Contributions to the biotechnological production of sweeteners from stevia rebaudiana bertoni. II. Induction of stevioside accumulation in cell cultures by variation of the nutrient medium and the analysis of small amounts of stevioside

Cell cultures of Stevia rebaudiana in general do not contain stevioside when grown on their basal nutrient medium. No stable and prolonged stevioside accumulation was achieved by varying the medium components. However, in some cases a transient state was observed where more stevioside was synthesized than catabolized. The dynamic behaviour of product synthesis which led to accumulation of stevioside during only a short period was demonstrated. For accurate determination of very small amounts of stevioside, a combined TLC-HPLC method was used [1].     

Enrichment of the rebaudioside A concentration in Stevia rebaudiana extract with cyclodextrin glycosyltransferase from Bacillus licheniformis DSM 13

Stevia rebaudiana is a sweet herbaceous perennial plant, which is frequently used in the preparation of plant-based sweeteners. The demand for such sweeteners continues to increase due to purposeful nutrition and modern-day metabolic syndromes. More than 20 types of steviol glycosides provide a sweet taste, which are more than 300 times sweeter than sucrose. They are formed of two main components, namely stevioside and rebaudioside A. Only a handful of studies have dealt with Stevia rebaudiana leaf extracts, the conversion of pure stevioside into the preferred rebaudioside A is more common. The aim of this study was to enrich the rebaudioside A content of Stevia rebaudiana leaf extract using enzymatic bioconversion by applying fermented cyclodextrin glycosyltransferase from Bacillus licheniformis DSM13. Two differently processed plant materials, namely dried and lyophilized Stevia rebaudiana plants, were extracted and compared. Following the bioconversion, the rebaudioside A content was on average doubled. The maximum increase was fivefold with a 70–80% conversion of the stevioside.     

Catalytic properties of β-cyclodextrin glucanotransferase from alkalophilic<Emphasis Type="Italic">Bacillus</Emphasis> sp. BL-12 and intermolecular transglycosylation of stevioside

The catalytic properties of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilicBacillus sp. BL-12 specific for the intermolecular transglycosylation of stevioside were investigated. The molecular mass of purified β-CGTase by ultra-filtration and β-cyclodextrin polymer affinity chromatography was estimated to be 90 kDa, which is high compared to other known bacterial CGTases. The optimal pH and temperature were 9.0 and 50°C, respectively, and thermal stability at 40°C was elevated 10-fold in the presence of 1% maltodextrin. The kinetic parameters of the new β-CGTase from alkalophilicBacillus sp. BL-12 indicate that it is more suitable for transglycosylation than the cyclization reaction. Maltodextrin was the most suitable glycosyl donor for transglycosylation of stevioside. The transglycosylation of stevioside was carried out using 60 units of CGTase per gram of maltodextrin, 20 g/L stevioside as the glycosyl acceptor, and 50 g/L maltodextrin as the gycosyl donor at 40°C for 6 h, and a conversion yield of stevioside as high as 76% was obtained.     

Stevioside biosynthesis by callus,root, shoot and rooted-shoot cultures in vitro

Leaf explants of Stevia rebaudiana Bertoni (Compositae), an herb which produces the sweet ent-kaurene glycoside stevioside, were cultured in Murashige and Skoog medium with vitamins, sucrose (30 g l^–1), agar (0.9% w/v) and supplemented with naphthaleneacetic acid (NAA, 0.5 mg l^–1) and benzylaminopurine (BAP, 0.5 mg l^–1). These conditions yielded friable callus cultures. Differentiation of the callus tissue was then achieved by eliminating the agar and modulating the medium's hormone concentrations. Thus, medium containing increased auxin concentration (1.0 mg l^–1) and no cytokinin or increased cytokinin (1.0 mg l^–1) and no auxin yielded root or shoot cultures respectively. Supplementation of the shoot medium with NAA (1.0 mg ml^–1) induced shoot cultures to grow roots thereby differentiating into rooted-shoot cultures. Only the rooted-shoot cultures tasted sweet. Feedings of [2-¹⁴C]acetic acid to callus, shoot or rooted-shoot cultures demonstrated that only the rooted-shoot cultures are capable of de novo biosynthesis of the aglycone moiety of stevioside (steviol). In addition, [methyl-³H(N)steviol feedings to shoot or rooted-shoot cultures illustrated that both types of cultures are capable of the glycosylation reaction. The ability of these tissues to glycosylate steviol to stevioside was also demonstrated employing crude enzyme preparations derived from shoot or rooted-shoot cultures. These results suggest that stevioside biosynthesis is a function of tissue differentiation since both roots and leaves are required for cultured S. rebaudiana to biosynthesize stevioside from acetate, while the final biosynthetic steps can be performed at all levels of differentiation.     

