Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.     

Studies on kinetics and thermostability of a novel acid invertase from Fusarium solani

The present investigation deals with purification and thermal characterization of an acid invertase produced by Fusarium solani in submerged culture. The maximum enzyme activity (9.90 U mL(-1)) was achieved after 96 h of cultivation at pH 5.0 and 30 degrees C in a basal medium containing molasses (2%) as the carbon and energy source supplemented with 1% peptone. Invertase was purified by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephadex G-200. The purified enzyme was proven to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass of the enzyme was 65 kDa. The optimum pH and temperature for activity were 2.6 and 50 degrees C, respectively. The Km value for sucrose was 3.57 mM with an activation energy of 4.056 kJ mol(-1). Enthalpies of activation (DeltaH) were decreased while entropies (DeltaS) of activation increased at higher temperatures. The effects of alpha-chymotrypsin and 4 M urea were tetraphasic with periodic gain and loss of enzyme activity. A possible explanation for the thermal inactivation of invertase at higher temperatures is also discussed.     

Evolution of invertase activity in honey over two years

Invertase activity is a good parameter for evaluating honey freshness. Invertase activity evolution was determined on 57 fresh, unheated, commercially purchased Galician (northwestern Spain) floral honey samples. All honeys were stored in darkness at room temperature for up 24 months and analyzed each 6 months so as to determine the invertase activity evolution tendency for the first time. Invertase activity analysis was carried out according to Siegenthaler's method and in a simple assay, the latter showing a good precision (coefficient of variation between 0.35 and 0.66%). Initial invertase activity mean value was 163.9 (48.4-251.0) micromol of 4-nitrophenyl-alpha-D-glucopyranoside hydrolyzed/kg of honey/min. After application of the SPSS statistical package, the values of invertase activity showed five types of temporal behavior: exponential (56% of samples), linear (25% of samples), logarithmic (11% of samples), inverse (5% of samples), and quadratic (3% of samples). Linear regression equations were used to predict the invertase activity at 6, 12, 18, and 24 months from the initial Galician honeys' invertase activities; no statistical differences were found between experimental data and the activities calculated from the linear regression equations.     

孕穗期低温对小麦幼穗发育及产量的影响

为了探究孕穗期低温胁迫对小麦幼穗蔗糖代谢和内源激素含量的影响,以温度敏感性不同的小麦品种为材料,采用盆栽试验,在小麦孕穗期于人工气候室内模拟低温胁迫,测定小麦幼穗穗轴维管束数目、蔗糖代谢相关酶活性及基因表达特征、内源激素含量以及产量构成因素。结果表明,孕穗期低温胁迫后,小麦幼穗穗轴维管束数目减少,幼穗蔗糖含量增加,蔗糖磷酸合成酶(SPS)活性增强;蔗糖合成酶(SS)活性在2℃处理下升高,在0℃和-2℃处理条件下活性降低;随着处理温度的降低,尿苷二磷酸-葡萄糖焦磷酸化酶(UGP)活性与转化酶(Inv)活性下降幅度逐渐增大。孕穗期低温胁迫后,小麦幼穗小花中细胞壁转化酶基因IVR1和液泡转化酶基因IVR5表达量受到抑制,幼穗中生长素(IAA)与赤霉素(GA)含量下降,脱落酸(ABA)含量增加,小麦穗粒数和千粒重均显著下降。综上,孕穗期低温胁迫导致小麦幼穗穗轴维管束数目减少,幼穗中蔗糖积累,蔗糖代谢相关酶活性、基因表达及内源激素含量发生显著变化,严重影响了小麦产量的形成。本研究结果为小麦抗逆减灾和高产稳产研究提供了理论依据。     

Glass transition and water effects on sucrose inversion by invertase in a lactose-sucrose system

