Employing chimeric xylanases to identify regions of an alkaline xylanase participating in enzyme activity at basic pH

The xylanase A (XynA) from the alkaliphilic Bacillus halodurans C-125 and the xylanase B (XynB) from Clostridium stercorarium F9 were subdivided into four fragments at highly homologous regions present in their primary structures: an amino-terminal region (A or a), a region containing the putative proton donor (P or p), a region containing the putative catalytic nucleophile (N or n), and a carboxyl-terminal region (C or c). Six chimeric xylanases were constructed by the selective substitution of the four fragments using an overlapping PCR technique. Two of the six xylanases, APnc and Apnc (regions originating from XynA are denoted by upper case letters and those from XynB are denoted by lower case letters), were produced in Escherichia coli while the other four xylanases were obtained only as inclusion bodies. The APnc and Apnc chimeric enzymes were purified by column chromatography using Ni-NTA agarose and DEAE-Toyopearl. The respective pH and temperature stabilities of the purified enzymes were observed from pH 5.6 to 11.6 and up to 45 degrees C for APnc, and from pH 5.6 to 11.2 and up to 45 degrees C for Apnc. Thus, these enzymes were slightly less stable than the parental xylanases. An assessment of the pH-activity relationships for the chimeric xylanases employed p-nitrophenyl-beta-D-xylobioside as the substrate in determinations of the k(cat) values. The pK(a1) values for the APnc and Apnc chimeric enzymes were 4.3 and 4.2, respectively, which were almost identical to those for the parental xylanases. In contrast, the pK(a2) values obtained for APnc and Apnc were 9.1 and 8.5, respectively; these values fall between those for the parental xylanases, XynA (9.4) and XynB (7.8). These results indicate that the main regions necessary to maintain the high pK(a2) value of XynA locate in the A and P sections.     

A kinetic study on pH-activity relationship of XynA from alkaliphilic Bacillus halodurans C-125 using aryl-xylobiosides

Xylanase A from alkaliphilic Bacillus halodurans C-125 was expressed in Escherichia coli and purified by affinity and anion exchange chromatographies. It exhibited a strong substrate inhibition using xylan as the substrate. Its K(i) value increased with an increase in pH. The effect of pH on the enzyme activity was determined using two aryl-xylobiosides as substrates, and it was found that the enzyme had a flat k(cat)-pH curve in the pH range of 5.8-8.8. This range was different from that obtained with 0.45% xylan as previously reported (Honda, H. et al., Agric. Biol. Chem., 49, 3165-3169, 1985). The substrate inhibition was presumed to cause the difference. It has been clarified that the use of aryl-xylobiosides as substrates yields more accurate kinetic results than that of xylan.     

The substrate specificity and susceptibility to wheat inhibitor proteins of Penicillium funiculosum xylanases from a commercial enzyme preparation

Filamentous fungi produce a range of carbohydrate‐degrading enzymes, in particular hemicellulases, that are often exploited by the baking, brewing and feed industries. Controlled xylan degradation is a key component in several industrial processes, in which the efficiency of xylanases (endo‐1,4‐β‐D ‐xylan hydrolases) depends on both their substrate specificity and their susceptibility to inhibitors present in the plant material. The three major xylanases present in a commercial hemicellulase product derived from a Penicillium funiculosum fermentation broth were characterized with respect to their substrate specificity and interaction with three known xylanase inhibitor proteins (XIP‐I, TAXI I and TAXI II) present in wheat flour. XYNA (48 kDa) and XYNB (22 kDa) have significant sequence similarity with family 7 cellobiohydrolases and family 11 xylanases respectively (previous studies), while XYND (36 kDa) has similarity with family 10 xylanases (this paper). All three enzymes hydrolysed various heteroxylan substrates, including birchwood xylan and soluble wheat arabinoxylan, at 30 °C and pH 5.5. XYNA was strongly inhibited by XIP‐I, TAXI I and TAXI II (K_i = 106, 46 and 46 nM respectively); XYNB was only significantly inhibited by TAXI I (K_i = 165 nM ). In each case the inhibition of XYNA and XYNB was competitive. XYND was only inhibited by XIP‐I but, in contrast to XYNA and XYNB, the apparent inhibition was dependent on the order that xylanase, inhibitor and substrate were added to the incubation mixture; the inhibition was weak when incubations were initiated by addition of xylanase (to a mixture of substrate and XIP‐I), but was very strong when enzyme and XIP‐I were pre‐incubated before addition of substrate. These findings demonstrate that the susceptibility of individual xylanases to inhibitor proteins present in cereals is likely to be a critical factor in determining their efficiency in various commercial applications. Copyright © 2004 Society of Chemical Industry     

