来自桔青霉的阿魏酸酯酶的分离纯化、理化性质

【目的】从桔青霉的发酵液中分离纯化了胞外阿魏酸酯酶(PcFAE)并进行了酶学性质的研究,初步探讨了PcFAE对麦糟的酶解作用。【方法】利用(NH4)2SO4沉淀、DEAE-Sepharose Fast Flow离子交换层析、Phenyl Sepharose6Fast Flow疏水层析纯化得到电泳纯的阿魏酸酯酶。【结果】从该菌株的发酵液中获得一阿魏酸酯酶,该酶亚基分子量约为31kDa,全酶分子量约为58kDa。其最适pH为6.0,最适温度为45℃-65℃,在pH5.0-6.0及25℃-55℃之间,酶保持了较好的稳定性。Mg2+、Fe2+、Mn2+、Ca2+和Na+对酶活有一定的促进作用,Zn2+对PcFAE酶活有一定的抑制作用,而Cu2+、亮抑肽素、抑肽酶有显著的抑制作用,Hg2+、苯甲基磺酰氟几乎完全抑制了酶活。EDTA对PcFAE活性无明显影响。PcFAE的kcat/Km对香豆酸甲酯、芥子酸甲酯、阿魏酸甲酯、咖啡酸甲酯的值分别为823、416、103、0,PcFAE对MpCA的催化效率最高。PcFAE作用于麦糟,当5U PcFAE/g麦糟时,其阿魏酸的释放量为7.2%。【结论】获得了一阿魏酸酯酶,其理化性质与至今报道的阿魏酸酯酶有所不同,为阿魏酸酯酶的开发提供了重要的实验依据。     

Synthesis of a fluorescent boronic acid which reversibly binds to cell walls and a diboronic acid which agglutinates erythrocytes

N-(5-dimethylamino-1-napthalene sulfonyl)-3-aminobenzene boronic acid (Dns-PBA) and N,N′-$\text{bis}$-3(dihydroxylborylbenzene)adipamide (Bis-PBA) were synthesized. The former is found to reversibly associate with $\text{Bacillus}$$\text{subtilis}$, apparently through boronate diester linkages with carbohydrates on the cell surface. The latter displays the lectin-like property of agglutinating red blood cells.     

An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase (Xy1D) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls.

Diferulate esters strengthen and cross-link primary plant cell walls and help to defend the plant from invading microbes. Phenolics also limit the degradation of plant cell walls by saprophytic microbes and by anaerobic microorganisms in the rumen. We show that incubation of wheat and barley cell walls with ferulic acid esterase from Aspergillus niger (FAE-III) or Pseudomonas fluorescens (Xy1D), together with either xylanase I from Aspergillus niger, Trichoderma viride xylanase, or xylanase from Pseudomonas fluorescens (XylA), leads to release of the ferulate dimer 5-5' diFA [(E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid]. Direct saponification of the cell walls without enzyme treatment released the following five identifiable ferulate dimers (in order of abundance): (Z)-beta-(4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy)-4-hydroxy-3-methoxycinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl) -7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid, 5-5' diFA, (E,E)-4, 4'-dihydroxy-3, 5'-dimethoxy-beta, 3'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -6-methoxy-1, 2-dihydronaphthalene-2, 3-dicarboxylic acid. Incubation of the wheat or barley cell walls with xylanase, followed by saponification of the solubilized fraction, yielded 5-5'diFA and, in some cases, certain of the above dimers, depending on the xylanase used. These experiments demonstrate that FAE-III and XYLD specifically release only esters of 5-5'diFA from either xylanase-treated or insoluble fractions of cell walls, even though other esterified dimers were solubilized by preincubation with xylanase. It is also concluded that the esterified dimer content of the xylanase-solubilized fraction depends on the source of the xylanase.     

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.     

