strain and its closest relatives. Based on the results of phenotypic, chemotaxonomic and phylogenetic studies, strain A-T 0308(T) (=BCC 21702(T) =NBRC 107571(T)) represents a novel species of the genus Sphaerisporangium, for which the name Sphaerisporangium krabiense sp. nov. is proposed.

Herbidospora sakaeratensis sp. nov., isolated from soil, and reclassification of Streptosporangium claviforme as a later synonym of Herbidospora cretacea.

An actinomycete strain, DMKUA 205(T), was isolated from a soil sample collected from the Sakaerat Biosphere Reserve in Nakhonratchasima Province, Thailand. The novel strain produced short chains of non-motile spores on the tips of long sporophores branching from the vegetative hyphae. The morphological and chemotaxonomic properties of this new isolate corresponded to those of members of the genus Herbidospora. Furthermore, 16S rRNA gene sequence analysis showed that the strain was closely related to members of the genus Herbidospora. Phenotypic properties and DNA-DNA relatedness values differentiated the new strain from its closest phylogenetic relatives Herbidospora yilanensis 0351M-12(T) (35-54 % DNA-DNA relatedness) and Herbidospora daliensis 0385M-1(T) (58-65 % relatedness). On the basis of phenotypic, genotypic and phylogenetic data, strain DMKUA 205(T) could be clearly distinguished from the type strains of H. yilanensis and H. daliensis. Therefore, strain DMKUA 205(T) represents a novel species, for which the name Herbidospora sakaeratensis sp. nov. is proposed. The type strain is strain DMKUA 205(T) ( = BCC 11662(T) = NBRC 102641(T)). In addition, the DNA-DNA hybridization results from this study revealed that Streptosporangium claviforme is a later synonym of Herbidospora cretacea.