Biosynthesis of levan and a new method for the assay of levansucrase activity

The polysaccharide levan was synthesized in a solidified agar medium containing sucrose as a source of fructose. The biosynthesis was achieved by the enzyme levansucrase (2,6-fructan–d-glucose 6-fructosyltransferase, EC 2.4.1.10), a small quantity of which was placed in circular wells cut in the agar gel. The enzyme slowly diffused through the agar–sucrose medium and the synthesis of levan was observed as circular white areas, the size of which was dependent on the time of incubation and the concentration of enzyme used.     

Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature

A thermoactive and thermostable levansucrase was purified from a newly isolated thermophilic Bacillus sp. from Thailand soil. The purification was achieved by alcohol precipitation, DEAE-Cellulose and gel filtration chromatographies. The enzyme was purified to homogeneity as determined by SDS-PAGE, and had a molecular mass of 56 kDa. This levansucrase has some interesting characteristics regarding its optimum temperature and heat stability. The optimum temperature and pH were 60 degrees C and 6.0, respectively. The enzyme was completely stable after treatment at 50 degrees C for more than 1 h, and its activity increased four folds in the presence of 5 mM Fe(2+). The optimum temperature for levan production was 50 degrees C. Contrary to other levansucrases, the one presented in this study is able to produce high molecular weight levan at 50 degrees C.     

Thermotolerant strain of Bacillus licheniformis producing lipase

A thermotolerant variant of Bacillus licheniformis strain H1 (isolated from Jordan valley soil), was highly active in degrading macromolecules, and possessed a lipase activity with a half life of 30 min at 70°C. This activity was produced during exponential growth. The extracellular crude lipase showed maximal activity at pH 10, and retained 65% of its stability at pH 12.The author is with the Department of Biological Sciences, University of Jordan, P.O. Box 2686, Amman 11181, Jordan     

Levan蔗糖酶及其在Levan果聚糖合成中的应用

Levan果聚糖是一类分子中含有大量β-(2,6)果糖苷键主链和少量β-(2,1)果糖苷键支链的聚糖。部分微生物来源的Levan果聚糖具有抗肿瘤、抗糖尿病、免疫增强、降血脂等重要的生物活性,在医药和功能食品方面具有巨大的应用潜能。由于微生物发酵液提取法产量相对较低,而化学法合成过程繁琐,Levan果聚糖的酶法合成备受关注。Levan蔗糖酶(Levansucrase,EC 2.4.1.10)属于糖苷酶家族GH68,是一类β-螺旋桨家族蛋白,其催化糖类合成遵循non-Leloir糖基转移酶机制,以蔗糖为底物转果糖基合成Levan果聚糖。部分微生物Levan蔗糖酶的分子结构及基因的表达调控已经得到阐明,Levan果聚糖的酶法合成得到广泛研究。本文综述了Levan蔗糖酶的催化机制、酶分子结构、酶基因表达调控以及酶在合成Levan果聚糖中的应用,以促进微生物Levan蔗糖酶及Levan果聚糖的研究和应用。     

Identification of a new Bacillus licheniformis strain producing a bacteriocin-like substance

The emergence of antibiotic resistance has spurred a great number of studies for development of new antimicrobials in the past decade. The purpose of this study was to screen environmental samples for Bacillus strains producing potent antimicrobial agents. A new strain, which showed strong antimicrobial activity against Staphylococcus aureus and Salmonella enterica ser. Pullorum, was isolated from soil and designated as B116. This new isolate was identified as Bacillus licheniformis by morphological, biochemical and genetic analyses. The production of bacteriocin-like substance (BLS) started at early exponential phase and achieved highest level at early stationary phase. The BLS was precipitated by ammonium sulfate and its molecular mass was determined as ～4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Culture supernatant of the new isolate exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, including Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Micrococcus luteus, Escherichia coli, and Salmonella spp. The BLS was resistant to heat, acid and alkaline treatment. Activity of the BLS was totally lost after digestion by pronase and partially lost after digestion by papain and lipase. The new isolate and relevant BLS are potentially useful in food and feed applications.     

Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity

Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues.     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

产生物表面活性剂的地衣芽孢杆菌种子培养条件研究

本文对产生物表面活性剂的油藏地衣芽孢杆菌种子培养条件进行优化.通过测定种子培养液中菌体浓度和发酵液的菌浓及表面张力,研究温度、通气量、接种龄、接种量对种子生长和发酵产生物表面活性剂的影响.确定了种子适宜培养条件为装液量100 mL/250 mL,12层纱布封口,于50 ℃、180 r/min摇床培养14 h.以10％接种量接种发酵,发酵24 h的发酵液表面张力降至最低,为22.6 mN/m.在该条件下培养种子,可缩短种子培养时间,实现提前接种发酵并高产生物表面活性剂.     

Regulation of glutamine synthetase. V. Partial purification and properties of glutamine synthetase from Bacillus licheniformis

The glutamine synthetase of Bacillus licheniformis has been obtained at about 15% purity. Sucrose gradient centrifugation gave a molecular weight value of approximately 612,000. Both l- and d-glutamate can be utilized as substrates in the biosynthetic reaction, although the l isomer was five times more active. The requirement for adenosine triphosphate (ATP) can be partially replaced by guanosine or inosine triphosphates, but not by cytidine or uridine triphosphates. The Mn(++) was required for activity, and the requirement cannot be satisfied with Mg(++). Maximal activity of the biosynthetic reaction was observed when ATP and Mn(++) were present in equimolar amounts. An excess of either reactant gave less activity. However, other purine and pyrimidine nucleotides, when added in combination with ATP, can partially substitute for ATP in attaining the equimolar ratio of nucleotide to Mn(++). A complex of ATP and Mn(++) is the preferred form of substrate. The B. licheniformis enzyme catalyzes the glutamyl transfer reaction but at a much slower rate than the Escherichia coli glutamine synthetase. Either adenosine diphosphate (ADP) or ATP can activate the glutamotransferase, although ADP is more active.     

Elevated temperature and chemical modification selectively abolishes levan forming activity of levansucrase of Zymomonas mobilis

A levansucrase (SacB) of Zymomonas mobilis was purified to electrophoretic homogeneity from a recombinant Escherichia coli. The 55 kDa enzyme hydrolysed -fructosides but not -glucosides and catalysed levan formation from sucrose as well as raffinose. The optimum temperature for polymerase activity (30°C ) was lower than that for hydrolase activity (50°C ). In contrast to other levansucrases, polymerase activity of levansucrase was inhibited by para- chloromercuribenzoate (1 mM) but with little or no effect on hydrolase activity. Selective modulation of polymerase activity by this inhibitor will be useful in revealing the mechanism of levansucrase catalysis.     

Microbial production of levansucrase for synthesis of fructooligosaccharides and levan

A newly isolated thermophilic bacterial strain from Tunisian thermal source was identified as Bacillus sp. and was selected for its ability to produce extracellular levansucrase. Following the optimization of carbon source, nitrogen source, temperature and initial pH of the growth medium in submerged liquid cultures. In fact, sucrose was found to be a good inducer of levansucrase enzymes. The optimal temperature and pH of the levansucrase were 50°C and 6.5, respectively and its activity increased four folds in the presence of 50mM Fe(2+). This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for more than 1h at pH 6.5. The half-life of the enzyme was 1h at 90°C. Crude enzyme of Bacillus sp. rich in levansucrase was established for the synthesis of fructooligosaccharides and levan. Bacillus sp. could therefore be considered as a satisfactory and promising producer of thermostable levansucrases. Contrary to other levansucrases, the one presented in the current study was able to produce high levels of levan with high molecular weight at 50°C and having an important effect as a hypoglycemic agent which was demonstrated in our previous publications (Dahech et al., 2011 [25]) and as a hypo-cholesterolemic agent which will be investigated in further research.     

Efficient production of levan using a recombinant yeast Saccharomyces cerevisiae hypersecreting a bacterial levansucrase

Journal of Industrial Microbiology & Biotechnology - Levan is a fructose polymer with diverse applications in the food and medical industries. In this study, levansucrase from Rahnella...     

Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis

We have amplified the previously cloned and sequenced genes of the bacitracin exporter (bcr), a member of the ATP-binding transport protein family, within the chromosome of the bacitracin producing Bacillus licheniformis. Amplification of the transporter genes was followed by greatly increased bacitracin resistance. Antibiotic production was enhanced at a low level of bcr genes amplification. An enlarged increase in the copy number of the bcr genes negatively affects the overall growth of bacteria.     

Crystallography of Bacillus subtilis levansucrase

Levansucrase, an exocellular enzyme, has been isolated from a high producer mutant, the BS5C4 constitutive strain, of Bacillus subtilis. Three crystalline forms have been obtained, all three belonging to the orthorhombic space group P2₁2₁2₁. The most suitable form for a three-dimensional structure investigation has cell dimensions, $\text{a = 68}\text{A}\text{?}$, $\text{b = 125}\text{A}\text{?}$, $\text{c = 54}\text{A}\text{?}$. There is one molecule in the asymmetric unit.     

Penicillinase-releasing protease of Bacillus licheniformis: purification and general properties.

The membrane penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein which differs from the exoenzyme in that it has an additional sequence of 24 amino acid residues and a phosphatidylserine at the NH2 terminus. In exponential-phase cultures, the conversion of membrane penicillinase to exoenzyme occurs at neutral and alkaline pH. An enzyme that will cleave the membrane penicillinase to yield the exoenzyme is present (in small amounts) in exponential-phase cells and is released during their conversion to protoplasts. The enzyme is found in the filtrate of a stationary-phase culture of the uninduced penicillinase-inducible strain 749 and has been purified to apparent homogeneity from this source. The protease has an approximate molecular weight of 21,500 and requires Ca2+ ions for stabilization. It has a pH optimum of 7.0 to 9.5 for hydrolysis of casein and for the cleavage of membrane penicillinase. Both activities are inhibited by diisopropylfluorophosphate; hence, the enzyme is a serine protease. This enzyme may be entirely responsible for the formation of exopenicillinase by this organism, since the other neutral and alkaline proteases of strain 749 have little, if any, activity in releasing exopenicillinase. The enzyme has been termed penicillinase-releasing protease.     

Growth promotion of groundnut by IAA producing rhizobacteria Bacillus licheniformis MML2501

B. licheniformis MML2501 which was isolated from groundnut rhizosphere soil showed increased populations on spermozphere colonisation and significantly increased the seed germination and other growth parameters in groundnut under in vitro conditions. B. licheniformis MML2501 did not solubilise phosphate but produced indole acetic acid (IAA), with a maximum of 23 μg/ml under optimised conditions such as pH 7.0, temperature 35°C, tryphtophan at a concentration of 16 mM and at 200 rpm shaken conditions. The production of IAA by B. licheniformis MML2501 was further confirmed by TLC and HPLC analyses, in which the R_f value and retention time of 0.66 and 4.1 min respectively, match with that of the authentic IAA. Seed treatment of B. licheniformis MML2501 in groundnut showed a significant increase in seed germination, other growth parameters and yield parameters under potted plant experiments.     

A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile transfructosylation reactions

This work disclosed the broad transglycosylation capability of the levansucrase from Bacillus licheniformis 8-37-0-1 for the first time. The levansucrase was firstly purified from the strain 8-37-0-1 and found to be a monomer of ∼51 kDa with KETQDYKKSY as the N-terminus. Then, the gene encoding the enzyme was cloned and it contained an ORF of 1449 nucleotides, encoding a 482 amino-acid protein with a predicted 29 amino-acid signal peptide. The deduced mature protein without the signal showed the same N-terminus to the purified enzyme. The mature enzyme was subsequently expressed in Escherichia coli. The recombinant enzyme showed similar biochemical properties to the native one. It produced maximal yield of 7.1 mg/mL levan (M_r 9.6 × 10⁶) from 0.8 M sucrose (pH 6.5) at 40 °C for 24 h in vitro. When using sucrose as the donor, the enzyme displayed a wide range of acceptor specificity and was able to transfer fructosyl to a series of sugar acceptors including hexose, pentose, β- or α-disaccharides, along with the difficult branched alcohols that have not been investigated before. Chemical structures of the resultant products were analyzed by MS and NMR spectra.