Enzymatic changes are often detrimental to quality of low-moisture foods. In the present study, effects of glass transition and water on sucrose inversion in a lactose-sucrose food model were investigated. Amorphous samples were produced by freeze-drying lactose-sucrose (2:1)-invertase (20 mg invertase/49.4 g of carbohydrate) dissolved in distilled water. Sorption isotherms were determined gravimetrically at 24 degrees C. Sucrose hydrolysis was determined by monitoring glucose content using a test kit and the amounts of fructose, glucose, and sucrose using HPLC. The glass transition temperatures, T(g), at various water contents were measured using differential scanning calorimetry (DSC). The BET and the GAB sorption models were fitted to experimental data up to a(w) 0.444 and 0.538, respectively. Water sorption and DSC results suggested time-dependent crystallization of sugars at a(w) 0.444 and above. Significant sucrose hydrolysis occurred only above T(g), concomitantly with crystallization. Sucrose hydrolysis and crystallization were not likely in glassy materials.     

土壤转化酶活性的测定方法

许多研究者^{[1,2,3,4,5,6]}的工作指出,土壤酶的活性与土壤肥力存在着一定的关系。     

Antioxidant activity and emulsion-stabilizing effect of pectic enzyme treated pectin in soy protein isolate-stabilized oil/water emulsion

The antioxidant activity of pectic enzyme treated pectin (PET-pectin) prepared from citrus pectin by enzymatic hydrolysis and its potential use as a stabilizer and an antioxidant for soy protein isolate (SPI)-stabilized oil in water (O/W) emulsion were investigated. Trolox equivalent antioxidant capacity (TEAC) was found to be positively associated with molecular weight (M(w)) of PET-pectin and negatively associated with degree of esterification (DE) of PET-pectin. PET-pectin (1 kDa and 11.6% DE) prepared from citrus pectin after 24 h of hydrolysis by commercial pectic enzyme produced by Aspergillus niger expressed higher α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, TEAC, and reducing power than untreated citrus pectin (353 kDa and 60% DE). The addition of PET-pectin could increase both emulsifying activity (EA) and emulsion stability (ES) of SPI-stabilized O/W emulsion. When the SPI-stabilized lipid droplet was coated with the mixture of PET-pectin and pectin, the EA and ES of the emulsion were improved more than they were when the lipid droplet was coated with either pectin or PET-pectin alone. The amount of secondary oxidation products (thiobarbituric acid reactive substances) produced in the emulsion prepared with the mixture of SPI and PET-pectin was less than the amount produced in the emulsion prepared with either SPI or SPI/pectin. These results suggest that PET-pectin has an emulsion-stabilizing effect and lipid oxidation inhibition ability on SPI-stabilized emulsion. Therefore, PET-pectin can be used as a stabilizer as well as an antioxidant in plant origin in SPI-stabilized O/W emulsion and thus prolong the shelf life of food emulsion.     

Expression and characterization of sweet potato invertase in Pichia pastoris

An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.     

Changes in invertase activities and reducing sugar content in sweetpotato stored at different temperatures

Cured sweetpotato roots were stored at different temperatures (4.5, 15.6, and 24 degrees C) for 7 weeks and assayed for invertase activities and reducing sugar levels during two separate years. Invertase activities and reducing sugar concentration significantly increased in the roots kept at low temperature. Of the three types of invertases assayed, acid invertase specific activity was the highest. Acid invertase was the most influential in determining reducing sugar levels in stored sweetpotato. Cultivar differences were found in invertase specific activities and reducing sugar concentration. Reducing sugar content was highly correlated to acid and total invertase activity, regardless of cultivar.     

Color formation in dehydrated modified whey powder systems as affected by compression and T(g)

Whey powders have attracted attention for use in the food industry. The Maillard reaction is a major deteriorative factor in the storage of these and other dairy food products. The objective of the present work was to further study the Maillard reaction as related to the physical structure of the matrix, either porous or mechanically compressed, or to storage above the T(g) of anhydrous whey systems. Sweet whey (W), reduced minerals whey (WRM), whey protein isolate (WPI), and whey protein concentrate (WPC) were stored in ovens at selected temperatures. Colorimetric measurements were performed with a spectrocolorimeter, thermal analyses (TGA) by means of a thermobalance, and glass transition temperature studies by DSC. The browning order in the vials and in the compressed systems followed the order W > WRM> WPC > WPI. k(w2), the slope of the second linear segment of the TGA curve, was related to the loss of water due to nonenzymatic browning (NEB). Browning development was in good relationship with this loss of weight. In the glassy state, the compressed systems developed higher rates of browning and weight loss (assigned to NEB reactions) than the porous systems. Reaction rates in both (porous and compressed) systems became similar as (T - T(g)) increased.     

