Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis

The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the K_m values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their k_cat/K_m values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose.     

婴儿围产期感染乙型肝炎病毒后各项血清学指标的动态变化

本文观察了102名新生儿出生后至18月龄的HBV血清学指标的动态变化,婴儿分成乙型肝炎疫苗按种组(63人)和对照组(39人), 观察期间HBsAg始终阴性的70名婴儿,出生后6、12和18月龄的抗-HBc阳性率依次为90％、30％和4.3％;而HBsAg阳转的27名婴儿,18月龄时抗-HBc全都阳性,但仅有6名婴儿在6月龄时测出IgM抗-HBc,疫苗接种组婴儿出生后1、3、6、12和18月龄的抗-HBs阳性率,依次为28.6％、76.2％、77。8％、82.5％和82.5％;对照组婴儿18月龄时抗-HBs阳性率仅为12.8％。     

马铃薯试管诱导结薯方法的改进

本研究对马铃薯试管诱导结薯的方法作了一些改进。用带有4—5个节的马铃薯无病毒茎段,在加有生长因子的MS液体培养基中作浅层静置培养,2—3周后,可长成3~2—4~2个枝条。此时,将诱导结薯的液体培养基加入同一容器内,培养1—3周后,小植株的多数茎节上开始出现气生小块茎。再经过3—4周的培养,小块茎成熟并具有发芽能力,可以收获作为无病毒超级原种加以应用。这种改进了的方法可以大大缩短无病毒小薯的生产周期,节约成本、提高产量。讨论了激素等培养条件对小块茎形成的影响和将试管诱导结薯技术应用于马铃薯无病毒种薯生产的前景。     

茶梅体细胞胚胎发生和植株的再生

在山茶属植物中,我们曾报道过金花茶和云南山茶组织培养形成胚状体和再生植株。茶梅(Camellia sasanqua)也是山茶属的一种重要的观赏植物,其植株矮小,花色鲜艳且具香味,除供观花外,还可做成不同造型的盆景供观赏,在园艺上也可用作茶花嫁接的砧木。茶梅组织培养通过体细胞胚状体产生植株,至今尚未见报道。现把部份实验结果报道如下。     

Purification and characterization of a novel monomeric glutathione peroxidase from rat liver

A novel glutathione peroxidase, which is active toward hydroperoxides of phospholipid in the presence of a detergent, has been purified to homogeneity from a rat liver postmicrosomal supernatant fraction by ammonium sulfate fractionation and three different column chromatographies. From a DE52 column, glutathione peroxidase active toward phosphatidylcholine dilinoleoyl hydroperoxides was eluted in one major and two minor peaks. The enzyme in the major peak was found to be separated from the "classic" glutathione peroxidase and glutathione S-transferases and further purified by Sephacryl S-200 and Mono Q column chromatographies. The purified enzyme was found to be homogeneous on polyacrylamide gel electrophoresis under nondenaturing conditions as well as that in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 22,000, and that by gel filtration was comparable, indicating that the enzyme protein is a single polypeptide. The purified enzyme was found to catalyze the reduction of phosphatidylcholine dilinoleoyl hydroperoxides to the corresponding hydroxy derivatives. The isoelectric point of the enzyme was found at pH 6.2, and the optimum pH for the enzyme activity was 8.0. The enzyme was active toward cumene hydroperoxide, H2O2, and 1-monolinolein hydroperoxides in the absence of a detergent. The enzyme activity toward phospholipid hydroperoxides was minute in the absence of a detergent but was remarkably enhanced by the addition of a detergent. From these results, the presently purified enzyme is obviously different from the classic glutathione peroxidase and also from phospholipid hydroperoxide glutathione peroxidase purified from pig heart (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70), though considerably similar to the latter.     

A bombesin-like peptide from skin of Sanguirana varians

Bombesin-like peptides (BLPs) are a family of neuro-endocrinic peptides that mediates a variety of biological activities. A BLP named bombesin-SV from the skin secretions of the frog, Sanguirana varians, was purified and characterized. Its amino acid sequence is pEMIFGAPMWALGHLM-NH2 determined by Edman degradation and mass spectrometry. The cDNA encoding bombesin-SV precursor was cloned from skin cDNA library of S. varians. The precursor is composed of 67 amino acid residues including a copy of mature bombesin-SV. Two predicted enzymatic processing sites (-Lys-Arg-) are flanked at the mature bombesin-SV sequence. In the in vitro myotropic contraction experiment, the synthesized bombesin-SV displayed the stimulating effect toward rat stomach strips, suggesting that bombesin-SV acts as agonist as most other BLPs do.     

