Conditions for Production of Microbial Cell Flocculant by Aspergillus sojae AJ7002

Cultivation is reported on Aspergillus sojae AJ7002 which synthesized an extracellular bio-flocculant. Growth studies in shaking flasks and fermentors were conducted to obtain higher flocculant production. The highest level of polymer accumulation was attained after 48–72 hr cultivation at 30–34°C. The favorable substrates for polymer formation were casein, yeast extract, polypepton and amino acids, such as glutamic acid and alanine. The addition of saccharides to the medium was found to reduce the pH of the culture broths, and hence inhibit the accumulation of flocculant in the culture broth. The finding that the product was a single substance from the early stage of fermentation suggested that the polymer was not a product of cell autolysis. The components of the polymer which were produced by Asp. sojae did not vary even if the medium composition or culture condition changed. The addition of 2-ketogluconic acid, which is one of the constituents of the polymer increased the flocculating activity of the culture medium.     

Purification and Characterization of the Antioxidative Substance Produced by Aspergillus sojae K

An antioxidative substance produced by Aspergillus sojae K is absolutely soluble in water and strongly inhibits autoxidation of Na-ascorbate. The substance, produced extracellularly in the culture ftuid by the mold, was purified by ion exchange chromatography, gel filtration, and HPLC. The substance was purified 34-fold with an activity recovery of 38% from culture fluid. Purity of the substance was confirmed with a single peak through HPLC using an analytical column as well as a single spot on TLC. The purified substance consists of equimolar aspartic acid and glycine, indicating that the substance is a peptide. From mass spectral analysis the molecular weight was 710, but the precise sequence of the amino acids is not clear. The substance is stable at 70°C, but about 80% of the activity was lost at 80°C after 60 min. Besides, the substance is completely stable at pH 3–14. This substance efficiently suppressed the oxidation of fish oil.     

Purification and Chemical Properties of a Flocculant Produced by Paecilomyces

A new flocculant for microbial cells was purified by ethanol precipitation and gel chromatography from the culture fluid of Paecilomyces sp. I-1. Isoelectric focusing of the flocculant (PF-101) showed a single band at pH 8.5, and its molecular weight was estimated to be over 300,000 daltons by the ultra-filtration method. The results of elemental analysis, the IR spectrum and investigation of the acid hydrolysate by gas and liquid chromatography and colorimetrie analysis suggested that PF-101 was a polysaccharide composed of galactosamine. About 80% of the galactosamine residues were N-unsubstituted and 8% were N-acetylated. Studies on deaminative cleavage, periodate oxidation and Smith degradation suggested that the galactosamine residues were mainly linked by α (→4)-linkaees.     

Purification,Crystallization and Properties of Tryptophanase from Proteus rettgeri

An inducible tryptophanase was crystallized from the cell extract of Proteus rettgeri grown in a medium containing l-tryptophan. The purification procedure included ammonium sulfate fractionation, heat treatment, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystals were obtained from solutions of the purified enzyme by the addition of ammonium sulfate.

The crystalline enzyme preparation was homogeneous by the criteria of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be approximately 210,000.

The crystalline enzyme catalyzed the degradation of l-tryptophan into indole, pyruvate and ammonia in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from 5-hydroxy-l-tryptophan, 5-methyl-l-tryptophan, S-methyl-l-cysteine and l- cysteine. l-, d-Alanine, l-phenylalanine and indole inhibited pyruvate formation from these substrates.     

Chemical and Enzymatic Properties of Acid-cellulase Produced by Aspergillus niger

Some properties of a purified acid-cellulase produced by Aspergillus niger were investigated. The acid-cellulase was stable at the pH range between 4.0 and 10.0 and exhibited the highest activity toward glycol cellulose at pH 2.5. The optimum temperature of activity was measured to be 50 C, while the enzyme was inactivated above 40‘C by heating for 1 hr. Insoluble cellulose such as filter paper was difficult to be attacked by the enzyme.

Mg²⁺ and Mn²⁺ ions inhibited the activity, while Co²⁺ ion caused a slight activation.

The nitrogen content of the enzyme protein was determined to be 14.37%. The enzyme contained 378 residues of amino acids rich in acidic amino acids, 12 residues of glucosamine and 10 residues of arabinose per molecule. N-terminus was not detected by DNP-method.     

