两种耐热磷酸化酶的构建及高效催化合成红景天苷

目的:使用表达耐热蔗糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Spase和耐热纤维二糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Cpase,发酵培养后粗酶液作为催化剂,以价格低廉的蔗糖为原料合成红景天苷。方法:分别构建耐热蔗糖磷酸化酶和耐热纤维二糖磷酸化酶大肠杆菌重组菌,然后将重组菌、蔗糖、酪醇和磷酸混合,得到反应混合物,使反应混合物在45℃下转化,而产生红景天苷。结果:在耐热蔗糖磷酸化酶酶液1200 U/L、耐热纤维二糖磷酸化酶酶液500 U/L、蔗糖110 g/L、酪醇30 g/L和磷酸50 m M的浓度下,反应条件为pH 7.0、温度45℃、转速50转/分、反应时间32小时后,红景天苷浓度达到23.7 g/L。结论:本研究使用蔗糖磷酸化酶和纤维二糖磷酸化酶联合催化的工艺,成功地高收率合成了红景天苷。同时,本研究构建的耐热磷酸化酶酶活高,处理简单,为拓展糖苷类似物的合成提供了一种新的方法。     

Correlation between the Antitumor Activity of Schizophyllan and Its Triple Helix

trans-3-Methylthioacrylamide (3-MTAA-NH₂) was isolated as colorless needles from the culture broth of Streptomyces sioyaensis, a siomycin-producer. This substance is considered to be not only a new metabolite from methionine but also a new substance. The isolation and identification of 3-MTAA-NH₂, as well as the cultural conditions for production, were investigated. A variety of other Streptomyces also produced 3-MTAA-NH₂ from methionine.     

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

Cellodextrins are linear β‐1,4‐gluco‐oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP‐controlled, bottom‐up synthesis from expedient substrates is desired for their bulk production. Here, a three‐enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short‐chain (soluble) cellodextrins (DP range 3–6). The cascade reaction involves iterative β‐1,4‐glucosylation of glucose from α‐glucose 1‐phosphate (αGlc1‐P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1‐P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L^?1 is obtained under near‐complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)^?1. Through a simple two‐step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.     

Synthesis of amylose acetates and amylose sulfates with high structural uniformity

Amylose triacetate (ATA) dissolved in DMSO was partially deacetylated by 1,6-hexamethylendiamine, 1,8-octamethylendiamine, 1,12-dodecylmethylendiamine and 1,2-cyclohexyldiamine (mixture of cis and trans isomers) at 80 degrees C. The reaction kinetics of the deacetylation were studied. Differences were found in the course of the reaction depending on the type of alkylene diamine (linear or cyclic). The isolated amylose acetates were dissolved in DMF and subsequently sulfated with sulfamic acid. In the course of the sulfation, the acetyl groups acted as protective groups and were completely cleaved after reaction. The amylose acetates and sulfates obtained were studied by 13C NMR spectroscopy and elemental analysis. It could be shown, that the deacetylation of ATA with the described alkylene diamines as well as the subsequent sulfation are highly regioselective. By proceeding this reaction scheme it is possible to synthesize 6-amyloseacetate, 2,6-di-amyloseacetate and 2-amylosesulfate with a high structural uniformity.     

Bioengineering and Application of Novel Glucose Polymers

Abstract

Glucan phosphorylase, branching enzyme, and 4-α-glucanotransferase were employed to produce glucose polymers with controlled molecular size and structures. Linear or branched glucan was produced from glucose-1-phosphate by glucan phosphorylase alone or together with bracnhing enzyme, where the molecular weight of linear glucan was strictly controlled by the glucose-1-phosphate/primer molar ratio, and the branching pattern by the relative branching enzyme/glucan phosphorylase activity ratio. Cyclic glucans were produced by the cyclization reaction of 5-αglucanotransferases and branching enzyme on amylose and amylopectin. Molecular size and structure of cyclic glucan was controlled by the type of enyzyme and substrate chosen and by the reaction conditions. This in vitro approach can be used to manufacture novel glucose polymers with applicable value.     

In situ synthesis of amylose/single-walled carbon nanotubes supramolecular assembly

A supramolecular assembly of amylose and single-walled carbon nanotubes (SWNTs) was synthesized in situ through vine-twining polymerization. Raman analysis indicated that the amylose-SWNTs supramolecular assembly was formed after the polymerization and SEM images displayed the twisted ribbons in the SWNTs wrapped by amylose. The dispersion stability of the SWNTs in aqueous solutions was improved by the wrapping of short-chain amylose molecules around the SWNTs.     