Loop结构引入半胱氨酸对木聚糖酶XynASP热稳定性的影响

为了探究Loop结构引入半胱氨酸对GH11家族木聚糖酶热稳定性的影响，以溶糖曲霉（Aspergillus saccharolyticus）JOP 1030-1木聚糖酶XynASP Loop结构为研究对象，通过定点突变技术，将第95位点天冬酰胺（Asn）突变成半胱氨酸（Cys），获得突变体XynN95C，并在大肠杆菌（Escherichia coli）BL21（DE3）中诱导表达。酶学性质分析结果表明，突变体XynN95C最适温度为50 ℃，酶活性半衰期t1/240 ℃为38 min，相比野生型XynASP分别提高了5 ℃、18 min，最适pH从6.0降至5.0。另外，XynN95C的金属离子耐受性较强，经Fe3+处理1 h，相对酶活性为93.9%，较野生型（65.9%）明显提高。由此得出，在Loop结构引入半胱氨酸可有效提高木聚糖酶的热稳定性，这为GH11家族木聚糖酶的热稳定性改造又提供一新思路。     

A rapid colorimetric assay for the extracellular lipase of Pseudomonas fluorescens B52 using beta-naphthyl caprylate

Conditions necessary for the determination of crude extracellular lipase activity from Pseudomonas fluorescens B52 using beta-naphthyl caprylate (beta-NC), an 8-carbon ester, as the substrate were examined. Maximum enzyme synthesis occurred at 20 degrees C in pyruvate mineral salts medium containing 1 mM-CaCl2. Bile salts were necessary for enzyme activity; 6 mM-Na taurocholate or Na deoxycholate gave maximum activity but the latter compound was inhibitory at higher concentrations. Activity was optimal in N-Tris[hydroxymethyl]methyl-2-aminoethane sulphonic acid buffer pH 8.0 at 40 degrees C. A comparison of beta-NC with beta-naphthyl butyrate (a 4-carbon ester) and beta-naphthyl myristate (a 14-carbon ester) showed that beta-NC was the best substrate; Km values of 0.0415, 0.141 and 0.200 mM and Vmax values of 67.2, 20.1 and 5.28 mumol ml-1 h-1 were obtained for those substrates respectively. The enzyme was inhibited 50% in its activity against beta-NC by 0.009 mM-EDTA, 0.0007% (w/v) mixed alkyltrimethylammonium bromide and 0.00275% (w/v) Triton X-100. The biochemical properties determined using beta-NC as substrate are consistent with those reported for the lipases of other strains of Ps. fluorescens using natural substrates.     

A152G突变对GH43家族麦氏交替单胞菌木聚糖酶XynZT-2的影响

前期研究发现GH43家族近缘物种相同位点存在天然突变.为了探究保守位点区域的基因突变对酶催化性能的影响,对来源于麦氏交替单胞菌(Alteromonas macleodii)的木聚糖酶XynZT-2利用软件计算、随机突变及天然存在的突变进行分子改造,定点突变第152位丙氨酸为甘氨酸(A152G),将原酶与突变酶基因转化到...     

Quantifying the dimensions of nanoscale organic surface layers in natural waters

Nanoscale surface films are known to develop on surfaces exposed to natural waters and have potential impacts on many environmental processes. A new method using atomic force microscopy is presented which physically removes the developed film in a defined area and then quantifies the difference in height between the film and the area where the film has been removed. The difference gives the absolute thickness of the surface film, which has not previously been measured. Suwannee River humic acid was exposed to substrates, and the surface film thickness as a function of pH and exposure time was measured. Discrete and very small colloids in the range 1-5 nm were observed as expected, and these sat on a coherent surface film, notthe original mica substrate. Low pH values of 2 gave rise to relatively thick surface films of about3 nm, although these films were not continuous at higher pH values. At pH 4.8, the film thickness increased with exposure time up to about 5 h and did not subsequently increase. The maximum film thickness measured was about 1 nm at that pH. The method is applicable to the measurement of many environmental surfaces, although resolution will depend on the substrate and film roughness.     

