Expression and Activity Analysis of Fructosyltransferase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The fructosyltransferase gene was isolated and cloned from Aspergillus oryzae. The gene was 1368 bp, which encoded a protein of 455 amino acids. To analyze the activity of the expressed fructosyltransferase, the pET32a-fructosyltransferase recombined plasmid was transformed into Escherichia coli BL21. The fructosyltransferase gene was successfully expressed by Isopropyl-β-d-thiogalactoside (IPTG) induction. The molecular weight of the expression protein was about 45 kDa. The optimal conditions of protein expression were 25?°C, 0.1 mM IPTG, and 8 h of inducing time. The optimal concentration of urea dealing with inclusion body was 2.5 M. The expressed protein exhibited a strong fructosyl transfer activity. These results showed that the expressed fructosyltransferas owned transferase activity, and could catalyze the synthesis of sucrose-6-acetate.     

Heterologous expression and characterization of a novel halotolerant,thermostable, and alkali-stable GH6 endoglucanase from <Emphasis Type="Italic">Thermobifida halotolerans</Emphasis>

A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462^T, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 % of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application.     

Purification and characterisation of 1F-fructosyltransferase from the roots of asparagus (asparagus officinalis L.)

A fructosyltransferase that transfers a terminal d-fructosyl group from a (2→1)-β-linked fructosaccharide to HO-1 of another d-fructosyl group has been purified from an extract of asparagus roots by successive chromatography with DEAE-cellulose, octyl-Sepharose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The disc-electrophoretically homogeneous enzyme was free from β-d-fructofuranosidase, sucrose:sucrose 1-fructosyltransferase, and 6^G-frutosyltransferase activity, and catalysed the d-fructosyl transfer from 1-kestose more rapidly to saccharides of the neokestose series [1^F(1-β-d-fructofuranosyl)_m-6^G(1-β-d-fructofuranosyl)_nsucrose] than to those of the 1-kestose series [1^F(1-β-d-fructofuranosyl)_nsucrose]. The enzyme was tentatively termed 1^F-fructosyltransferase. The general properties of the enzyme were as follows: mol. wt., ~64,000; optimum pH, ~5.0; stable at pH 5.0–5.5 at 45° for 20 min; stable at 30–45° for 10 min; inhibited by Hg²⁺, p-chloromercuribenzoate, and Ag⁺.     

Purification and characterisation of 6G-fructosyltransferase from the roots of asparagus (Asparagus officinalis L.)

A fructosyltransferase that catalyses the transfer of the terminal (2 → 1)-β-linked d-fructosyl group of fructo-oligosaccharides [1^F(1-β-d-fructofuranosyl)_msucrose, m > 0] to HO-6 of the glucosyl group of similar saccharides [1^F(1-β-d-fructofuranosyl)_nsucrose, n > 0] has been purified (760-fold) from an extract of the roots of asparagus (Asparagus officinalis L.) by successive fractionation with ammonium sulfate, treatment with calcium phosphate gel, and then chromatography on octyl-Sepharose, DEAE-cellulose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The enzyme, tentatively termed 6^G-fructosyltransferase, was homogeneous in disc electrophoresis, had a mol. wt. of ~69,000 and an optimum pH of ~5.5, was stable at pH 5.0–6.0 on heating for 20 mins at 45° and for 10 min at 20–37°, and was inhibited by Hg²⁺, p-chloromercuribenzoate, and Ag⁺.     

Nonhongiella spirulinensis gen. nov., sp. nov., a bacterium isolated from a cultivation pond of Spirulina platensis in Sanya, China

A Gram-negative, aerobic, motile rod strain, designated Ma-20^T, was isolated from a pool of marine Spirulina platensis cultivation, Sanya, China, and was subjected to a polyphasic taxonomy study. Strain Ma-20^T can grow in the presence of 0.5–11 % (w/v) NaCl, 10–43 °C and pH 6–10, and grew optimally at 30 °C, pH 7.5–9.0 in natural seawater medium. The polar lipids were composed of phosphatidylethanolamine, three unidentified phospholipids and three unidentified polar lipids. The respiratory quinone was ubiquinone 8 (Q-8) and the major fatty acids were C_18:1ω6c/C_18:1ω7c (summed feature 8, 32.84 %), C_16:1ω6c/C_16:1ω7c (summed feature 3, 30.76 %), C_16:0 (13.54 %), C_12:03-OH (4.63 %), and C_12:0 (4.09 %). The DNA G+C content of strain Ma-20^T was 58 mol %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Ma-20^T belonging to Gammaproteobacteria, it shared 88.46–91.55 and 89.21–91.26 % 16S rRNA gene sequence similarity to the type strains in genus Hahella and Marinobacter, respectively. In addition to the large 16S rRNA gene sequence difference, Ma-20^T can also be distinguished from the reference type strains Hahella ganghwensis FR1050^T and Marinobacter hydrocarbonoclasticus sp. 17^T by several phenotypic characteristics and chemotaxonomic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic properties, strain Ma-20^T is suggested to represent a novel species of a new genus in Gammaproteobacteria, for which the name Nonhongiella spirulinensis gen. nov., sp. nov. is proposed. The type strain is Ma-20^T (=KCTC 32221^T=LMG 27470^T).     