纤维二糖磷酸化酶和纤维寡糖磷酸化酶的研究与应用

自然界中一些厌氧的纤维素降解菌能够产生纤维二糖磷酸化酶(Cellobiose Phosphorylase,CBP)和纤维寡糖磷酸化酶(Cellodextrin Phosphorylase,CDP)磷酸化裂解纤维二糖和纤维寡糖.CBP和CDP属于糖苷水解酶94家族(Glycoside Hydrolase Family 9...     

Enzymatic regioselective synthesis of sucrose acrylate esters

Using a protease (at 100 g l^–1) from Bacillus licheniformis, enzymatic acryloylation of sucrose (1 M) with vinyl acrylate (4 M) was carried out in anhydrous pyridine and yielded sucrose acrylate esters with more than 90% of sucrose converted in 24 h. After 5 days of reaction, the ester products consisted of 70% sucrose monoacrylate and 30% sucrose diacrylate. The monoester product was a sucrose 1-acrylate and the diester products consisted of sucrose 6,1-diacrylate and sucrose 6,1-diacrylate in the ratio of 3:2.     

应用乙酰短杆菌酶法合成2'-脱氧腺苷

胸苷和腺嘌呤在乙酰短杆菌作用下转化成胸腺嘧啶和目标产物2’-脱氧腺苷。50mL的三角瓶装液10mL,40mmol/L的底物,50mmol/L的磷酸盐缓冲液(pH7)进行反应,5%游离乙酰短杆菌在55℃下,经1h转化率就可达到65.6%。     

Diffusion of KCl in an amylose film

A method has been developed measuring the diffusion coefficient of KCl in amylose films. The films were soaked in potassium chloride solutions, then immersed in pure water and conductivity measured as a function of time. Different concentrations of the soaking solution were used and the measurements were made at several temperatures. The diffusion coefficient of KCl was found to be independent of the soaking solution KCl concentration, but found to increase with increasing temperature. The diffusion coefficient values were about one quarter of those found in water and varied from 4.8×10⁻¹⁰ to 11×10⁻¹⁰ m² s⁻¹. The activation energy of diffusion was close to that found in water. Two values for the activation energy were obtained, 20.1 and 14 kJ mol⁻¹, indicating a change in the film structure at 45 °C. Amylose films swelled equally in KCl-solutions and water. The thickness of amylose films doubled and the increase in mass was 100–200% corresponding the decrease of amylose content from about 87 to 37%, when the conditions changed from normal humidity conditions to water.     

Extrusion processing of high amylose potato starch materials

Thermoplastic starch was prepared by mixing native high amylose potato starch and normal potato starch in a Buss co-kneading extruder at starch to glycerol ratios of 100:45 and 100:30. The materials were also conditioned to different moisture contents at different relative humidities at 23 °C. After the mixing, the compounds were extruded into sheets with a Brabender laboratory extruder. The thermoplastic high amylose materials exhibited a higher melt viscosity than the normal potato starch materials when conditioned at 53% relative humidity. Increasing the moisture content in HAP from 27% to 30% (by weight) lowered the melt viscosity to the same level as that of normal potato starch with a moisture content of 28%. In general, the high amylose materials were more difficult to extrude than the thermoplastic material based on normal starch. The main extrusion problems encountered with the high amylose starch were unstable flow, insufficient melt tenacity and clogging of the die. By increasing the moisture content, increasing the compression ratio of the screw and increasing the rotation rate of the screw, the problems were reduced or eliminated. However, only with a starch to glycerol ratio of 100:45 was an acceptable extrusion result obtained. Extruded sheets of such high amylose materials had a stress at break of about 5 MPa at room temperature and 53% relative humidity, whereas the corresponding value for normal potato (thermoplastic) starch was 3 MPa. The elongation at break was also higher in the case of the high amylose material. The results are discussed in terms of residual crystallinity of the starch materials.     

Effects of amylose/amylopectin ratio on starch-based superabsorbent polymers

Biodegradable superabsorbent polymers (SAPs) were prepared by grafting acrylamide onto starches then crosslinking with N,N′-methylene-bisacrylamide. This work focused on the effects of the amylose/amylopectin ratio of starches from the same source (corn) on the grafting reactions and performance of the resultant starch-based SAPs. To characterise each SAP, the acrylamide groups grafted onto the starch were detected by FTIR; grafting ratio and grafting efficiency were evaluated by a gravimetric method; and graft position and the length of the grafted segment were investigated by NMR. The relationships between the microstructures of the starches, and the graft reactions and performance of the SAPs were studied based on the amylose content in the starches. It was found that under the same reaction conditions, the grafting ratio and efficiency increased with increasing amylose content, which corresponds with water absorption ratio. NMR results indicated that the acrylamide group mainly grafted onto C6, and that the length of the grafted segment decreased with increasing amylopectin content in general, and in particular for waxy starch. The high molecular weight and branched structure of amylopectin reduced the mobility of the polymer chains and increased viscosity, which could explain the graft reactions and performance of the starch-based SAPs.     

