Cloning and characterization of a sucrase from Leuconostoc mesenteroides

A sucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The cloned enzyme did not show dextransucrase or sucrose phosphorylase activity. HPLC and GC-MS analyses of the sucrase products indicated the presence of fructose and glucose in equimolar amounts. IPTG induction did not increase sucrase activity in E. coli indicating that the cloned gene may be transcribed from its own promoter. To our knowledge, this is the first sucrase cloned from L. mesenteroides that has invertase activity.     

Induction of honeybee haemolymph sucrase activity by high levels of dietary sucrose

• 1.1. Continuous feeding of adult honeybees with a 2 M sucrose syrup results in an early increase of both trehalase and sucrase activities in haemolymph, followed by a trough at day 5, then a second phase of increasing sucrase activity.
• 2.2. In the presence of chloramphenicol as an inhibitor of protein synthesis, the induction of trehalase activity is depressed but the very early increase of sucrase is not; by contrast, the second phase of increase in sucrase activity is delayed and depressed by the inhibitor.
• 3.3. These results suggest that in the earlier phases of induction, the sucrase molecules constitute a part of the trehalase complex already present whereas the second phase corresponds to the synthesis of a more specific sucrase.
• 4.4. New carbohydrates (di and trisaccharides) are produced which might be due to the transglycosylation properties of the induced enzymes.

     

Cloning of mini-mu bacteriophage in cosmids: in vivo packaging into phage lambda heads

A technique has been developed that permits the packaging of mini-Mu-carrying cosmids into phage lambda heads. This procedure has several advantages over packaging into Mu helper capsids: the amounts of DNA to be packaged can be increased, the packaging efficiency is improved, and the stability of transducing lysates is high.     

Cloning of sucrase genes from Streptococcus mutans in bacteriophage lambda

Abstract An extracellular peroxidase was purified by chromatofocusing column chromatography from the growth medium of ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium Burds BKM-1767. The enzyme was electrophoretically pure with an M _r of 45 000–47 000. It contained an easily dissociable heme, and required Mn²⁺ ions for activity. In the presence of hydrogen peroxide and Mn²⁺ it oxidized compounds such as vanillylacetone, 2,6-dimethyloxyphenol, curcumin, syringic acid, guaiacol, syringaldazine, divanillylacetone, and coniferyl alcohol. It did not oxidize veratryl alcohol. In reactions requiring Mn²⁺ and O₂, but not hydrogen peroxide, the enzyme oxidized glutathione, dithiothreitol, and NADPH with production of hydrogen peroxide. The hydrogen peroxide produced could be used as a co-substrate by ligninases such as those that oxidize veratryl alcohol, or by the peroxidase itself to oxidize lignin model compounds.     

米曲霉GX0015蔗糖酶基因克隆及生物信息学分析

在亚洲,低聚果糖的工业生产通常利用米曲霉或黑曲霉发酵蔗糖而来,而曲霉含有水解蔗糖和低聚果糖的蔗糖酶。因此要生产高纯度低聚果糖,必须抑制蔗糖酶的水解活性。本研究以工业生产低聚果糖的米曲霉菌株GX0015为研究材料,采用RT-PCR技术,克隆获得蔗糖酶基因(GenBank登录号:EU181219)。利用生物信息学手段对蔗糖酶基因进行分析:该酶为525个氨基酸残基组成的亲水性膜外蛋白;功能域分析结果显示:该酶具有信号肽序列,糖苷酶32家族N端特征序列和糖苷酶32家族特征序列;并具有糖苷酶32家族酶活性中心的NDPNG、RDP和EC保守序列。米曲霉蔗糖酶与酵母菌的转化酶在进化树上的位置最近。     

Physical characterization of mini-mu and mini-D108

Several derivatives of phages Mu and D108 have been isolated that carry an internal deletion generated by one of the IS1 components of a Tn9 transposon located in the A, B, or S gene of the prenatal phage. The deletions remove most of the lytic functions of the phage but leave intact either genes A and B or gene A and the left and the right end of the phages. These deleted derivatives, called mini-Mu and mini-D108, were physically characterized by electron microscopy and digestion with restriction enzymes. Mini-Mu and mini-D108, which carry an antibiotic resistance marker, are described and some of their genetic properties are summarized in the paper by Toussaint et al. (1981).     

