Cloning and characterization of a novel α-galactosidase from Bifidobacterium breve 203 capable of synthesizing Gal-α-1,4 linkage

A novel α-galactosidase gene ( aga2 ) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p -nitrophenyl-α- d -galactopyranoside ( p NPG). The K _m value for p NPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-α-1, 4-Gal-α-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-α-1,4 linkage, a novel galactosidic link formed by microbial α-galactosidase. In the presence of p NPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.     

The phosphoproteome of Aspergillus nidulans reveals functional association with cellular processes involved in morphology and secretion

We describe the first phosphoproteome of the model filamentous fungus Aspergillus nidulans. Phosphopeptides were enriched using titanium dioxide, separated using a convenient ultra‐long reverse phase gradient, and identified using a “high‐high” strategy (high mass accuracy on the parent and fragment ions) with higher‐energy collisional dissociation. Using this approach 1801 phosphosites, from 1637 unique phosphopeptides, were identified. Functional classification revealed phosphoproteins were overrepresented under GO categories related to fungal morphogenesis: “sites of polar growth,” “vesicle mediated transport,” and “cytoskeleton organization.” In these same GO categories, kinase‐substrate analysis of phosphoproteins revealed the majority were target substrates of CDK and CK2 kinase families, indicating these kinase families play a prominent role in fungal morphogenesis. Kinase‐substrate analysis also identified 57 substrates for kinases known to regulate secretion of hydrolytic enzymes (e.g. PkaA, SchA, and An‐Snf1). Altogether this data will serve as a benchmark that can be used to elucidate regulatory networks functionally associated with fungal morphogenesis and secretion. All MS data have been deposited in the ProteomeXchange with identifier PXD000715 ( http://proteomecentral.proteomexchange.org/dataset/PXD000715 ).     

微粒添加对棘白菌素B发酵过程的影响

阿尼芬净是一种新型的抗真菌药物,能够抑制各种致病念珠菌在活体内外的活性。棘白菌素B(Echinocandin B,ECB)是合成阿尼芬净的关键前体,其发酵单位的高低直接关系到阿尼芬净的价格及市场前景。文中考察了构巢曲霉在摇瓶发酵生产ECB的过程中,添加滑石粉、Al2O3、玻璃珠等微粒对其发酵单位的影响。发现微粒的粒径和添加浓度是菌体形态和ECB产量的关键影响因素,添加20 g/L滑石粉(d50=14.2μm)和7颗玻璃珠(d=6 mm)可使ECB发酵产量分别比对照提高33.2%和41.7%,达到1 262.9 mg/L和1 344.1mg/L。结果表明微粒的添加可以显著地改善丝状微生物发酵过程中的菌丝形态,提高其产物的发酵产量,为丝状微生物发酵过程的优化提供了一种重要手段。     

β-N-Acetylglucosaminidase from Aspergillus nidulans which degrades chitin oligomers during autolysis

A hexosaminidase from autolyzed cultures of Aspergillus nidulans was purified 196 fold and characterized as a beta-N-acetylglucosaminidase (EC 3.2.1.30). The enzyme has a MW of 190000, a pI of 4.3, and optimum pH of 5.0 and is unstable at temperatures above 50 degrees C. The enzyme is a glycoprotein with 19.5% sugars, mannose being the principal component. It binds strongly to chitin. The enzyme hydrolyzes different substrates. The Ki with the competitive inhibitor 2-acetamido-2-deoxy-D-gluconolactone was independent of the substrate used. The enzyme was inhibited by Hg2+, Ag+, acetate and other organic anions. The kinetics of hydrolysis of chitin oligosaccharides from 2 to 6 units was studied by HPLC. This enzyme is an exoenzyme which degraded chitin oligomers gradually with the production of N-acetylglucosamine. The hydrolysis of N-N'-diacetylchitobiose was inhibited non-competitively by glucosamine and N-acetylglucosamine. In mixtures of chitin oligosaccharides, the hydrolysis of chitobiose was competitively inhibited by each of the other oligomers.     