苦杏仁醇溶蛋白酶解抗氧化肽的制备工艺优化

以苦杏仁醇溶蛋白为原料,采用甲醛电位滴定法以水解度和自由基清除率为评价指标对风味蛋白酶、碱性蛋白酶、木瓜蛋白酶、中性蛋白酶和胃蛋白酶五种生物酶在最适条件下对苦杏仁醇溶蛋白的酶解效果进行酶的优选。在单因素实验的基础上,以酶添加量、底物浓度、p H、时间为自变量,通过四因素三水平Box-Bohnken响应面分析法对生物酶解工艺进行优化。结果表明碱性蛋白酶为酶解苦杏仁醇溶蛋白的最优选择,预测最优工艺条件为时间:4.12 h、酶添加量5208.93 U/g、底物浓度3.21%、p H 9.22,根据最终试验目的,选取条件为:时间:4.0 h、酶添加量5000.0 U/g、底物浓度3.0%、p H 9.0。通过验证试验得出响应值DH%为26.74%±0.54%,DPPH自由基清除率为97.86%±0.58%,与预测值相差较小,故此条件下酶解苦杏仁醇溶蛋白抗氧化肽为最优工艺。     

Functional Characterization of the GH10 and GH11 Xylanases from Streptomyces olivaceoviridis E-86 Provide Insights into the Advantage of GH11 Xylanase in Catalyzing Biomass Degradation

We functionally characterized the GH10 xylanase (SoXyn10A) and the GH11 xylanase (SoXyn11B) derived from the actinomycete Streptomyces olivaceoviridis E-86. Each enzyme exhibited differences in the produced reducing power upon degradation of xylan substrates. SoXyn10A produced higher reducing power than SoXyn11B. Gel filtration of the hydrolysates generated by both enzymes revealed that the original substrate was completely decomposed. Enzyme mixtures of SoXyn10A and SoXyn11B produced the same level of reducing power as SoXyn10A alone. These observations were in good agreement with the composition of the hydrolysis products. The hydrolysis products derived from the incubation of soluble birchwood xylan with a mixture of SoXyn10A and SoXyn11B produced the same products as SoXyn10A alone with similar compositions. Furthermore, the addition of SoXyn10A following SoXyn11B-mediated digestion of xylan produced the same products as SoXyn10A alone with similar compositions. Thus, it was hypothesized that SoXyn10A could degrade xylans to a smaller size than SoXyn11B. In contrast to the soluble xylans as the substrate, the produced reducing power generated by both enzymes was not significantly different when pretreated milled bagasses were used as substrates. Quantification of the pentose content in the milled bagasse residues after the enzyme digestions revealed that SoXyn11B hydrolyzed xylans in pretreated milled bagasses much more efficiently than SoXyn10A. These data suggested that the GH10 xylanases can degrade soluble xylans smaller than the GH11 xylanases. However, the GH11 xylanases may be more efficient at catalyzing xylan degradation in natural environments (e.g. biomass) where xylans interact with celluloses and lignins.     

Substrate specificity of a tripeptidase as a metalloenzyme purified from Lactococcus lactis subsp. lactis biovar. diacetylactis ATCC 13675

The peptidase purified to homogeneity from Lactococcus lactis subsp. lactis biovar. diacetylactis ATCC 13675 was considered to be an aminotripeptidase (EC 3.4.11.4) from the results of substrate specificity. The K(m) value showed a tendency to decrease with the number of alanine residues, but to increase with the number of glycine residues in the substrate tripeptide. The effects of divalent metal ions on enzyme activity were considerably different depending on the tripepride used as a substrate. In the case of Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+, there was apparent correlation between enzyme activities observed in the presence of metal ions and following metal ion replacement. The Zn2+-replaced enzyme showed almost the same K(m) and k(cat) values as the native enzyme, suggesting the enzyme to be a zinc metallopeptidase. The K(m) of the divalent metal-replaced enzyme increased in the order of Co2+, Zn2+, and Mn2+. As a result of replacement with Co2+ an enzyme having 2.3-fold higher activity compared to the native enzyme for GGF as a substrate was obtained. Thus, the change in substrate specificity observed following metal replacement may suggest a highly specific interaction between the enzyme, metal and substrate, leading to the activity expression and stability of the tripeptidase.     

