Isolation and characterization of two types of β-1,3-glucanases from the common sea hare Aplysia kurodai

Two types of β-1,3-glucanases, AkLam36 and AkLam33 with the molecular masses of 36 kDa and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare Aplysia kurodai. AkLam36 was regarded as an endolytic enzyme (EC 3.2.1.6) degrading laminarin and laminarioligosaccharides to laminaritriose, laminaribiose, and glucose, while AkLam33 was regarded as an exolytic enzyme (EC 3.2.1.58) directly producing glucose from polymer laminarin. AkLam36 showed higher activity toward β-1,3-glucans with a few β-1,6-linked glucose branches such as Laminaria digitata laminarin (LLam) than highly branched β-1,3-glucans such as Eisenia bicyclis laminarin (ELam). AkLam33 showed moderate activity toward both ELam and LLam and high activity toward smaller substrates such as laminaritetraose and laminaritriose. Although both enzymes did not degrade laminaribiose as a sole substrate, they were capable of degrading it via transglycosylation reaction with laminaritriose. The N-terminal amino-acid sequences of AkLam36 and AkLam33 indicated that both enzymes belong to the glycosyl hydrolase family 16 like other molluscan β-1,3-glucanases.     

Purification,characterization and sequencing of the major β-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major β-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes β-1,3-glucans as laminarin and yeast β-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched β-1,3-glucans or mixed β-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.     

6种植物次生物质对斜纹夜蛾解毒酶活性的影响

草食性昆虫取食植物时遇到宿主植物中大量次生物质的化学防御,研究昆虫适应植物毒素的反防御策略具有重要的科学意义。分别添加0.01%肉桂酸、0.01%水杨酸、0.01%花椒毒素、0.02%槲皮素、0.05%黄酮和0.1%香豆素等6种植物次生物质的人工饲料饲养斜纹夜蛾(Spodoptera litura)五龄幼虫48 h后,测定斜纹夜蛾幼虫中肠和脂肪体中谷胱甘肽S-转移酶(GSTs)、羧酸酯酶(CarE)、P450的酶含量及头部乙酰胆碱酯酶(AChE)的活性,利用半定量RT-PCR检测中肠和脂肪体中CYP4M14和CYP4S9的基因表达水平。结果表明:取食肉桂酸和香豆素后,斜纹夜蛾中肠中CarE的酶活性分别提高了1.67和1.37倍,取食6种次生物质均能显著提高斜纹夜蛾脂肪体中GSTs酶活性。取食肉桂酸和香豆素48 h后,脂肪体中P450酶含量比对照增加2.93和14.50倍。取食肉桂酸、花椒毒素、槲皮素和香豆素后,斜纹夜蛾头部AchE酶活性与对照相比提高了1.53、1.80、2.36和1.56倍。6种次生物质均可诱导脂肪体中CYP4M14基因表达,槲皮素、肉桂酸和香豆素强烈诱导CYP4S9在脂肪体中表达。表明,斜纹夜蛾具有利用植物次生物质诱导其解毒酶的能力,进而提高其对毒素的抗性。     

A novel cysteine proteinase inhibitor from seeds of Enterolobium contortisiliquum and its effect on Callosobruchus maculatus larvae

This study focused on the characterization of a novel cysteine proteinase inhibitor from Enterolobium contortisiliquum seeds targeting the inhibition of the growth of Callosobruchus maculatus larvae, an important cosmopolitan pest of the cowpea Vigna unguiculata during storage. The inhibitor was isolated by ion-exchange besides of size exclusion chromatography. EcCI molecular mass is 19,757 Da, composed of two polypeptide chains. It strongly inhibits papain (Ki_app 0.036 nM) and proteinases from the midguts of C. maculatus (80 μg mL^?1, 60% inhibition). The inhibitory activity is reduced by 40% after a heat treatment at 100 °C for 2 h. The protein displayed noxious activity at 0.5% and 1% (w/w) when incorporated in artificial seeds, reducing larval mass in 87% and 92%, respectively. Treatment of C. maculatus larvae with conjugated EcCI-FIT and subsequent biodistribution resulted in high fluorescence intensity in midguts and markedly low intensity in malpighian tubules and fat body. Small amounts of labeled proteins were detected in larvae feces. The detection of high fluorescence in larvae midguts and low fluorescence in their feces indicate the retention of the FITC conjugated EcCI inhibitor in larvae midguts. These results demonstrate the potential of the natural protein from E. contortisiliquum to inhibit the development of C. maculatus.     