High-performance liquid chromatographic and tandem mass spectrometric quantitation ofN7-methyldeoxyguanosine in methylated calf thymus DNA

Quantitation ofN7-methyldeoxyguanosine (N7-MedG) produced in thein vitro N-methyl-N-introsourea (NMU) action on calf thymus DNA has been achieved by enzymatic degradation, liquid chromatographic separation and desorption chemical ionization tandem mass spectrometry. In conjunction with the resolving power of HPLC in the separation of isomers, desorption chemical ionization tandem mass spectrometry has been utilized in determining modified nucleosides at low levels using a stable-isotope labeled compound as an established by an independent HPLC analysis of methylated calf thymus DNA. A sensitive and specific methodology for the quantitation ofN7-MedG at the picomole level using HPLC combined with tandem mass spectrometry without radioisotope labeling process is presented. The potential of the liquid chromatographic tandem mass spectrometric analysis shows the detection ofN7-MedG as a possible marker for human exposure to methylating agentsin vitro.     

Biological effects of stevioside on the survival of Escherichia colistrains and plasmid DNA

Stevioside is widely used daily in many countries as a non-caloric sugar substitute. Its sweetening power is higher than that of sucrose by approximately 250–300 times, being extensively employed as a household sweetener, or added to beverages and food products. The purpose of this study was to ascertain stevioside genotoxic and cytotoxic potentiality in different biological systems, as its use continues to increase. Agarose gel electrophoresis and bacterial transformation were employed to observe the occurrence of DNA lesions. In addition to these assays, Escherichia coli strains were incubated with stevioside so that their survival fractions could be obtained. Results show absence of genotoxic activity through electrophoresis and bacterial transformation assays and drop of survival fraction of E. coli strains deficient in rec A and nth genes, suggesting that stevioside (i) is cytotoxic; (ii) could need metabolization to present deleterious effects on cells; (iii) is capable of generating lesions in DNA and pathways as base excision repair, recombination and SOS system would be important to recover these lesions.     

连作改变土壤性状对甜叶菊产量和品质的影响

为了解连作障碍的产生机理，对甜叶菊(Stevia rebaudiana)连作后的土壤性状变化进行了研究，并探讨土壤性状与叶片干质量和甜菊糖苷之间的相关性。结果表明，连作2 a和3 a的土壤pH值、有机质、速效磷、脲酶、过氧化氢酶、蔗糖酶、磷酸酶和甜叶菊叶片干质量及甜菊糖苷组分含量均无显著差异。连作4 a后，土壤pH值、全氮和速效钾含量显著下降，分别比对照降低了10.07%、14.38%和24.79%，土壤电导率(EC)和速效磷含量显著增加，是对照的2.57和1.70倍；土壤脲酶、蔗糖酶、磷酸酶活性、微生物量碳和微生物量氮在连作4 a降到最低，比对照分别降低了63.68%、72.03%、47.43%、78.35%和41.07；多酚氧化酶则在连作4a达到最高，是对照的4.22倍；与对照相比，连作4a的叶片干质量和甜菊苷含量降低了29.51%和16.00%，莱鲍迪苷A含量则增加了22.19%。叶片干质量及甜菊糖苷含量与土壤性状间存在着相关性。因此，连作通过改变土壤性状影响甜叶菊产量和品质，生产中最大连作年限不宜超过3 a。     

Simple and efficient enzymatic transglycosylation of stevioside by β-cyclodextrin glucanotransferase from <Emphasis Type="Italic">Bacillus firmus</Emphasis>

Stevioside was subjected to 1,4-intermolecular transglycosylation using β-cyclodextrin glucanotransferase (β-CGtase) produced from an alkalophilic strain of Bacillus firmus. The reaction was carried out by traditional, ultrasound-assisted and microwave-assisted techniques. Reaction under microwave conditions was faster and was completed in 1 min yielding two 1,4 transglycosylated products, 4′-O-alpha-d-glycosyl stevioside (I) and 4′′-O-alpha-d-maltosyl stevioside (II) in 66% and 24%, respectively. The optimum transglycosylation occurred by using stevioside (1.24 mmol), β-CD (1.76 mmol) and β-CGtase (2 U/g) under microwave assisted reaction (MAR) in 5 ml sodium phosphate buffer (pH 7) at 50°C and 80 W power. MAR is therefore potentially a useful and economical method for faster transglycosylation of stevioside. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of the composition of the fibrillar part of Gracilaria verrucosa (Gracilariales,Rhodophyta) cell wall in order to prepare protoplasts

The cell wall of Gracilaria verrucosa is composed of two fractions: a matrix made of agar and a skeleton whose composition is unknown. This fibrillar part was isolated using both physical and chemical techniques. Total hydrolysis followed by gas-liquid chromatography allowed us to establish the sugar composition. Enzymatic degradations were carried out with cellulases, xylanases, agarases and pectinases. Efficiencies of the enzymatic digestions were monitored by both chemical analysis and electron microscopy. Pectinases had no effect. The fibrillar part was composed mainly of a cellulosic network that was unmasked by the xylanase action and degraded after cellulase digestion. The results suggest that a cocktail composed of agarases and cellulases can be used successfully to prepare protoplasts from Gracilaria verrucosa.     