Carbohydrates and carbohydrate esters of ferulic acid released from cell walls of Lolium multiflorum by treatment with cellulolytic enzymes

41% of the cell walls from mature leaf blades of Lolium multiflorum were digested by treatment during 14 days with C₁ enzyme (cellulase) which had been purified by gel filtration and ion-exchange chromatography. Cellobiose was the main sugar released from the walls, together with some glucose and higher oligosaccharides. Considerable amounts of carbohydrate esters of ferulic and p-coumaric acids were also released. When the C₁ enzyme was further purified by isoelectric focusing, only 8% of the cell walls were digested. Purified C_x (CM-cellulase) containing β-glucosidase digested 51% of the cell walls in 16 hours: the major component detected in the soluble products was glucose together with some β (1 → 4)-xylobiose, xylose and arabinose. Higher oligosaccharides and carbohydrate esters of ferulic and p-coumaric acids were also present. It was shown that these acids were present in the cell walls mainly in the trans-configuration.     

Cellulosomal carbohydrate-binding module from Clostridium josui binds to crystalline and non-crystalline cellulose,and soluble polysaccharides

To understand the lignocellulose degradation activity of the Clostridium josui cellulosome, a carbohydrate-binding module of the scaffoldin CjCBM3 was characterized. CjCBM3 shows binding to crystalline cellulose, non-crystalline cellulose and soluble polysaccharides. The binding isotherm of CjCBM3 to acid-swollen cellulose is best fitted by the Langmuir two-site model, suggesting that there are two CjCBM3 binding sites on acid-swollen cellulose with different affinities. The second site shows lower affinity and larger binding capacity, suggesting that the cellulosome is directly targeted to the cellulose surface with high affinity, where larger amounts of the cellulosome bind to cellulose with low affinity.     

The structure of plant cell walls: v. On the binding of xyloglucan to cellulose fibers

Cell wall strength is decreased by both auxin treatment and low pH. In a recently proposed model of the plant cell wall, xyloglucan polymers are hydrogen-bonded to cellulose fibrils, forming the only noncovalent link in the network of polymers which cross-link the cellulose fibers. The decreased strength of the cell wall seen upon lowering the pH might be due to an effect of hydrogen ions on the rate of xyloglucan creep along cellulose fibers. This paper investigates binding of xyloglucan fragments to cellulose. At equilibrium, the per cent of nine- and seven-sugar xyloglucan fragments which are bound to cellulose is sensitive to both temperature and the concentration of nonaqueous solvents. However, neither the per cent of xyloglucan fragments bound to cellulose at equilibrium, nor the rate at which the xyloglucan fragments bind to cellulose, is sensitive to changes in hydrogen ion concentration. These results support the hypothesis that, within the cell wall, xyloglucan chains are connected to cellulose fibers by hydrogen bonds, but these results suggest that this interconnection between xyloglucan and cellulose is unlikely to be the point within the wall which regulates the rate of cell elongation.     

Proteins from the lichenXanthoria parietina which bind to phycobiont cell walls. Correlation between binding patterns and cell wall cytochemistry

Summary A protein fraction was isolated from the lichenXanthoria parietina which bound to the appropriate cultured phycobiont, but not to the freshly isolated symbiotic alga. The protein also appeared to discriminate between five other strains of cultured phycobionts from different lichens; phycobionts isolated from lichens in the familyTeloschistaceae bound the protein whereas phycobionts isolated from lichens in other families did not. Using cytochemical techniques, it was shown that protein binding ability was correlated with high levels of acidic polysaccharide in the cell wall, and the presence of a protein coat on the cell wall surface of the phycobiont. The possible role of this protein in recognition between lichen symbionts is briefly discussed.     