枯草芽孢杆菌核黄素操纵子rib operon的克隆与表达

目的:构建产核黄素的枯草芽孢杆菌基因工程菌.方法:以穿梭载体pEB03构建核黄素操纵子的表达质粒载体pGJB13和pGJB14,与质粒pMX45分别转化产核黄素的枯草芽孢杆菌GJ07,并通过发酵摇瓶实验检测核黄素的产量.结果:得到产核黄素的工程菌GJ13 、GJ14和GJ08,在以蔗糖为碳源的发酵条件下,GJ08可产核黄素820mg/L,提高了约55%.结论:得到了产核黄素的高产菌种G J08.     

A novel sucrose/H+ symport system and an intracellular sucrase in Leishmania donovani

The flagellated form of pathogenic parasitic protozoa Leishmania, resides in the alimentary tract of its sandfly vector, where sucrose serves as a major nutrient source. In this study we report the presence of a sucrose transport system in Leishmania donovani promastigotes. The kinetics of sucrose uptake in promastigotes are biphasic in nature with both high affinity K(m) (K(m) of ～ 75 μM) and low affinity K(m) (K(m)～ 1.38 mM) components. By contrast the virulent amastigotes take up sucrose via a low affinity process with a K(m) of 2.5mM. The transport of sucrose into promastigotes leads to rapid intracellular acidification, as indicated by changes in the fluorescence of the pH indicator 2',7'-bis-(2-carboxyethyl)-5-(6) Carboxyfluorescein (BCECF). In experiments with right side-out plasma membrane vesicles derived from L. donovani promastigotes, an artificial pH gradient was able to drive the active accumulation of sucrose. These data are consistent with the operation of a H(+)-sucrose symporter. The symporter was shown to be independent of Na(+) and to be insensitive to cytochalasin B, to the flavonoid phloretin and to the Na(+)/K(+) ATPase inhibitor ouabain. However, the protonophore carbonylcyanide P- (trifluromethoxy) phenylhydrazone (FCCP) and a number of thiol reagents caused significant inhibition of sucrose uptake. Evidence was also obtained for the presence of a stable intracellular pool of the sucrose splitting enzyme, sucrase, in promastigote stage parasites. The results are consistent with the hypothesis that L. donovani promastigotes take up sucrose via a novel H(+)-sucrose symport system and that, on entering the cell, the sucrose is hydrolysed to its component monosaccharides by an intracellular sucrase, thereby providing an energy source for the parasites.     

Cloning and sequencing of a plasmid-mediated erythromycin resistance determinant from Staphylococcus xylosus

A 2.3-kb DNA fragment cloned from plasmid pCH200, the largest (52 kb) of four plasmids detected in Staphylococcus xylosus, was found to confer resistance to 14-membered ring macrolides in Bacillus subtilis and Staphylococcus aureus. DNA-sequence analysis of the fragment revealed the presence of an open-reading frame, the deduced product of which was identical to one of the two ATP-binding domains encoded by the macrolide/streptogramin-B-resistance gene msrA of Staphylococcus epidermidis. The observation that a polypeptide homologous to the C-terminus of MsrA is capable of mediating erythromycin resistance in the absence of the N-terminal region is of significance both to the evolution and functional activity of members of the ATP-binding transport super-gene family.     

The intracellular sucrase (SacA) of Zymomonas mobilis is not involved in sucrose assimilation

The intracellular sucrase SacA from Zymomonas mobilis was purified to homogeneity from a recombinant E. coli strain containing the SacA gene under an expression system. The protein was monomeric with a molecular mass of 58 kDa. The sucrase activity was maximal at 25 °C and thermal stability of the purified protein was low (50% recovery after 30 min at 46 °C ). The activation energy was low at 33 kJ mol^–1. Maximum activity was at pH 6.5. Activity was strongly inhibited (>99%) by SH blocking reagents and reducing agents slightly (10–60%) increased the activity of purified SacA. The sucrase showed a low K _M (42 mM) and k _cat (125 s^–1) which indicated its very low efficiency for sucrose hydrolysis. A mutant strain of Z. mobilis not able to grow on sucrose was isolated. This strain (ZM4S) lacked the two sucrases SacB and SacC but SacA was present in the intracellular fraction. Therefore, SacA alone is unable to allow growth Z. mobilis on sucrose.     

Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.