Enzymatic determination of stevioside in Stevia rebaudiana

A simple enzymatic method is described for the determination of stevioside in Stevia rebaudiana. The method is based on the hydrolysis of stevioside with crude hesperidinase. The reaction is followed by monitoring the production of glucose with a glucose oxidase-peroxidase-2, 2′-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) system. The results for the stevioside content in S. rebaudiana leaves correlate with those obtained by other methods. The stevioside content in S. rebaudiana plants showed large variation.     

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP‐glycosyltransferase 76G1 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3‐β‐d ‐glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, the Reb A to stevioside ratio was drastically increased from 0.30 in wild‐type (WT) plants up to 1.55 in transgenic lines without any significant changes in total SGs content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S‐transferase‐tagged SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of SrUGT76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.     

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae     

Contributions to the biotechnolgical production of sweeteners from stevia rebaudiana bertoni. III. Accumulation of secondary metabolites by means of a precursor and by elicitation of cell cultures

Gibberellin A₃ fed to cell suspension cultures of Stevia rebaudiana showed a fast conversion to stevioside. The product was detected within one day after gibberellin addition and achieved its maximum concentration after one week. However, using special production media (without precursor or elicitor), stevioside was produced only two to seven weeks after inoculation [1]. Elicitation of suspension cultures was performed with Stevia specific and non-specific fungi and with yeast extract. Although production of some secondary metabolites was induced, stevioside was not synthesized.     

樟子松固沙林更新迹地草本植物多样性及其对土壤理化性质的影响

以科尔沁沙地南缘的彰武县章古台万亩林为研究对象,野外取样调查和实验室检测分析相结合,以樟子松固沙林采伐迹地为对照,研究不同植被恢复类型下樟子松固沙林更新迹地生境的改良效果,探讨植被重建后植物多样性及土壤理化性质的响应。结果表明:1)樟子松(Pinus sylvestris)固沙林更新迹地植被重建后,草本植物物种多样性增加,林下植物偶见种数目增多,13种草本植物在8个样地中未重复出现;2)与采伐迹地相比,不同植被重建类型土壤理化性质均有所改善,土壤容重、田间持水量、有机质、全氮、碱解氮、速效钾在彰武小钻杨(Populus xiaozhuanica)、五角枫(Acer mono)林地改良效果较好,全钾在红刺榆林(Hemiptelea davidii)地含量明显提高、山杏(Armeniaca sibirica)林地的有效磷相比采伐迹地明显提高,且均表现为上层改良效果优于下层;3)不同植被恢复类型土壤理化性质间存在显著差异,随着物种多样性的增加,土壤理化性质逐渐改善,土壤容重、田间持水量、有机质、全磷与植物多样性具有显著的相关性,土壤理化性质的与植物多样性相互作用,共同促进生态系统正向演替。研究结果为控制科尔沁沙地土壤沙化,加速该区生态系统的恢复与重建提供理论依据。     

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.     

Cyclic nucleotide metabolism during amphibian forelimb regeneration

Summary Concentrations of the cyclic nucleotides in regenerating limb tissues change in a manner which suggests that they might mediate neural or endocrine influences upon specific developmental events. Since modulation of the role of cAMP within this process can be achieved through cAMP phosphodiesterase, enzymatic activity, relative intracellular distribution, and the kinetic parameters of this enzyme were examined at several stages of limb regeneration in adultNotophthalmus viridescens. Both forms of the phosphodiesterase displayed decreased activity about the time of bud formation. Total phosphodiesterase activity was reduced between 66% and 85% (as compared to intact limbs) between wound healing and palette stages. Relative intracellular distribution (soluble vs. particulate), however, remained essentially constant, 93%–98% soluble for the highK _m form and 61%–71% soluble for the lowK _m form of the enzyme, throughout this process. The apparentK _m of the highK _m form increased more than 2-fold during wound healing then fell to approximately 10% (0.7–1.1 M) of the value of intact limbs (8.3 M) during dedifferentiation and bud formation. A return to pre-amputational levels was subsequently achieved. In contrast, the apparentK _m of the lowK _m form increased (from 0.064 to 0.86 M) during dedifferentiation and began decreasing thereafter. These results are consistent with the hypothesis that one or more mechanisms are operating to modify either the quantity, activity, or physical characteristics of the cAMP phosphodiesterases and that such changes are instrumental in regulating endogenous concentrations of cAMP in limb tissues during regeneration.     