Clostridium swellfunianum sp. nov., a novel anaerobic bacterium isolated from the pit mud of Chinese Luzhou-flavor liquor production

A novel Gram-positive, strictly anaerobic, spore-forming, rod-shaped bacterium, designated strain S11-3-10^T, was isolated from the pit mud used for Chinese Luzhou-flavor liquor production. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a monophyletic clade with the closely related type strains of Clostridium cluster I and was most closely related to Clostridium amylolyticum JCM 14823^T (94.38 %). The temperature, pH, and NaCl range for growth was determined to be 20–45 °C (optimum 37 °C), 4.0–10.0 (optimum pH 7.3), and 0–3.0 % (w/v), respectively. The strain was able to tolerate up to 7.5 % (v/v) ethanol. Yeast extract or peptone was found to be required for growth. Acids were found to be produced from glucose, mannose and trehalose. The major end products from glucose fermentation were identified as ethanol, acetate and hydrogen. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified phospholipids and polar lipids. The major fatty acids (>5 %) were identified as iso-C_15:0, C_16:0, C_16:0 dma, C_14:0, anteiso-C_15:0 and iso-C_13:0. No respiratory quinone was detected. The diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid and the whole-cell sugars were found to include galactose and glucose as major components. The DNA G+C content was determined to be 36.4 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic evidence, the isolate is considered to represent a novel species of the genus Clostridium for which the name Clostridium swellfunianum sp. nov. is proposed. The type strain is S11-3-10^T (=DSM 27788^T = JCM 19606^T = CICC 10730^T).     

Sucrose:sucrose fructosyltransferase and fructan:fructan fructosyltransferase from allium cepa

Sucrose: sucrose fructosyltransferase and fructan:fructan fructosyltransferase were isolated from the inner leaf bases of bulbing onion plants (Allium cepa) and separated by gel filtration on Bio-Gel P-150. Sucrose:sucrose fructosyltransferase produced only one trisaccharide, 1^F-fructosylsucrose, from sucrose. Fructan:fructan fructosyltransferase produced tetrasaccharide and higher polymers from trisaccharide. The trisaccharide found in the greatest concentration in onion, 6^G-fructosylsucrose, was produced from 1^F-fructosylsucrose by fructan:fructan fructosyltransferase and was not a product of sucrose:sucrose fructosyltransferase.     

<Emphasis Type="Italic">Algoriella xinjiangensis</Emphasis> gen. nov., sp. nov., a new psychrotolerant bacterium of the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>

An aerobic, Gram-stain negative, non-spore-forming and psychrotolerant bacterium, designated strain XJ109^T, was isolated from a sewage water sample collected from Xinjiang Uigur Autonomous Region, China. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain XJ109^T represents a novel member of the family Flavobacteriaceae. The strain showed 95.5 % similarity with the 16S rRNA gene sequence of Empedobacter brevis LMG 4011^T, 95.4 % with Chishuiella changwenlii BY4^T, 95.3 % with Empedobacter falsenii NF 993^T and 92.3 % with Weeksella virosa DSM 16922^T. Strain XJ109^T showed the common phenotypic and chemotaxonomic characteristics of the family Flavobacteriaceae, containing menaquinone-6 (MK-6) as the predominant respiratory quinone and iso-C_17:0 3OH and iso-C_15:0 as the major fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, one unidentified phospholipid and two unidentified lipids. The genomic DNA G+C content was 38.0 mol%. Strain XJ109^T was positive for catalase and oxidase activities, and it was observed to grow at 4–30 °C (optimal 16–20 °C), pH 6.5–10.0 (optimal 7.0–7.5) and in media containing 0–2.0 % (w/v) NaCl (optimal 0.5 %). On the basis of the polyphasic evidence presented, strain XJ109^T is considered to represent a novel genus and species of the family Flavobacteriaceae, for which the name Algoriella xinjiangensis gen. nov., sp. nov. is proposed. The type strain is XJ109^T (=CGMCC 1.10229^T=JCM 16590^T).     