Partial purification of cell wall α-galactosidases and α-arabinosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

Two protein fractions with activity as α-galactosidase (EC 3.2.1.22) and α-arabinosidase (EC 3.2.1.55), respectively, were identified in the proteins of cell wall of Cicer arietinum L. cv. Castellana extracted with 3 M LiCl. These fractions were partially purified by gel filtration chromatography (Bio Gel P-150), increasing the specific arabinosidase activity 57-fold and the α-galactosidase activity 6-fold. Other protein fractions with glucosidase (EC 3.2.1.21) and glucanase (EC 3.2.1.6) activity also appeared. According to earlier authors, α-arabinosidases and α-galactosidases are related to alterations in linkages occurring in cell walls, since the enzymes are able to hydrolyze isolated wall polymers. However, our preparations hydrolyze intact cell walls only to a very limited extent, such that their participation in the autolytic processes of cell walls can be ruled out.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Coinduction of α-L-arabinofuranosidase and α-D-galactosidase formation in Trichoderma reesei RUT C-30

Abstract Formation of α-L-arabinosidase can be induced in Trichoderma reesei by growing the fungus on L-arabinose or dulcitol, and by adding L-arabinose, L-arabitol, D-galactose, or dulcitol ot non-growing mycelia. The same conditions also stimulated the formation of α-D-galactosidase, but not that of various other enzymes involved in hemicellulose degradation. The optimal inducer concentration with all compounds was 4 mM for both enzymes. Using L-arabinose and D-galactose, the induction efficiency was highest at pH 6.5, whereas induction by arabitol and dulcitol was more efficient at low pH (2.5). The addition of 50 mM glucose did not repress α-L-arabinosidase or α-D-galactosidase formation. These findings suggest coregulation of two hemicellulose side-chain cleaving enzymes in T. reesei .     

Characterization of spinach leaf α-l-arabinofuranosidases and β-galactosidases and their synergistic action on an endogenous arabinogalactan-protein

Two β-galaclosidases (β-Galase-I and -II, EC 3.2.1.23) and two α-l -arabinofuranosidases (α-l -Arafase-I and -II. EC 3.2.1.55). were purified from mesophyll tissues of spinach (Spinacia oleracea L.), using chromatography on DEAE-cellulose, lactose-conjugated Sepharose CL-4B, and Sephadex G-100, or on hydroxylapatite and Sephadex G-150. The apparent molecular mass (M_r) of β-Galase-I and -II, respectively, were estimated to be 38 000 and 58 000 on SDS-PAGE and 64 000 and 60 000 on gel-permeation chromatography, indicating that the former was a dimeric protein. The isoelectric points of β-Galase-I and -II were 6.9 and 5.2, respectively. Both enzymes hydrolyzed maximally p-nitrophenyl (PNP) β-galactoside at pH 4.3, and were activated about 2-fold in the presence of BSA (100 μg ml^?1). The activity of both enzymes was inhibited strongly by heavy metal ions and p-chloromercuriberszoate (p-CMB). d -Galactono-(1→4)-lactone and d -galactal served as potent competitive inhibitors for the enzymes. β-Galase-I and -II could be distinguished from each other in their relative rates and kinetic properties in the hydrolysis of aryl β-galactosides as well as of lactose and galacto-oligosaccharides. In particular. β-Galase-I exhibited a preferential exowise cleavage of β-1,6-galactotriose and β-1.3-galactan. α-l -Arafase-l (M_r 118000) and -II (M, 68 000) were optimally active on PNP α-l -arabinofuranoside at pH 4.8 and gave K_m values of 1.2 and 2.2 mM. respectively. l -Arabino-(1 → 4)-lactone. Ag⁺, and SDS acted as inhibitors for the isozymes. α-l Arafase-I was characterized by its activity to hydrolyze PNP β-d -xylopyranoside besides PNP α-l -arabinofuranoside. inhibition by d -xylose and d -glucono-(1 → 5)-lactone. and less sensitivity to Hg²⁺. Cu²⁺, and p-CMB. Sugar beet arabinan was hydrolyzed rapidly by α-l Arafase-II at one-half the rate for PNP α-l arabinofuranoside, while the polysaccharide was less susceptible to α-l Arafase-I. A spinach leaf arabinogalactan-protein was practically resistant to the action of β-Galases, but its susceptibility to the enzymes increased remarkably after prior hydrolysis with α-l Arafase-Il.     

Endochitinase from Aspergillus nidulans implicated in the autolysis of its cell wall

An endochitinase from centrifuged autolyzed cultures of Aspergillus nidulans has been purified 100 times. The enzyme has Mw 27,000, pI of 4.8 units, pH optimum around 5 pH units. It is unstable at temperature greater than 70 degrees C and does not have a cation requirement. It is inhibited by Hg2+, Cu2+, Ca2+ and Ag+ and it does not have muramidase activity. The enzyme depolymerizes chitin rapidly with production of high molecular weight polysaccharides, and then slowly degrades these with production of N,N'-diacetylchitobiose. The enzyme hydrolyzes N,N',N'-triacetylchitotriose with production of N,N'-diacetylchitobiose and N-acetylglucosamine and this hydrolysis is inhibited by other chitin oligomers and N-acetylglucosamine. This enzyme hydrolyzes in the same way the chitin obtained from the cell wall of Aspergillus nidulans.     