一株曲霉的一种中温糖化酶的纯化及其部分酶学特性

本研究从广西花坪自然保护区采集的土壤中筛选获得了一株产糖化酶丝状真菌菌株57-45,通过形态学观察和真菌内转录间隔区(internal transcribed spacer,ITS)序列比对分析,将其初步鉴定为曲霉属(Aspergillus sp.)的一个种。纯化真菌57-45所产的一种胞外糖化酶经过硫酸铵分级沉淀、疏水层析和阴离子交换层析三步蛋白质纯化步骤,得到在SDS-PAGE上约60kD的单一蛋白质条带,薄层层析分析表明该纯化的蛋白质水解可溶性淀粉的产物只有葡萄糖,证明该纯化的蛋白质为糖化酶。纯化的糖化酶Km值为1.9mg/mL,Vmax为4148μmol/(min·mg),最适作用pH5.5,最适作用温度50℃,在同步糖化发酵中有应用的潜力。金属离子Fe3+、Zn2+、Cu2+对酶活有较强的抑制作用,EDTA对酶活有较强的促进作用。本文结果将为进一步研究曲霉糖化酶的酶学特性提供新的材料。     

Draft Genome Sequencing and Comparative Analysis of Aspergillus sojae NBRC4239

We conducted genome sequencing of the filamentous fungus Aspergillus sojae NBRC4239 isolated from the koji used to prepare Japanese soy sauce. We used the 454 pyrosequencing technology and investigated the genome with respect to enzymes and secondary metabolites in comparison with other Aspergilli sequenced. Assembly of 454 reads generated a non-redundant sequence of 39.5-Mb possessing 13 033 putative genes and 65 scaffolds composed of 557 contigs. Of the 2847 open reading frames with Pfam domain scores of >150 found in A. sojae NBRC4239, 81.7% had a high degree of similarity with the genes of A. oryzae. Comparative analysis identified serine carboxypeptidase and aspartic protease genes unique to A. sojae NBRC4239. While A. oryzae possessed three copies of α-amyalse gene, A. sojae NBRC4239 possessed only a single copy. Comparison of 56 gene clusters for secondary metabolites between A. sojae NBRC4239 and A. oryzae revealed that 24 clusters were conserved, whereas 32 clusters differed between them that included a deletion of 18 508 bp containing mfs1, mao1, dmaT, and pks-nrps for the cyclopiazonic acid (CPA) biosynthesis, explaining the no productivity of CPA in A. sojae. The A. sojae NBRC4239 genome data will be useful to characterize functional features of the koji moulds used in Japanese industries.     

Structure and Chemical Analysis of Major Specialized Metabolites Produced by the Lichen Evernia prunastri

We performed comparative profiling of four specialized metabolites in the lichen Evernia prunastri, collected at three different geographic locations, California and Maine, USA, and Yoshkar Ola, Mari El, Russia. Among the compounds produced at high concentrations that were identified in all three specimens, evernic acid, usnic acid, lecanoric acid and chloroatranorin, evernic acid was the most abundant. Two depsidones, salazinic acid and physodic acid, were detected in the Yoshkar‐Ola collection only. The crystalline structure of evernic acid (2‐hydroxy‐4‐[(2‐hydroxy‐4‐methoxy‐6‐methylbenzoyl)oxy]‐6‐methylbenzoate) (hmb) revealed two crystallographically and conformationally distinct hmb anions, along with two monovalent sodium atoms. One hmb moiety contained an exotetradentate binding mode to sodium, whereas the other exhibited an exohexadentate binding mode to sodium. Embedded edge‐sharing {Na₂O₈}_n sodium‐oxygen chains connected the hmb anions into the full three‐dimensional crystal structure of the title compound. The crystal used for single‐crystal X‐ray diffraction exhibited non‐merohedral twinning. The data suggest the importance of the acetyl‐polymalonyl pathway products to processes of maintaining integrity of the lichen holobiont community.     

Preparation of Aroma Compounds by Microbial Transformation of Isophorone with Aspergillus niger

( ± )-cis-γ-Irone (1a), ( ± )-cis-dihydro-γ-irone (2a) and their trans- isomers (1b, 2b) were synthesized via 3,3-(Claisen) or 2,3-sigmatropic rearrangement of 1-hydroxymethyl-3,3,4- trimethyl-1-cyclo he xene (8) derivatives as each key step.     