盐胁迫对枸杞叶片糖代谢及相关酶活性的影响研究

试验研究盐胁迫对枸杞叶片糖代谢及相关酶活性的影响结果表明,随NaCl浓度升高和处理时间的延长,枸杞叶片多糖和可溶性糖含量显著增加(P<0.05),蔗糖含量呈上升趋势,而淀粉含量显著下降(P<0.05),还原糖含量呈下降趋势。随NaCl浓度的增加,枸杞叶片中中性转化酶活性显著降低(P<0.05);3g/kg、6g/kg NaCl处理对酸性转化酶活性影响较小,3g/kg NaCl处理降低蔗糖合成酶而对蔗糖磷酸合成酶活性基本无影响;6g/kg NaCl处理蔗糖合成酶活性随时间先降后升,而对蔗糖磷酸合成酶活性影响较小;但9g/kg NaCl处理显著降低酸性转化酶、蔗糖磷酸合成酶活性(P<0.05)。     

Influence of extreme K:Na ratios and high substrate salinity on plant metabolism of crops differing in salt tolerance

The relation between activity of soluble acid invertase and sucrose content in leaves of young bushbeans (salt sensitive) and sugarbeets (salt tolerant) and ion‐specific effects of salinity environment were investigated. For comparison the response of isolated invertase from both plant species to ion combination and ion concentration was investigated. The plants were grown in water culture under controlled conditions.

In busbean leaves invertase activity decreased while sucrose increased. K⁺ with Cl^‐ as counterion was most effective “in vivo”;. However, there was little change in invertase activity or sucrose content in sugarbeet leaves. Independent of the origin of the enzyme, invertase activity was not affected by either ion concentration or ion combination “in vitro”;. Acid invertase might be a key enzyme in the utilization of carbohydrates. The ionic effect on acid invertase activity and carbohydrate content in intact plant tissue could be a possible indicator of salt tolerance of crops.     

不同配肥方案对塿土酶活性和小麦产量的影响

以塿土为研究对象,探讨不同有机无机肥料配比对土壤酶活性和作物产量的影响。结果表明,不同有机无机肥料配比条件下,蔗糖酶、脲酶和碱性磷酸酶活性的动态变化特征没有明显改变;但对蔗糖酶、脲酶和碱性磷酸酶活性的影响显著,显著影响小麦干物质量和子粒产量。2/3CN+3/5OM处理蔗糖酶活性最高,达29.51 mg/g,分别比3/3CN、3/3CN+1/5OM、1/3CN+5/5OM和5/5OM处理提高33%、32%、89%和146%;2/3CN+3/5OM处理脲酶活性最高为0.75 mg/g,与其余处理差异达显著水平。2/3CN+3/5OM处理碱性磷酸酶活性则与其它处理差异不显著;但小麦子粒产量为5.7 t/hm2,比其它处理分别提高22%、18%、27%和45%。     

Impact of preferential interactions on thermal stability and gelation of bovine serum albumin in aqueous sucrose solutions