嗜热细菌Clostridium thermocellum阿魏酸酯酶基因的克隆、异源表达及酶学特性分析

【目的】阐明嗜热细菌Clostridium thermocellum Xyn Z蛋白的阿魏酸酯酶催化域的酶学特性,为其在生物质能源及其它发酵工业中的应用奠定基础。【方法】分别构建了C.thermocellum Xyn Z的阿魏酸酯酶催化域(FAE)及该阿魏酸酯酶催化域和碳水化合物结合域(FAE-CBM6)编码基因的原核表达载体,并在大肠杆菌菌株BL21(DE3)中异源表达,在此基础上分析比较了温度、pH、底物、金属离子及CBM6结合域对阿魏酸酯酶活性的影响。【结果】重组FAE酶及FAE-CBM6酶发挥催化活性的适宜pH值为5.0-9.0,适宜温度为50-70°C,它们对不同金属离子的响应有差异。【结论】在同一反应条件下,FAE-CBM6酶的酶活均比FAE高,说明CBM6结合域的存在对于阿魏酸酯酶活性有促进作用。     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious.     

The purification and characterization of 4-hydroxy-3-methoxycinnamic (ferulic) acid esterase from Streptomyces olivochromogenes.

A 4-hydroxy-3-methoxycinnamic acid (ferulic acid) esterase has been purified from the extracellular broth of cultures of Streptomyces olivochromogenes after growth on oat splet xylan. The purification procedure utilizes ion exchange on DEAE-BioGel A, anion exchange on Mono Q, gel filtration and hydrophobic interaction chromatography. The purified enzyme appeared as a single band on SDS-PAGE, with an apparent Mr of 29,000. Two bands, at pI7.9 and 8.5, were observed on isoelectric focusing. With methyl ferulate as substrate, the pH and temperature optima were 5.5 and 30 degrees C respectively, with a Km of 1.86 mM and Vmax of 0.3 mumols min-1 mg-1. The purfied enzyme released ferulic acid from de-starched wheat bran only in the presence of xylanase.     

Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus

Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.     

A truncated herpes simplex virus origin binding protein which contains the carboxyl terminal origin binding domain binds to the origin of replication but does not alter its conformation.

We have studied the DNA binding properties of a polypeptide consisting of the carboxyl terminal 37% of UL9, the herpes simplex virus type 1 (HSV-1) origin of replication binding protein. Using a Sindbis virus expression system, we expressed and partially purified this truncated form of UL9 (UL9CT) which contains the site-specific DNA binding domain. UL9CT specifically recognized UL9 binding sites on a 200 base pair DNA fragment containing the HSV origin ori(s) and appeared to bind as a dimer to each site. DNAse I footprint analysis showed that UL9CT protected the two high affinity binding sites of ori(s), but unlike full-length UL9, UL9CT did not induce a conformational change in the origin. Addition of anti-UL9CT antibody to the UL9CT-origin complex, however, caused a conformational change in the origin to be evident. Our results suggest that a domain, or domains, in the amino terminus are necessary for a UL9-induced origin conformational change to occur and that UL9-UL9 interactions between binding sites are involved.     

Characterization of a modular enzyme of exo-1,5-alpha-L-arabinofuranosidase and arabinan binding module from Streptomyces avermitilis NBRC14893

A gene encoding an alpha-L: -arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45 degrees C and was stable over the pH range of 5.0-6.5 at 30 degrees C. The enzyme hydrolyzed p-nitrophenol (PNP)-alpha-L: -arabinofuranoside but did not hydrolyze PNP-alpha-L: -arabinopyranoside, PNP-beta-D: -xylopyranoside, or PNP-beta-D: -galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-alpha-L: -arabinofuranosyl-alpha-L: -arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-alpha-L: -arabinofuranosyl-alpha-L: -arabinofuranoside and methyl 3-O-alpha-L: -arabinofuranosyl-alpha-L: -arabinofuranoside were not. These data suggested that the enzyme only cleaves alpha-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-alpha-L: -arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.     

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.     

p-Coumaroyl and feruloyl arabinoxylans from plant cell walls as substrates for ruminal bacteria.

Growth of the ruminal bacteria Ruminococcus flavefaciens FD1, Selenomonas ruminantium HD4, and Butyrivibrio fibrisolvens 49 was limited by ester-linked feruloyl and p-coumaroyl groups. The limitation of growth on phenolic acid-carbohydrate complexes varied with individual bacteria and appeared to be influenced by ability to hydrolyze carbohydrate linkages.