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3–1,4-Glucans through Distinct Mechanisms

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with β-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a β-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to β-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to β-1,3–1,4-glucans while substantially decreasing the affinity for decorated β-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified β-1,3–1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant.     

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.     

Comparison of brush border membrane vesicles prepared by three methods from larval Manduca sexta midgut

Brush border membrane vesicles (BBMV) were prepared from freshly isolated posterior larval Manduca sexta midguts by differential calcium precipitation, differential magnesium precipitation and differential ultrasonication. BBMV were also prepared from frozen posterior larval M. sexta midguts by differential calcium precipitaion and differential magnesium precipitation. The yield of BBMV by both differential precipitation methods was 5–6 times greater than that by the differential ultrasonification method. Enrichments of the brush border membrane marker enzymes alkaline phosphatase, γ-glutamyl transferase, and aminopeptidase were similar in all preparations. The polypeptide composition of all preparations was also similar. The specific activity of mitochondrial and microsomal marker enzymes was higher in BBMV prepared from freshly isolated midguts by the differential precipitation methods than in BBMV prepared by the ultrasonication method. The specific activity of cytochrome-c oxidase was 2.5–7 times higher in BBMV prepared from frozen midguts than in BBMV prepared from fresh tissue.     

Purification and Partial Characterization of a Novel β-1,3-Endoglucanase from Streptomyces rutgersensis

A β-1,3-endoglucanase produced by Streptomyces rutgersensis was purified to a homogeneity by the fractional precipitation with ammonium sulfate, ion exchange chromatography on Q-Sepharose and hydrophobic chromatography on Butyl Sepharose. A typical procedure provided 11.74-fold purification with 12.53 % yield. SDS-PAGE of the purified protein showed one protein band. The exact molecular mass of the enzyme obtained by mass spectrometry was 41.25 kDa; the isoelectric point was between pH 4.2–4.4. The optimal β-glucanase catalytic activity was at pH 7 and 50 °C. An enzyme was only active toward glucose polymers containing β-1,3 linkages and hydrolyzed Saccharomyces cerevisiae cell wall β-glucan in an endo-like way: reaction products were different molecular size β-glucans, which were larger than glucose.     

The effect of acquired microbial enzymes on assimilation efficiency in the common woodlouse,Tracheoniscus rathkei

Summary The digestive tract of the common woodlouse, Tracheoniscus rathkei Brandt (Isopoda: Oniscoidea), contains digestive enzymes active against -1,4-glucans, which are the chief storage polysaccharides of vascular plants, algae, fungi, and animals, and -1,3-glucans, which are present in algae and fungi. Digestive tract extracts also exhibit significant activity toward xylan and carboxymethyl-cellulose but negligible activity toward microcrystalline cellulose, substrates representative of the major structural polysaccharides of vascular plants. Low activity was detected toward pectin, and no activity was detected toward chitin. Activity toward xylan is due in part to microbial enzymes acquired from the leaf litter which was the isopod's normal food. Although ingested microbial xylanases are stable and active in the gut fluid, they do not make a quantitatively significant contribution to the isopod's ability to assimilate the hemicellulosic component of its diet. However, the assimilation of carbon from labeled plant fiber is enhanced in isopods which have acquired a cellulase by ingestion of leaf litter amended with a commercial preparation of the cellulase complex from the fungus, Penicillium funiculosum. This result demonstrates the potential contribution of acquired enzymes to the digestion of plant fiber in terrestrial detritivores. We urge caution, however, in assigning an important digestive function to ingested enzymes on the basis of evidence that only indicates that such enzymes are present in the gut fluid without additional evidence that their presence results in an enhancement of digestive efficiency.     