The influence of sucrose (0--40 wt %) on the thermal denaturation and gelation of bovine serum albumin (BSA) in aqueous solution has been studied. The effect of sucrose on heat denaturation of 1 wt % BSA solutions (pH 6.9) was measured using ultrasensitive differential scanning calorimetry. The unfolding process was irreversible and could be characterized by a denaturation temperature (T(m)), activation energy (E(A)), and pre-exponential factor (A). As the sucrose concentration increased from 0 to 40 wt %, T(m) increased from 72.9 to 79.2 degrees C, E(A) decreased from 314 to 289 kJ mol(-1), and ln(A/s(-1)) decreased from 104 to 94. The rise in T(m) was attributed to the increased thermal stability of the globular state of BSA relative to its native state because of differences in their preferential interactions with sucrose. The change in preferential interaction coefficient (Delta Gamma(3,2)) associated with the native-to-denatured transition was estimated. The dynamic shear rheology of 2 wt % BSA solutions (pH 6.9, 100 mM NaCl) was monitored as they were heated from 30 to 90 degrees C, held at 90 degrees C for either 15 or 120 min, and then cooled to 30 degrees C. Sucrose increased the gelation temperature due to thermal stabilization of the native state of the protein. The complex shear modulus (G) of cooled gels decreased with sucrose concentration when they were held at 90 degrees C for 15 min because the fraction of irreversibly denatured protein decreased. On the other hand, G of cooled gels increased with sucrose concentration when they were held at 90 degrees C for 120 min because a greater fraction of irreversibly denatured protein was formed and the strength of the protein-protein interactions increased.     

Corn protein-based thermoplastic resins: effect of some polar and amphiphilic plasticizers.

Homogeneous blends of corn gluten meal (CGM) and "polar" plasticizers (water, glycerol) or "amphiphilic" plasticizers [octanoic and palmitic acids, dibutyl tartrate and phthalate, and diacetyl tartaric acid ester of mono-diglycerides (DATEM)] were obtained by a hot-mixing procedure. The glass transition temperature (T(g)) of the blends was measured by modulated differential scanning calorimetry and dynamic mechanical thermal analysis, as a function of plasticizer type and content (0-30%, dwb). The plasticizing efficiency (i.e., decrease of T(g)) at equal molar content was found to be proportional to the molecular weight and inversely proportional to the percent of hydrophilic groups of the plasticizer. The migration rate of the plasticizers in the polymer was related to their physicochemical characteristics. It was assumed that polar substances interacted with readily accessible polar amino acids, whereas amphiphilic ones interacted with nonpolar zones, which are buried and accessible with difficulty. The temperature at which a thermoplastic resin of plasticized CGM could be formed was closely connected to the T(g) of the blend.     

不同施肥方式对夏玉米碳水化合物代谢关键酶活性的影响

以郑单958为材料,研究了4种不同施肥方式[种沟施肥 (T1) 、种沟施肥＋小口期追氮(T2)、种沟施肥＋拔节期追氮＋大喇叭口期追氮(T3)、控释肥(T4)]对夏玉米碳水化合物代谢中几种关键酶活性及产量性状的影响。结果表明,在本试验所采用的施肥方式中, T3处理不仅有利于提高植株地上部干重、叶面积指数、叶片中蔗糖磷酸合成酶活性,使源端保持较强的同化物供应能力;而且可以提高子粒蔗糖合成酶和酸性转化酶的活性,有利于库端保持较高的同化物转化能力。该处理产量性状得到改善,获得较高的穗粒数、千粒重和单位面积产量。     

钾素对食用型甘薯糖代谢相关酶活性的影响

为了探讨钾素提高甘薯块根可溶性糖含量的生理基础。选用典型的食用型甘薯品种北京553,设置不同施钾处理,于2009～2010年2个生长季在山东农业大学农学试验站进行试验。采用甘薯块根膨大过程中定期取样的方法,测定块根可溶性糖和淀粉含量及相关酶活性、功能叶蔗糖含量及相关酶活性。结果表明,与对照比较,施用钾肥能显著提高块根产量、可溶性糖及各糖组分含量,其中K2O用量为24 g/m2处理增幅最大,为最适用量。进一步研究发现,适宜供钾处理显著提高了功能叶磷酸蔗糖合成酶活性和蔗糖含量,生育期内平均增幅分别为10.31%和34.13%,同时提高了块根中蔗糖合成酶、不溶性酸性转化酶的活性,生育期内平均增幅为16.47%和3.66%,在提高源端光合产物供应的同时促进蔗糖在库端的卸载,促进块根中淀粉和可溶性糖的积累;适宜供钾处理还提高了块根中-和-淀粉酶的活性,生育期内平均增幅分别为26.06%和14.64%,促进淀粉向可溶性糖转化。此外,适宜供钾处理还显著提高了生长前期和后期块根中可溶性酸性转化酶活性、以及生长后期块根中蔗糖-蔗糖果糖基转移酶活性,促进了葡萄糖、果糖和果聚糖在块根中的积累。在甘薯收获期,块根可溶性糖和淀粉含量分别提高了13.52%和3.02%。即钾肥能够增加块根中蔗糖的供应量、促进块根对蔗糖的吸收、促进淀粉水解,是其提高块根可溶性糖含量的生理原因。     