Selected Properties of Polyphenol Oxidase Obtained from Ispir Sugar Bean

Polyphenol oxidase enzyme was isolated from Ispir sugar bean by ammonium sulphate precipitation and its biochemical properties were investigated. For this purpose, K_M and V_max values for optimum conditions of pH, temperature, and ionic strength were determined for catechol, catechin, and chlorogenic acid as substrates. Enzyme activities were measured spectrophotometrically at 420 nm using the same substrates at optimum conditions. K_M values were found to be 2.4875, 1.3154, and 2.2487 M for catechol, catechin, and chlorogenic acid, respectively. V_max values were 3.1480, 0.6130, and 0.5039 EU/ml.min for the same substrates, respectively. These results indicated that catechol was used as a subsrate for inhibition studies. For catechol substrate, dithiothreitol, glutathione, thiourea, and L-cysteine chlorid were inhibitors. For these inhibitors, K_i constants were calculated from Lineweaver-Burk plots and inhibiton types were estimated. Moreover, I₅₀ values were also determined. The most effective inhibitor was found to be glutathione.     

ELECTROPHORETIC STUDY OF SUBSTRATE AND pH DEPENDENCE OF ENDOPROTEOLYTIC ENZYMES IN GREEN MALT*

The endoproteolytic enzymes of malt influence several different aspects of malt and beer quality. For this reason, we are extracting and characterizing these enzymes from green malt. The proteolytic activity of a Morex green malt extract was highest at pH 3.8 with haemoglobin substrate but gelatin hydrolysis was maximal from pH 4.7 to 6.0. Endoproteolytic hydrolysis of a 55% isopropanol-soluble reduced hordein fraction was about three times slower than gelatin hydrolysis but was relatively constant over the pH range from 3.8 to 6.5, although the activity did decrease at more acidic (3.0) or basic (7.0) pH values. To study the green malt proteinases in detail, a non-denaturing electrophoretic system was developed in which substrate proteins—either gelatin, edestin or hordein—were incorporated into an electrophoretic gel. After electrophoresis and incubation of the gels at pH 3.8, 4.7, 5.5, or 6.5 to allow enzymatic hydrolysis, the separated activities were determined by using protein staining to determine where the incorporated substrate had been hydrolysed. Using this system, seven proteolytic activity bands were detected. Five of the bands were maximally active at pH 3.8 and their activities dropped quickly as the pH increased. The other two bands, which migrated more slowly, hydrolysed gelatin more rapidly than they did the other substrates tested. Their gelatinolytic activities increased as the pH was raised (by 3- to 6-fold in the pH range tested). The electrophoretic system described has proven very useful for studying the proteinases of germinating barley seeds. The results indicate that much past research on malt proteinases may not be particularly relevant to the malting and brewing industries because it was conducted under pH conditions and with substrates that are likely quite different from those in the seed during the barley germination process. By using electrophoresis to separate proteinases before analysis, we can now study their individual characteristics and thus can conduct studies more relevant to malting and brewing.     

Purification and characterization of malate dehydrogenase from Corynebacterium glutamicum

The malate dehydrogenase (MDH) (EC 1.1.1.37) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 8) matched the sequence (residues 2 to 9) of the MDH from C. glutamicum (GenBank accession no. CAC83073). The molecular mass of the native enzyme was 130 kDa. The protein was a homotetramer, with a 33-kDa subunit molecular mass. The enzyme was almost equally active both for NADU and NADPH as coenzyme on the bases of the k(cat) values at pH 6.5 which is the optimum pH for the both coenzymes. Plotting of the logarithms of the 1/Km, k(cat), and k(cat)/K(m) values with respect to oxalacetate against pH lead to speculation that imidazolium is possibly a functional group in the active center of the enzyme. Citrate activated the enzyme in the oxidation of malate to oxalacetate and inhibited it in the reverse reaction.     