An unusual chimeric amylosucrase generated by domain-swapping mutagenesis

Amylosucrase (ASase; EC 2.4.1.4) synthesizes α-1,4-glucans using sucrose as a sole substrate. The aim of this study was to compare the enzymatic properties of four recombinant ASase genes to determine the underlying mechanisms thereof. Following cloning and expression in Escherichia coli, we determined that the ASase enzyme from Deinococcus geothermalis (DGAS) had the highest thermostability whereas ASase from Neisseria polysaccharea (NPAS) showed the greatest polymerization activity. Chimeric ASases were constructed using dgas and npas genes by overlap extension polymerase chain reaction. Two of the six chimeric ASases generated, NPAS-B′ and DGAS-B, showed ASase activity using sucrose as the sole substrate. However, DGAS-B was not able to produce longer α-1,4-glucans; the highest degree of polymerization was <12. In the kinetic study, not only the substrate binding affinity but also the production rate of DGAS-B was greater than those of DGAS. Molecular dynamic computational simulation suggested that DGAS-B could not synthesize longer glucan chains because of the change in flexibilities of loops 4, 7, and 8 as compared to those of DGAS.     

Correlation of cellulose synthesis in vivo and in vitro during the encystment of Acanthamoeba

Acanthamoeba castellanii differentiates when placed in a starvation medium. The mature cysts formed are characterized by a cellulosic wall synthesized from endogenous sources during encystment. A particulate enzyme system whose specific activity increases some 30-fold during encystment catalyzes the formation of an alkali-soluble and an alkali-insoluble β-(1 → 4)-glucan (cellulose). The activity in vitro of this enzyme extracted from populations of cells during encystment correlates with the formation in vivo of the mature cyst and the alkali-insoluble β-glucan of the cyst wall. The conclusion is based on the following observations:

1.

1. Both alkali-soluble and alkali-insoluble β-glucans similar to the enzymatic products of the isolated β-glucan synthetase occur in cyst walls.     

Water-soluble glucans from true cardamom (Elettaria cardamomum White at Maton) seeds

Water-soluble polysaccharides from seeds of true cardamom (Elettaria cardamomum White at Maton, family Zingiberaceae) have been studied. The study has shown the presence of neutral and acidic components in these polysaccharides. Three polysaccharides (380, 166, and 27 kDa) have been isolated from the neutral fraction. According to the structural analysis data, they represent α-glucans with different degrees of branching (7.1–46.1%); α-(1→4)-D-glucopyranose residues of their backbone chains are substituted at the C6 position with single α-D-glucopyranose residues. Polysaccharides with such structures have a wide range of biological activity. The presence of branched α-glucans in E. cardamomum seeds has been demonstrated.     

Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island,Indian Ocean

The digestive ability of four sympatric land crabs species (the gecarcinids, Gecarcoidea natalis and Discoplax celeste and the anomurans, Birgus latro and Coenobita perlatus) was examined by determining the activity of their digestive enzymes. The gecarcinids are detritivores that consume mainly leaf litter; the robber crab, B. latro, is an omnivore that preferentially consumes items high in lipid, carbohydrate and/or protein; C. perlatus is also an omnivore/detritivore. All species possess protease, lipase and amylase activity for hydrolysing ubiquitous protein, lipid and storage polysaccharides (glycogen and starch). Similarly all species possess enzymes such as N-acetyl-β-d-glucosaminidase, the cellulases, endo-β-1,4-glucanase and β-glucohydrolase and hemicellulases, lichenase and laminarinase for the respective hydrolysis of structural substrates chitin, cellulose and hemicelluloses, lichenan and laminarin. Except for the enzyme activities of C. perlatus, enzyme activity could not be correlated to dietary preference. Perhaps others factors such as olfactory and locomotor ability and metabolic status may determine the observed dietary preferences. The digestive fluid of C. perlatus possessed higher endo-β-1,4-glucanase, lichenase and laminarinase activities compared to that of the other species. Thus, C. perlatus may be efficient at digestion of cellulose and hemicellulose within plant material. Zymography indicated that the majority of protease, lipase, phosphatase, amylase, endo-β-1,4-glucanase, β-glucohydrolase and N-acetyl-β-d-glucosaminidase isozymes were common to all species, and hence were inherited from a common aquatic ancestor. Differences were observed for the phosphatase, lipase and endo-β-1,4-glucanase isozymes. These differences are discussed in relation to phylogeny and possible evolution to cope with the adoption of a terrestrial diet.     