三峡库区不同林龄柑橘土壤酶活性的演变

土壤酶驱动土壤生态系统养分的循环和控制生态系统的功能。本研究以生长年限为10、20年和30年的柑橘林0~20 cm和20~40 cm土层土壤为研究对象,主要探讨了土壤脲酶、转化酶和过氧化氢酶活性随着柑橘林龄的延长和土壤深度的增加的变化规律。结果表明,随着柑橘林龄的延长,0~20 cm土层土壤过氧化氢酶活性10年和20年样地之间无显著性差异,但都显著大于30年的样地;而0~20 cm土层土壤转化酶和脲酶活性逐渐提高,20年时达到最大值,其后又降低。随着土壤深度的增加,过氧化氢酶、转化酶和脲酶活性在3个林龄的柑橘林中都呈现显著的下降趋势。相关性分析的结果表明,土壤转化酶活性、脲酶活性都与土壤有机碳和微生物量碳氮之间都具有显著的正相关性,而过氧化酶氢活性与土壤理化特性及其微生物量之间都无显著的相关性。主成分分析结果进一步显示,土壤转化酶活性、脲酶活性、有机碳和微生物生物量碳氮均在第一主成分中具有较大的载荷,对第一主成分的贡献最大。以上结果表明脲酶和转化酶活性能够做为柑橘土壤质量变化的敏感指标。     

若尔盖湿地土壤酶活性和理化性质与微生物关系的研究

本文旨在研究若尔盖湿地土壤退化过程中四种典型土壤(泥炭土、沼泽土、草甸土、风沙土)中脲酶、过氧化氢酶、转化酶、多酚氧化酶活性及其与理化性质、微生物数量的相关性分析。结果表明:随着土壤深度增加,脲酶、转化酶活性逐渐降低,过氧化氢酶、多酚氧化酶活性逐渐升高;随着土壤退化程度的加剧,脲酶和转化酶活性总体呈下降趋势;泥炭土、沼泽土、草甸土中过氧化氢酶活性显著高于风沙土;多酚氧化酶活性随退化程度加剧呈上升趋势,但在沼泽土中活性最低。泥炭土、沼泽土、草甸土中转化酶及风沙土中过氧化氢酶与土壤理化性质、微生物数量的相关性较显著。因此土壤酶活性(脲酶、转化酶)、理化性质(含水量、有机质、全氮、全磷、全钾)、微生物数量能较好地指示当地土壤退化状况。     

Determination of molecular weight of agars and effect of the molecular weight on the glass transition

A novel procedure to determine the molecular weight (MW) and MW distributions for various agars is described. The MW values of commercial agars, an agarose, an agaropectin, and hydrolyzed agaroses were determined by size exclusion chromatography-low angle laser light scattering, using 4.0 M guanidine hydrochloride as eluent to avoid gelation. The MW for the commercial agars was between 106 400 and 243 500 with polydispersity between 1.283 and 6. 600. The MW of the agarose separated from a commercial agar was lower than that of the agaropectin. To prepare agaroses with different MW values, the obtained agarose was hydrolyzed. The MW of the agarose decreased with hydrolysis time, and the polydispersity, on the contrary, increased. The glass transition temperature (T(g)) of agarose with different MW values and that of agaropectin were measured by differential scanning calorimetry. The T(g) of the agarose was higher than that of the agaropectin with higher MW. The T(g) of agarose increased with MW.