Carotenoid Production by a Novel Isolate of <Emphasis Type="Italic">Microbacterium paraoxydans</Emphasis>

This study reports extraction and characterization of carotenoid pigments from Microbacterium paraoxydans, a non-photosynthetic bacterium, cultivated in Luria–Bertani (LB) medium. The isolate was identified to be moderately halo- and osmo-tolerant capable of withstanding high (~ 6%) salt and sugar (30% w/v sucrose, 20% w/v glucose) concentrations after a brief period of adaptation. The pigments were characterized using a combination of UV–Vis spectral analysis with the λ_max at 407, 436 and 466 nm and ESI–MS with an m/z value at 536.44. The absorption profile of the pigments and their nature was influenced by carbon, nitrogen source and presence of salt in the growth medium. Highest level of pigment (~ 16 g kg dry wt cells^?1) was produced in NH₄Cl supplemented LB medium. The pigment displayed free radical scavenging, anticancer activity, characteristic of the plant carotenoids. Based on the accumulation of pigments under different conditions, a biochemical pathway for synthesis of neurosporene was proposed.     

Solirubrobacter taibaiensis sp. nov., isolated from a stem of Phytolacca acinosa Roxb.

A white-coloured bacterium, designated strain GTJR-20^T, was isolated from a stem of Phytolacca acinosa Roxb. collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. The novel isolate was found to grow optimally at 28–30 °C, at pH 7.5–8.0 and in the absence of NaCl. Cells were observed to be Gram-stain positive, strictly aerobic, rod-shaped and non-motile. The predominant respiratory quinone was identified as MK-7(H₄) and the major cellular fatty acids were identified as iso-C_16:0 (35.8 %), C_18:1 ω9c (17.7 %), C_17:1 ω6c (11.0 %), C_17:1 ω8c (7.8 %) and C_18:3 ω6c (6, 9, 12) (7.2 %). The DNA G+C content was determined to be 71.6 mol %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GTJR-20^T is a member of the genus Solirubrobacter and is closely related to Solirubrobacter phytolaccae GTGR-8^T (16S rRNA gene sequence similarity, 98.4 %), Solirubrobacter soli KCTC 12628^T (97.8 %), Solirubrobacter pauli KCTC 9974^T (97.7 %) and Solirubrobacter ginsenosidimutans KCTC 19420^T (97.6 %). No other recognized bacterial species showed more than 94.6 % 16S rRNA gene sequence similarity to the novel isolate. DNA–DNA relatedness values for strain GTJR-20^T with respect to its closely related neighbours S. phytolaccae GTGR-8^T, S. soli KCTC 12628^T, S. pauli KCTC 9974^T and S. ginsenosidimutans KCTC 19420^T were 48.3 ± 8.6, 21.3 ± 5.2, 36.8 ± 6.2 and 36.0 ± 5.5 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain GTJR-20^T is considered to represent a novel species of the genus Solirubrobacter, for which the name Solirubrobacter taibaiensis sp. nov. is proposed. The type strain is GTJR-20^T (=CCTCC AB 2013308^T = KCTC 29222^T).     

Paludibacter jiangxiensis sp. nov., a strictly anaerobic,propionate-producing bacterium isolated from rice paddy field

A mesophilic, obligately anaerobic, propionate-producing fermentative bacterium, designated strain NM7^T, was isolated from rural rice paddy field. Cells of strain NM7^T are Gram-negative, non-motile, non-spore-forming, short rods, and negative for catalase. The strain grew optimally at 37 °C (the range for growth 15–40 °C) and pH 7.0 (pH 5.0–7.5). The strain could grow fermentatively on various sugars, including arabinose, xylose, fructose, galactose, glucose, mannose, cellobiose, lactose, maltose, sucrose, pectin and starch. The main end products of glucose fermentation were acetate and propionate. Yeast extract was not required but stimulated the growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, and Fe(III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of genomic DNA was 42.8 mol%. The major cellular fatty acids were C_15:0, anteiso-C_15:0, C_16:0, and C_17:0. The most abundant polar lipid of strain NM7^T was phosphatidylethanolamine. 16S rRNA gene sequence analysis revealed that it belongs to the family Porphyromonadaceae of the phylum Bacteroidetes. The closest recognized species was Paludibacter propionicigenes (91.4 % similarity in 16S rRNA gene sequence). A novel species, Paludibacter jiangxiensis sp. nov., is proposed to accommodate strain NM7^T (=JCM 17480^T = CGMCC 1.5150^T = KCTC 5844^T).     