Purification and Characterization of Enterotoxin Produced by Aeromonas sobria

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.     

出芽短梗霉新菌株RM1603产普鲁兰多糖条件优化及多糖分析

目的:出芽短梗霉RM1603是一株高产胞外多糖新菌株,通过优化其产糖条件,鉴定其多糖结构,为进一步开发利用RM1603产胞外多糖奠定理论基础。方法:以RM1603为出发菌株,在单因素分析确定最佳氮源与无机盐的基础上,利用正交试验探究RM1603最佳发酵产糖条件;薄层层析及红外光谱分析确定胞外多糖产物结构。结果:出芽短梗霉菌RM1603的初始多糖产量为28.91g/L,发酵条件优化后产糖量达到65.213g/L,提高了约2.3倍;结构分析表明RM1603的多糖产物为普鲁兰多糖。结论:出芽短梗霉RM1603是一株具有较大产糖优势,极具开发潜力的高产普鲁兰糖新菌株;奠定了开发利用RM1603生产普鲁兰多糖的理论基础。     

植物同源盒基因的克隆与功能研究

同源盒基因在植物、动物、菌物的广泛存在说明这种结构在真核生物进化的早期就已出现,并暗示其具有重要功能。本文对植物同源盒基因的克隆与功能研究进行了综述,包括同源盒基因编码蛋白的结构特点、类型,并以玉米Knl、水稻OSH1及拟南芥STM为例,介绍了同源盒基因功能研究的现状。现有证据表明,同源盒基因与植物的发育过程有关。     

Structural features of the Nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity

The debranching enzyme Nostoc punctiforme debranching enzyme (NPDE) from the cyanobacterium Nostoc punctiforme (PCC73102) hydrolyzes the α‐1,6 glycosidic linkages of malto‐oligosaccharides. Despite its high homology to cyclodextrin/pullulan (CD/PUL)‐hydrolyzing enzymes from glycosyl hydrolase 13 family (GH‐13), NPDE exhibits a unique catalytic preference for longer malto‐oligosaccharides (>G8), performing hydrolysis without the transgylcosylation or CD‐hydrolyzing activities of other GH‐13 enzymes. To investigate the molecular basis for the property of NPDE, we determined the structure of NPDE at 2.37‐Å resolution. NPDE lacks the typical N‐terminal domain of other CD/PUL‐hydrolyzing enzymes and forms an elongated dimer in a head‐to‐head configuration. The unique orientation of residues 25–55 in NPDE yields an extended substrate binding groove from the catalytic center to the dimeric interface. The substrate binding groove with a lengthy cavity beyond the ?1 subsite exhibits a suitable architecture for binding longer malto‐oligosaccharides (>G8). These structural results may provide a molecular basis for the substrate specificity and catalytic function of this cyanobacterial enzyme, distinguishing it from the classical neopullulanases and CD/PUL‐hydrolyzing enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

啤酒酵母生产的重组水蛭素的纯化及脱色

对啤酒酵母生产的重组水蛭素变异体1(rHV1)进行多步骤的纯化。首先将分泌到培养上清液中的水蛭素进行硫酸铵沉淀和SephadexG-50凝胶过滤,再用Q-SepharoseHP阴离子交换层析分离,最后用HPLCSP-5PW阳离子交换柱脱色及HPLCC8柱反相层析。真空干燥后得到的水蛭素蛋白经SPS-PAGE、N端氨基酸序列分析、抗凝血酶活力分析鉴定,证明已获得高纯度的重组水蛭素HV1制剂,为利用基因工程方法生产重组水蛭素的规模化生产及临床应用提供了依据     

Purification and Characterization of Lipid Bioflocculant Produced by Rhodococcus erythropolis

The bioflocculant produced by Rhodococcus erythropolis S-1 was found to exist as huge assemblies, the molecular mass of which is over one million daltons, composed of many polypeptides and lipids in aqueous solution. We have isolated and purified this lipid bioflocculant by ultracentrifugation, extracting with 90% acetone, and two successive silica gel chromatographies from the culture broth. It was homogeneous on silica gel thin-layer chromatography. ¹H-NMR and HPLC studies showed that it was a kind of glycolipid that contained a C₁₆ methylene chain on the average and glucose in its chemical structure. The flocculating activity against kaolin clay suspension was dependent on the Ca²⁺ concentration.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Control of postprandial hyperglycaemia by galactosyl maltobionolactone and its novel anti-amylase effect in mice