Synthesis of amphiphilic amylose and starch derivatives

For non-food uses starch generally is modified in order to obtain products with properties suitable for various applications. In the present work, starch and amylose were hydrophobically modified through reactions with long-chain -alkyl epoxides (C₆ and C₁₂) in DMSO solution, in the presence of NaH as a catalyst. The molar substitution (MS) was calculated from NMR spectra. Derivatives with high as well as low MS values were obtained. In order to reach MS values above 1.5, the reaction had to be run for 150–300 h. Viscosity and GPC measurements indicated that the polysaccharides were degraded in DMSO under the influence of methyl sulfinyl anion, which presumably is the active catalyst.

The derivatives were also characterized by FTIR. The ratio between the peak areas for OH stretching and alkyl stretching vibrations, respectively, in the FTIR spectra, was found to be proportional to MS values determined from NMR spectra.

The solubility of the hydrophobically modified polysaccharide in various solvents was tested. Samples having C₁₂-alkyl side chains and MS>1 were soluble in toluene. The C₆ derivatives were water soluble up to a MS value of 0.3.     

Formation and characterization of superparamagnetic cross-linked high amylose starch

A gelatinized cross-linked high amylose starch matrix with magnetic properties was synthesized via in situ formation of iron oxides inside the polymer matrix. Precipitation and multiple oxidation of ferrous ions were performed. The samples were observed using transmission and scanning electron microscopy, showing morphological changes in the magnetic and polymer phases. The iron content analysis revealed a decay from one oxidation cycle to the next one if no fresh ferrous solutions are added before the multiple oxidation. X-ray diffractograms, magnetization curves and Mössbauer spectra were also recorded for the characterization of the magnetic phase. The products exhibit superparamagnetic properties due to the presence of ferrimagnetic nanoparticles, although some other iron compounds are also present.     

基因工程菌酶法合成抗癌新药6-甲基嘌呤-2′-脱氧核苷

6-甲基嘌呤-2′-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞。本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2′-脱氧尿苷为底物合成6-甲基嘌呤-2′-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%。用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%。HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR。     

基因工程菌酶法合成抗癌新药6-甲基嘌呤-2'-脱氧核苷

6-甲基嘌呤-2'-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞.本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2'-脱氧尿苷为底物合成6-甲基嘌呤-2'-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%.用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%.HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Dosage effects of the three Wx genes on amylose synthesis in wheat endosperm

Amylose synthesis in wheat endosperm is mainly controlled by the granule-bound starch synthase of about 60 kDa, the so-called waxy (Wx) protein. The Wx proteins are the product of the Wx genes at a triplicate set of single-copy homoeoloci located on chromosomes 7A (Wx-A1), 4A (Wx-B1) and 7D (Wx-D1). Using Chinese Spring and its aneuploid lines, including nullisomic-tetrasomics, tetrasomics, ditelosomics and deletion stocks, together with single-chromosome substitution lines for these chromosomes, the effects of varying the dosage of whole chromosomes and chromosome arms, as well as the effects of null alleles, upon amylose synthesis were investigated. Nullisomic 4A and the deletion of chromosome segments carrying the Wx-B1 gene reduced the amylose content by more than 3%. A reasonable agreement was found in the substitution lines. This confirms that the absence of the Wx-B1 gene, or else substitution of this gene by its null allele, has the most striking effect on decreasing amylose synthesis. The removal of chromosomes carrying either the Wx-A1 or the Wx-D1 gene reduces the amylose content by less than 2%. A similar reduction was revealed by substitution of these two genes by the null alleles. Double dosages of chromosomes 7A, 4A and 7D did not increase amylose content, while the tetrasomic chromosomes produced more of the respective Wx proteins. This suggests that a certain level of Wx gene activity or of the Wx proteins led to the maximum amount of amylose.     

Enzymatic activities of starch synthesis in potato tubers of different sizes

The objective of this study was to determine the relationship between tuber weight and enzymatic activities involved in tuber starch synthesis. As tuber weight increased, the activities of sucrose synthetase, UDPG pyrophosphorylase, and granular starch synthetase escalated, whereas the activities of soluble starch synthetase and ADPG pyrophosphorylase stayed constant and that of phosphorylase declined. This suggests that when samples are taken to determine specific enzymatic activities, the sampling procedure should ensure that results do not vary because of differences in the tuber weight or size distribution.