Basic subsite theory assumptions may not be applicable to hydrolysis of cellooligosaccharides by almond beta-glucosidase

Steady-state kinetic parameters for the hydrolysis of cellooligosaccharides by almond beta-glucosidase were evaluated at pH 5.0 and 25 degrees C in relation to the subsite theory (K. Hiromi, Biochem. Biophys. Res. Commun., 40, 1-6, 1970). The value of k0/Km decreased monotonously with increasing degree of polymerization (DP) of the substrates (DP = 2-6). Also, the Km and k0 values for cellotriose were smaller than those for cellobiose. These DP dependencies differ from those of most amylases and glucosidases studied so far, to which the subsite theory has been successfully applied. The subsite parameters could not be consistently obtained, which suggests that one or both of the two basic assumptions of the subsite theory might not be applicable to the hydrolysis of cellooligosaccharides by the enzyme. That is, the intrinsic rate of the hydrolysis may depend on the DP and/or there may be interaction between subsites for binding the glucose residues of a substrate.     

Physicochemical and Functional Properties of Soy Protein Substrates Modified by Low Levels of Protease Hydrolysis

ABSTRACT: Endo-protease treatments achieving low degrees of hydrolysis (DH 2% and 4%) were used to improve functional properties of hexane-extracted soy flour (HESF), extruded-expelled partially defatted soy flour (EESF), ethanol-washed soy protein concentrate (SPC), and soy protein isolate (SPI). These substrates had protein dispersibility indices ranging from 11% to 89%. Functional properties, including solubility profile (pH 3 to 7), emul-sification capacity and stability, foaming capacity and stability, and apparent viscosity were determined and related to surface hydrophobicity and peptide profiles of the hydrolysates. Protein solubilities of all substrates increased as DH increased. Emulsification capacity and hydrophobicity values of the enzyme-modified HESF and EESF decreased after hydrolysis, whereas these values increased for SPC and SPI. Emulsion stability was improved for all 4% DH hydrolysates. Hydrolyzed SPC had lower foaming capacity and stability. For substrates other than SPC, foaming properties were different depending on DH. Hydrolysis significantly decreased the apparent viscosities regardless of substrate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated differences in the molecular weight profiles of the hydrolysates. HESF and EESF, which had high proportions of native-state proteins, showed minor changes in the peptide profile due to hydrolysis compared with SPC and SPI.     

海洋微生物来源木聚糖酶研究进展

由于海洋微生物生存环境的特殊性，使其所产生的酶大部分具有突出的优势和特点，这些特性使得海洋微生物来源的酶在基础研究和工业应用等方面具有巨大价值，具有独特的应用前景。木聚糖酶作为一种重要的工业酶制剂，大部分来源于陆地，该文对木聚糖酶的海洋微生物来源、生产及应用现状进行综述，以期为木聚糖酶基因的研究提供参考。     

The processing of high-molecular-weight xylanase (XynE, 110 kDa) from Aeromonas caviae ME-1 to 60-kDa xylanase (XynE60) in Escherichia coli and purification and characterization of XynE60

A xylanase gene (xynE) encoding XynE (110 kDa) was cloned from a lambda phage genomic library of Aeromonas caviae ME-1 which is a multiple-xylanase-producing bacterium. Upon nucleotide sequence analysis, we found that xynE comprises 2823 by and encodes a protein of 941 amino acid residues (104,153 Da), which was similar to endo-beta-1,4-xylanases which are categorized to glycosyl hydrolase family 10. An Escherichia coli transformant that harbored pXED30 carrying xynE produced 110-, 84-, 72-, and 66-kDa xylanases in the cell-free extract, and 72- and 66-kDa xylanases in the culture supernatant. We purified the 66-kDa xylanase to electrophoretic homogeneity from a culture supernatant by a series of column chromatographies. The calculated molecular mass of the purified xylanase determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was 60,154.50 Da, and the xylanase was designated XynE60. Analysis of the N-terminal 10 amino acid residues and the determined molecular mass of XynE60 revealed that XynE60 is a product processed at the Gly26-Gly27, and Thr565-Ala566 sites of XynE by proteolytic cleavage. XynE60 showed optimal activity at 55 degrees C and pH 8.0, and was stable below 45 degrees C and at pH 7.0-8.5. The K(m) and V(max) of XynE60 were calculated to be 8.1 mg/ml and 6897 nkat/mg, respectively.     