α4 but Not α3 and α7 Nicotinic Acetylcholine Receptor Subunits Are Lost from the Temporal Cortex in Alzheimer's Disease

Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age-matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for alpha3, alpha4, and alpha7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to alpha3, alpha4, and alpha7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and alpha4-like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the alpha4 subunit) were reduced in AD cases compared with control subjects (p < 0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p < 0.05) for the former two parameters. [3H]Epibatidine binding and cytoplasmic alpha4-like immunoreactivity were significantly elevated in a subgroup of control subjects (n = 4) known to have smoked prior to death (p < 0.05). There were no significant changes in alpha3- or alpha7-like immunoreactivity associated with AD or tobacco use. The selective involvement of alpha4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.     

The effect of gene tubC on the vegetative growth of benomyl-resistant strains of Aspergillus nidulans

Abstract Two benomyl-resistant mutants, benD3 tubC41 and benD4 tubC42 , of Aspergillus nidulans were isolated after UV treatment. The tubC mutations permitted good conidiation of these strains in culture media containing benomyl and were responsible for increasing their benomyl resistance levels. This implies that β3-tubulin, a product of the tubC gene, in addition to being involved in fungal conidiation, participates in the vegetative growth of the fungus. The tubC gene was located in linkage group I.     

Role of the N-terminal α-helix in biogenesis of α7 nicotinic receptors

We studied the role of the α-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in α7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the α-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of α7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (α3β4 and α4β2) and 5-HT_3A receptors also abolished their expression at the membrane. We conclude that the N-terminal α-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.     

The occurrence of β-galactosidase and β-phosphogalactosidase in Lactobacillus casei strains

Abstract Several strains of Lactobacillus casei of different origins were compared and it was observed that lactose metabolism varied from one strain to the other. Certain strains contained a β-galactosidase, others a β-phosphogalactosidase and others contain both. It was shown that the activities present in these last strains are catalyzed by two proteins differing in their electrophoretic mobilities and M _r values. Genetic divergence of the studied strains is considered.     

应用实时荧光PCR技术检测构巢曲霉的初步研究

目的 根据构巢曲霉（Aspergillus nidulans）3-磷酸甘油醛脱氢酶（Glyceraldehyde-3-phosphate dehydrogenase,GAPDH）基因特异位点设计并合成Taqman探针及引物,建立构巢曲霉实时荧光 PCR检测方法。方法 应用lasergene7.1软件对构巢曲霉与13种常见曲霉主要包括黑曲霉（A.niger）、烟曲霉（A.fumigatus）、杂色曲霉（A.versicolor）、土曲霉（A.terrus）、黄曲霉（A. flavus）、温特曲霉（A.wentii）、寄生曲霉（A. parasiticus）、泡盛曲霉（A.awamori）、米曲霉（A. oryzae ）、棒曲霉（A.cavatus）、赤曲霉（A.ruber ）、亮白曲霉（A.ochraceus）及赭曲霉（A.ochraceus）GAPDH基因序列比对分析,在特异位点设计引物和探针,建立构巢曲霉实时荧光 PCR检测方法,并对该方法进行特异性及敏感性分析。结果 用曲霉属22种41株不同曲霉及其他属的12株病原真菌验证实验表明,所建立的荧光PCR方法特异性强;检测灵敏度可达4.03×10－12μg/ml的模板DNA。 结论 应用实时荧光PCR技术能够有效检测构巢曲霉,该方法具有特异、灵敏、快速等特点,可在实际工作中应用。     

Isolation of nuclei from Aspergillus nidulans protoplasts

Intact nuclei were isolated from the protoplasts of the filamentous fungus Aspergillus nidulans. The large amounts of protoplasts required for such nuclear preparations were produced from young mycelia grown in liquid culture. For final purification of the crude nuclear fraction, a Nycodenz density-gradient centrifugation was applied. The resulting nuclei were of good purity and morphological state, as demonstrated by fluorescence microscopy and electronmicroscopy. The weight ratio of DNA:RNA:protein was 1:3.0:10.8 in the nuclear fraction.     

pH regulation of penicillin production in Aspergillus nidulans

As shown by both bioassay and high-performance liquid chromatographic (HPLC) analysis, penicillin G production by Aspergillus nidulans is subject to regulation by the pH of the growth medium. Penicillin titres were highest at alkaline pH and in strains carrying mutations in the regulatory gene pacC which mimics the effects of growth at alkaline pH. They were lowest at acid pH and in strains carrying mutations in the palA, palB, palC, palE or palF genes which mimic the effects of growth at acid pH.