A genome scan for quantitative trait loci affecting three ear traits in a White Duroc × Chinese Erhualian resource population

Chinese Erhualian pigs have larger and floppier ears compared with White Duroc pigs (small, half- or fully-pricked ears). To identify quantitative trait loci (QTL) for ear weight and area as well as erectness, a genome-wide scan with 194 microsatellites was performed in a White Duroc × Chinese Erhualian resource population (>1000 F₂ animals). Twenty-three genome-wide significant QTL and 12 suggestive QTL were identified. All QTL for ear erectness and size detected in two previous studies, bar two on SSC6 and 9, were confirmed here. The 1% genome-wide significant QTL at 70 cM on SSC5 and at 58 cM on SSC7 have profound and pleiotropic effects on the three ear traits, with Erhualian alleles increasing weight and area but decreasing erectness. Notably, the 95% confidence interval of the QTL for weight and area on SSC7 spanned only 3 cM. New QTL reaching 1% genome-wide significance were found on SSC8 (at 37 cM) for all three ear traits, on SSC4 and 16 for weight and area, and on SSCX for area. Unexpectedly, Erhualian alleles at these loci were associated with lighter and smaller or erect ear. Some new suggestive QTL were also found on other chromosome regions. Almost all the QTL for weight and area had essentially additive effects, while the QTL for erectness on SSC2, 5 and 7 showed not only additive effects but also partial dominance effects of Erhualian alleles. The two most significant QTL on SSC7 and SSC5 could be promising targets for fine mapping and identification of the causative mutations.     

A New PAMPA Model Proposed on the Basis of a Synthetic Phospholipid Membrane

The purpose of this work was to investigate the synthetic phospholipid dependence of permeability measured by parallel artificial membrane permeability assay (PAMPA) method. Three phospholipids with hydrophobic groups of different lengths and phosphorylcholine as the hydrophilic group were concisely synthesized. Ten model drug molecules were selected because of their distinct human fraction absorbed (%FA) values and various pK_a characteristics. In vitro drug permeation experiments were designed to determine the effect of the incubation time (4–20 h), pH gradient (4.6–9.32) and carbon chain length (8, 10, 12) on the drug permeability through the synthetic phospholipid membrane in the PAMPA system. The results showed that intensive and significant synthetic phospholipids dependence of permeability influenced by the length of lipid’s hydrophobic carbon chain. The effective permeability constant (P_e) of each drug increased rapidly with time, then decreased slightly after reaching the maximum; the pH gradient changed the drug permeability according to the pH-partition hypothesis for drugs with diverse pK_a values; and longer hydrophobic chains in the synthetic phospholipid membrane improved the drug permeability, as observed for all test drugs at almost all incubation time points. This newly proposed PAMPA model considered the synthetic phospholipid membrane and showed good P_e-%FA correlation for the passive transport of drugs, making it a helpful supplementary method for PAMPA systems.     

The addition of Co2+ enhances the catalytic efficiency and thermostability of recombinant glucose isomerase from Thermobifida fusca

Glucose isomerase is an important industrial enzyme that catalyzes the reversible isomerization of glucose to fructose. In this study, the effect of cobalt ions (Co²⁺) on the catalytic efficiency and thermostability of recombinant glucose isomerase from Thermobifida fusca was analyzed. The activity of glucose isomerase from engineered Escherichia coli supplemented with 1 mM Co²⁺ (C-GI) reached 41 U/ml, 2.1-fold higher than enzyme prepared from E. coli without additive (GI). The purified C-GI also exhibited an increased specific activity (23.8 U/mg compared to 12.1 U/mg for GI) and a greater thermostability (half-life of 17 h at 75 °C, 11.3-fold higher than GI (1.5 h)). The optimal temperature for C-GI shifted from 80 °C to 85 °C and demonstrated higher activity over pH 7.0–9.0. The k_cat/K_m value of C-GI (89.3 M^?1 s^?1) for the isomerization of glucose to fructose was nearly 1.75-fold higher than that of GI. In addition, the engineered cells were immobilized with the method of flocculation-cross linking. The immobilized cells supplemented with 1 mM Co²⁺ (C-IGI) had a better operational performance than cells without additives (IGI); at the end of 6 cycles, the conversion rate of C-IGI was still 43.1%, meeting the conversion rate requirement.