Production of crude xylanase from Thermoascus aurantiacus CBMAI 756 aiming the baking process

In recent years, the baking industry has focused its attention on substituting several chemical compounds with enzymes. Enzymes that hydrolyze nonstarch polysaccharides, such as xylanase, lead to the improvement of rheological properties of dough, loaf specific volume, and crumb firmness. The purpose of this study was to find a better solid-state fermentation substrate to produce high levels of xylanase and low levels of protease and amylase, which are enzymes involved in bread quality, from Thermoascus aurantiacus CBMAI 756. Wheat bran, corncob, and corn straw were used as energy sources. The enzyme extract of corncob showed high xylanase activity (130 U/mL) and low amylase and protease activity (<1 and 15 U/mL, respectively). This enzyme profile may be more profitable for the baking industry, because it results in a slower degradation of gluten. Our results confirm this finding, because the enzyme obtained by fermentation in corncob resulted in a gluten with a higher specific volume than all the other substrates that were tested. The crude xylanase presented maximum activity at a pH of 5, and the optimum temperature was 75 °C. It was stable up to 70 °C for an hour and at a pH range from 4 to 10.     

Optimization of aeration and agitation rates to improve cellulase-free xylanase production by thermotolerant Streptomyces sp. Ab106 and repeated fed-batch cultivation using agricultural waste

Thermostable cellulase-free xylanase was produced by Streptomyces sp. Ab106 using agricultural waste, sugar cane bagasse, as the substrate at 50 degrees C and pH 7.0. The central composite face-centered experimental design was applied to evaluate the optimal agitation and aeration rates in a 5-l fermentor. The highest activity (16.0+/-0.5 IU/ml) was obtained at an aeration rate of 1 vvm and an agitation rate of 150 rpm (k(L)a = 351 h(-1)). Using the repeated fed-batch cultivation technique, the maximum xylanase activity of 32+/-1 IU/ml was obtained during the second cycle of repeated fed-batch culture.     

CHARACTERIZATION OF POLYPHENOLOXIDASE FROM WILD PEAR (PYRUS ELAEGRIFOLIA)

Wild pear polyphenoloxidase (PePPO) was extracted and purified using a Sepharose 4B- l -tyrosine- p -amino benzoic acid affinity column. Optimum conditions for pH, temperature and heat inactivation were determined. At the optimum pH and temperature, K _M and V _max values for PePPO with catechol and pyrogallol were determined. The V _max/ K _M showed that PePPO has the greatest activity toward catechol. Optimum pH for PePPO was pH 6.0 using catechol as substrate. Optimum temperatures of PePPO for pyragallol and catechol were 65 and 35C, respectively. Enzyme activity decreased because of heat denaturation with increasing temperature. Inhibition of PePPO was investigated using p -aminobenzoic acid, ethyleneglycol, l -cysteine, l -tyrosine, sodium azide, p -aminobenzenesulfonamide, β-mercaptoethanol and dithiothreitol and catechol as substrate. Competitive-type inhibition was obtained with ethyleneglycol, l -cysteine, l -tyrosine, p -aminobenzenesulfonamide and dithiothreitol. Uncompetitive inhibition was obtained with β-mercaptoethanol, sodium azide and p -aminobenzoic acid. These results show that the most effective inhibitor for PePPO was dithiothreitol and that the type of inhibition depended on the origin of PPO.

PRACTICAL APPLICATIONS

In this present work, the properties of polyphenoloxidase in Pyrus elaegrifolia , including optimum temperature, optimum pH, substrate specificity and response to inhibitors, were studied.