Large-scale degumming of ramie fibre using a newly isolated <Emphasis Type="Italic">Bacillus pumilus</Emphasis> DKS1 with high pectate lyase activity

A combined (enzymatic and chemical) process using a Bacillus pumilus strain (DKS1), isolated from the soil, was used to degum ramie bast fibres. After 24 h of incubation with the isolated pectinolytic strain using a low-cost medium, the weight loss of the ramie fibre was found to be 25% under small scale. High activity of pectate lyase was detected in the culture supernatants; 400 kg of ramie fibres was degummed with 24% weight loss in large-scale degumming under field conditions. No cellulase activity was found. Microbial intervention followed by mild (0.1%) alkali treatment showed high percentage of weight loss from the ramie fibre. Bacterial degumming followed by chemical treatment resulted in an increase of single fibre tenacity (cN/tex) by more than 20.81% as compared to non-degummed (decorticated) fibre samples. Scanning electron micrographs (SEM) and fluorescence microscope showed that after Bacillus pumilus DKS1 treatment the surface of the decorticated ramie fibre becomes very smooth. These results indicate the process provides an economical and eco-friendly method for the small scale as well as large-scale degumming of decorticated ramie fibre. This study has great relevance to the textile as well as paper industry.     

Ultrastructural differences between wall apices of growing and non-growing hyphae ofSchizophyllum commune

Summary Newly synthesized chitin at the hyphal apex ofSchizophyllum commune was shown to be highly susceptible to chitinase degradation and solubilization by dilute mineral acid. With time this chitin became gradually more resistant to these treatments. With a combination of the shadow-cast technique and electron microscopic autoradiography it could be shown that this process occurred as the newly synthesized chitin moved into subapical parts of growing hyphae but also in non-growing apices which had ceased growth after incorporation of theN-acetyl[6-³H]glucosamine. These results are in agreement with a model which explains apical morphogenesis by assuming that the newly synthesized wall material at the apex is plastic due to the presence of individual polymer chains but becomes rigidified because of subsequent physical and chemical changes involving these polymers.Dedicated to Dr. A.Quispel, Professor of Botany at the University of Leiden, on occasion of his retirement.     

The cell factory approach toward biotechnological production of high-value chitosan oligomers and their derivatives: an update

Chitin is one of the most abundant renewable resources, and chitosans, the partially deacetylated derivatives of chitin, are among the most promising functional biopolymers, with superior material properties and versatile biological functionalities. Elucidating molecular structure–function relationships and cellular modes of action of chitosans, however, it is challenging due to the micro-heterogeneity and structural complexity of polysaccharides. Lately, it has become apparent that many of the biological activities of chitosan polymers, such as in agricultural plant disease protection or in mediating scar-free wound healing, may be attributed to oligomeric break-down products generated by the action of chitosanolytic hydrolases present in the target tissues, such as human chitotriosidase. Consequently, the focus of current research is shifting toward chitosan oligomers so that the availability of well-defined chitosan oligosaccharides (COS) becomes a bottleneck. Well-known ways of producing COS use physical and/or chemical means for the partial depolymerization of chitosan polymers, typically leading to broad mixtures of COS varying in their degrees of polymerization (DP) and acetylation (DA), and with more or less random patterns of acetylation (PAs). Even after chromatographic separation according to DP and DA, such mixtures are of limited value to elucidate structure–function relationships and modes of action. More recently, enzymatic means using chitinases and/or chitosanases, and sometimes chitin deacetylases, have been proposed as these can be more tightly controlled and yield slightly better defined mixtures of COS. An alternative would be chemical synthesis of COS which in principle would allow for full structural control, but protocols for it are lengthy, costly, and not yet well developed, and yields are low. Synthetic biology now allows to develop today’s in vitro bio-refinery approaches into in vivo cell factory approaches for the biotechnological production of defined COS using recombinant microbial strains expressing chitin oligomer synthases and chitin oligomer deacetylases. In this review, we will describe the state-of-the-art of this cell factory approach, as a basis for upcoming developments. We will briefly describe traditional chemical protocols and enzymatic production of COS as a background to the more detailed presentation of what has been achieved through in vivo biosynthesis. We will only briefly describe those as a background to the more detailed presentation of what has been achieved through in vivo biosynthesis. We will also touch on the production of COS derivatives that has been achieved in this way, as these oligomers open up another plethora of potential applications when used as building blocks for defined biomaterials.     

Structures of the novel diterpene glycosides from Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, two new diterpenoid glycosides were isolated besides the known steviol glycosides including stevioside, rebaudiosides A–F, rubusoside, and dulcoside A. The structures of the two new compounds were identified as 13-[(2-O-6-deoxy-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (1), and 13-[(2-O-6-deoxy-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (2), on the basis of extensive NMR and MS spectral data as well as chemical studies.