Asymmetric reduction of 4-hydroxy-2-butanone to (R)-1,3-butanediol with absolute stereochemical selectivity by a newly isolated strain of Pichia jadinii

In this study, a novel strain of Pichia jadinii, HBY61, capable of the biocatalysis of 4-hydroxy-2-butanone (4H2B) to (R)-1,3-BD was isolated. HBY61 produced (R)-1,3-BD with high activity and absolute stereochemical selectivity (100 % e.e). Glucose and beef extract were found to be the key factors governing the fermentation, and their optimal concentrations were determined to be 84.2 and 43.7 g/L, respectively. The optimal bioconversion conditions of 4H2B catalyzed by HBY61 were pH 7.4, 30 °C, and 250 rpm with 6 % (v/v) glucose as the co-substrate. Accordingly, when 45 g/L of 4H2B was divided into three equal parts and added successively into the system at set time intervals, the maximum (R)-1,3-BD concentration reached 38.3 g/L with high yield (85.1 %) and strict 100 % enantioselectivity. Compared with previously reported yields for the biocatalytic production of (R)-1,3-BD, the use of strain HBY61 provided a high yield with excellent stereoselectivity.     

Characterization of a mutant glucose isomerase from <Emphasis Type="Italic">Thermoanaerobacterium saccharolyticum</Emphasis>

A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co²⁺ or Mg²⁺ for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.     

Flavobacterium kyungheensis sp. nov., isolated from soil of a ginseng field

A Gram-staining negative, strictly aerobic, motile by gliding, non-spore-forming, pale yellow pigmented and rod-shaped bacterium designated strain THG-107^T was isolated from soil of a ginseng field on Ganghwa Island in the Republic of Korea and its taxonomic position was investigated by using a polyphasic study. Growth of strain THG-107^T was found to occur at 4–37 °C (optimum, 20–30 °C), at pH 5.5–10 (optimum, pH 7.0) and in the presence of 0–1 % (w/v) NaCl (optimum, absence) on R2A agar. On the basis of 16S rRNA gene sequence similarity, strain THG-107^T was shown to belong to the family Flavobacteriaceae and was related to Flavobacterium denitrificans ED5^T (99.1 % similarity). The G+C content of the genomic DNA was determined to be 34.2 mol%. These results are consistent with characteristics of members of the genus Flavobacterium. The only isoprenoid quinone detected in strain THG-107^T was menaquinone-6 (MK-6) and the major polyamine was identified as homospermidine (82.9 %). The major polar lipid detected was phosphatidylethanolamine and the major cellular fatty acids were identified as iso-C_15:0 (26.3 %), iso-C_17:0 3OH (12.6 %) and summed feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c; 11.6 %). Flexirubin-type pigments were found to be present. Strain THG-107^T has β-glucosidase activity to convert ginsenosides Rb₁ and Rd into Gyp17 and F₂. DNA-DNA hybridization with F. denitrificans ED5^T was 52 %. Strain THG-107^T could be distinguished from F. denitrificans ED5^T and the other species of the genus Flavobacterium by its phylogenetic and genetic distinctiveness and by several phenotypic properties. Therefore, strain THG-107^T is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium kyungheensis sp. nov. is proposed (type strain THG-107^T = KACC 16219^T = LMG 26575^T).     

Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4

A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His₆-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70–75 °C and 11.0, respectively. The kinetic parameters K _m and V _max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min^?1 for l-alanine, and 9.95 mM and 702.6 μmol min^?1 for d-alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.     

Natronomonas gomsonensis sp. nov., isolated from a solar saltern

A halophilic archaeal strain, SA3^T, was isolated from sediment of a solar saltern in Gomso Bay, Republic of Korea. Cells of strain SA3^T were observed to be coccoid-shaped, to lyse in distilled water, Gram stain-negative and to form red-pigmented colonies. Strain SA3^T was found to require at least 18 % (w/v) NaCl for growth. Optimal growth was observed at 24 % (w/v) NaCl and 6 % (w/v) MgCl₂. The optimum pH and temperature for growth were determined to be pH 7.0 and 40 °C, respectively, while the strain was found to grow within pH and temperature ranges of 5.5–8.0 and 20–45 °C, respectively. The polar lipids were determined to consist of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, unidentified phosphoglycolipids and unidentified phospholipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SA3^T was most closely related to the members of the genus Natronomonas, Natronomonas moolapensis JCM 14361^T (95.2 %) and Natronomonas pharaonis JCM 8858^T (95.1 %). The genomic DNA G+C content (61.8 mol%) determined for strain SA3^T was slightly lower than those of N. moolapensis JCM 14361^T (63.4 mol%) and N. pharaonis JCM 8858^T (64.3 mol%). DNA–DNA hybridization values between N. moolapensis JCM 14361^T and N. pharaonis JCM 8858^T and strain SA3^T were <20 %. Based on phenotypic, chemotaxonomic and phylogenetic properties, we describe a new species of the genus Natronomonas, represented by strain SA3^T (=JCM 17867^T = KCTC 4088^T), for which we propose the name Natronomonas gomsonensis sp. nov.     