The ability to control carbohydrate digestion is useful in the treatment of diabetes mellitus and obesity. In the present study, we examined whether recently developed 4(2)-O-beta-D-galactosyl maltobionolactone (LG2O) having anti-amylase activity is able to control postprandial blood glucose concentration in mice. In addition, we tried to determine how LG2O regulates carbohydrate delivery in the gut lumen by conducting in vivo and in vitro studies. Male non-diabetic ddY mice and KK-A(y) mice, a spontaneously diabetic strain, had free access to a carbohydrate rich diet supplemented with LG2O (3 or 10 g/kg) for 0.5 hr, and blood glucose concentration was measured. LG2O suppressed any steep increase in postprandial blood glucose concentration in both ddY and KK-A(y) mice. Corresponding to the blood glucose response, LG2O also markedly suppressed any increase in postprandial plasma insulin concentration. After ingestion of the diet, LG2O produced a 1.5-3.5 fold increase in the gut contents and reducible sugar content in the small intestine but not in the stomach. Although alpha-amylase activity in the stomach was much lower compared with the activity in the small intestine, LG2O still strongly inhibited alpha-amylase activity in the stomach. In contrast, LG2O had little or no influence on alpha-amylase activity in the proximal intestine. From the in vitro carbohydrate digestion stimulation, LG2O at 7.5 mM decreased glucose production by 75% for dextrin, 25% for alpha-starch and 60% for raw starch. In conclusion, administration of LG2O inhibits carbohydrate digestion in the gut, and produces significant improvements in both blood glucose and insulin response following ingestion as part of the diet, and this evidence provides support for its therapeutic potential in treating diabetes mellitus and obesity.     

Inhibition of Proliferation of Apple Callus by Arginine and Serine

When l-arginine or l-serine at high concentrations found in the wintering plants was supplied to apple callus grown in modified White’s basal medium, concentration-dependent inhibition of proliferation was observed. Some derivatives of these amino acids also inhibited the proliferation of the callus.     

Purification and Characterization of a Xylanase Produced by Chaetomium thermophile NIBGE

Summary Xylanase was produced by growing Chaetomium thermophile NIBGE in a submerged liquid culture using wheat straw and urea as carbon and nitrogen sources respectively. The xylanase was purified to electrophoretic homogeneity after ammonium sulphate precipitation, anion exchange chromatography by FPLC and gel filtration. The molecular mass of this xylanase BII was 50 kDa. The pH and temperature optima were 6.5 and 70 °C respectively. The xylanase BII showed reasonable stability at high pH and 65 °C temperature. Some metal ions and EDTA caused little inhibition at low concentrations but complete inhibition was observed at concentrations higher than 2 mM. The K_m and V_max values with oat spelt xylan as the substrate were found to be 12.5 mg/ml and 83.3 IU/mg protein, respectively. Liberation of reducing sugars from commercial paper pulp samples suggest the feasibility of a biopulping process using this xylanase.     

Improved effects of okara atomized by a water jet system on α-amylase inhibition and butyrate production by Roseburia intestinalis

ABSTRACT

Improving the physicochemical properties of okara for various applications in foods is of great importance. Here, okara and microcrystalline cellulose (MCC) were atomized using a water jet (WJ) system. The WJ-treated okara and MCC dispersed homogeneously in water, and their median sizes in particle size distribution were 6.6 μm and 9.5 μm, respectively. The dispersions of WJ-treated okara and MCC showed high apparent viscosity and shear thinning behavior. Moreover, the inhibition of α-amylase activities by WJ-treated okara was more effective than that by untreated MCC and cellulose. Furthermore, the production of short-chain fatty acids by 32 dominant species of human gut microbes was determined. An increase in butyrate production by Roseburia intestinalis was observed in the presence of WJ-treated okara, but not in untreated okara or WJ-treated MCC. These results demonstrate that WJ system can be used on okara to increase inhibited α-amylase activities and butyrate production by gut microbiota.