Actinopolyspora dayingensis sp. nov., a novel halophilic actinomycete isolated from a hypersaline lake

A novel halophilic, filamentous actinomycete, designated TRM 4064^T, was isolated from a hypersaline habitat in Sichuan Province, China. Phylogenetic analysis based on an almost-complete 16S rRNA gene sequence of strain TRM 4064^T showed that it was most closely related to Actinopolyspora mortivallis (99.1 % sequence similarity). The sequence similarities between strain TRM 4064^T and other Actinopolyspora species with validly-published names were <97.0 %. However, it had relatively low mean values for DNA–DNA relatedness with the A. mortivallis DSM 44261^T (23.2 %). Optimal growth occurred at 37 °C, pH 7.0 and in the presence of 13 % (w/v) NaCl. The whole-cell sugar pattern consists of xylose, glucose, ribose and arabinose. The predominant menaquinones are MK-10(H₄) (38.2 %), MK-9(H₄) (25.1 %), MK-9(H₂) (28.6 %) and MK-8(H₄) (7.3 %). The major fatty acids are anteiso-C_17:0 (36.9 %) and iso-C_17:0 (19.3 %). The diagnostic phospholipids detected were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylinositol (PI) and two unknown phospholipids. The G+C content of the genomic DNA of the type strain is 66.3 mol%. Strain TRM 4064^T therefore represents a novel species of the genus Actinopolyspora, for which the name Actinopolyspora dayingensis sp. nov. is proposed. The type strain is TRM 4064^T (= KCTC 19979^T = CCTCC AA 2010010^T).     

Production and purification of a hyperthermostable chitinase from Brevibacillus formosus BISR-1 isolated from the Great Indian Desert soils

A strain of Brevibacillus formosus, capable of producing a high level of chitinase, was isolated and characterized for the first time from the Great Indian Desert soils. The production of extracellularly secreted chitinase was analyzed for its biocontrol potential and optimized by varying media pH, temperature, incubation period, substrate concentrations, carbon and nitrogen sources, etc. A twofold increase in chitinase production (798 IU/mL) was achieved in optimized media containing (g l^?1) chitin 2.0, malt extract 1.5, glycerol 1.0, ammonium nitrate 0.3 %, T-20 (0.1 %) and media pH 7.0 at 37 °C. The produced enzyme was purified using a three-step purification procedure involving ultra-filtration, ammonium sulphate precipitation and adsorption chromatography. The estimated molecular weight of the purified enzyme was 37.6 kDa. The enzyme was found thermostable at higher temperatures and showed a t _½ of more than 5 h at 100 °C. Our results show that the chitinase produced by B. formosus BISR-1 is thermostable at higher temperatures.     

The production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of fructosyltransferase and glucose oxidase

Summary A technique to produce high-content fructo-oligosaccharides by the mixed- enzyme system of fructosyltransferase and glucose oxidase was investigated. The mixed-enzyme reaction was carried out in a stirred tank reactor containing 40 %(w/v) sucrose with 10 unit of fructosyltransferase and 10 unit of glucose oxidase per gram sucrose for 25 h, at 40 °C and pH 5.5. Highly concentrated fructo-oligosaccharides up to 90 % was obtained by the mixed-enzyme system.     

Cloning, expression and characterization of a phosphoglucomutase/phosphomannomutase from sphingan-producing Sphingomonas sanxanigenens

Several strains of the genus Sphingomonas produce sphingans, extracellular polysaccharides used as thickeners, emulsifiers and gelling agents. The pgmG gene from Sphingomonas sanxanigenens, which encodes a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, was cloned and sequenced. The predicted amino acid sequence of the PgmG protein possessed 460 amino acids and a calculated molecular mass of 49.8 kDa, and it was 80 % identical to PGM/PMM from S. elodea. We overexpressed pgmG in Escherichia coli, and the purified protein displayed a K _m of 0.2 mM and a V _max of 1.3 μmol min^?1 mg^?1 with glucose 1-phosphate as substrate. The catalytic efficiency (K _cat/K _m) of PgmG was about 15-fold higher for glucose 1-phosphate than for mannose 1-phosphate. Overexpression of pgmG in S. sanxanigenens resulted in a 17 ± 0.3 % increase in sphingan production to ~12.5 g l^?1.