Properties of glucosyltransferase and glucan transferase from spinach

A glucosyl and a glucosyl-glucan transferase activity from spinach (Spinacia oleracea L. var. Matador) leaves have been partially purified and characterized. The latter activity (fraction 1 after diethylaminoethylcellulose chromatography) is responsible for the transfer of glucosyl as well as of maltosyl, maltotriosyl, and higher homologous residues to glucose giving rise to maltose and the correspondingly larger molecules. This fraction also shows β-amylase activity. The transfer takes place only to glucose; maltose, as well as other α-1,4-glucans, serve as donors. The enzyme fraction 2 is amylase-free and catalyzes only the transfer of glucosyl moieties, again with high acceptor specificity to glucose. Maltose and larger α-1, 4-glucans, with the exception of maltotriose and maltotetraose, act as donors. The physiological function of these enzymes may be the formation of oligosaccharide primers for starch synthetase or phosphorylase.     

松嫩草原不同演替阶段大型土壤动物功能类群特征

大型土壤动物处于整个土壤食物网的最顶端,其各功能类群控制着其他动物所需资源的有效性,是土壤生态系统的重要组成部分。为了查明松嫩草原大型土壤动物的功能类群特征,在2006年5—10月期间,逐月对松嫩草原羊草、羊草+虎尾草、虎尾草、碱茅、碱蓬和光碱斑6个演替阶段大型土壤动物的功能类群组成、结构、多样性等特征进行研究。依据其食性将该区土壤动物划分为杂食性、植食性、捕食性和腐食性4个功能类群其中,杂食性土壤动物个体密度所占比例最多为39.16%,植食性土壤动物的类群数所占比例最多为50.00%,腐食性土壤动物个体密度和类群数所占比例均最小,分别为8.09%和12.82%。各功能类群土壤动物个体密度和类群数的相关性不显著(P0.05)。从水平结构来看,总体上各功能类群土壤动物在羊草群落和羊草+虎尾草群落个体密度和类群数较多,在无植被的光碱斑生境土壤动物的个体密度和类群数较少,植食性土壤动物的个体密度和类群数、杂食性土壤动物个体密度、腐食性土壤动物个体密度和类群数随着群落演替发生显著的变化(P0.01)。植食性和腐食性土壤动物个体密度和类群数相关性显著(P0.05)。垂直结构上,0—10 cm土层和20—30 cm土层除捕食性土壤动物个体密度以外,其它各功能类群土壤动物个体密度随着群落演替发生显著的变化(P0.05或P0.01);10—20 cm土层,除腐食性土壤动物个体密度以外,其它各功能类群土壤动物个体密度随着群落演替发生显著的变化(P0.05或P0.01)。0—10cm土层,植食性和杂食性土壤动物个体密度(P0.05)相关性显著;10—20 cm土层,植食性和腐食性土壤动物个体密度(P0.05)相关性显著。不同演替阶段对各功能类群土壤动物的多样性影响程度有所不同。4种功能类群土壤动物在羊草群落和光碱斑之间相似性指数较低,个体数量组成在演替初期的羊草群落和演替后期的光碱斑差异比较大。以上研究结果表明,松嫩草原不同退化演替阶段能够降低大型土壤动物功能类群组成和结构复杂性。     

A critical review on the impacts of β-glucans on gut microbiota and human health

The β-glucans are the glucose polymers present in the cells walls of yeast, fungi and cereals. β-Glucans are the major compositions of various nutritional diets such as oats, barley, seaweeds and mushrooms. Various biological activities of β-glucans have been reported such as anticancer, antidiabetic, anti-inflammatory and immune-modulating effects. The importance of β-glucans in food processing industries such as bread preparation, yogurt and pasta have been well elucidated. In recent findings on food science research gut microbiota plays a significant role and vastly studied for its intermediate role in regulating health. Several reports have suggested that β-glucans should have a significant impact on the gut microbiota changes and in turn on human health. The review was aimed to accumulate the evidence on types of β-glucans, their functional properties and the mechanism by how the β-glucans regulate the gut microbiota and human health. The various in vitro, in vivo and clinical studies, have been summarized, in particular, the changes happening upon the β-glucans supplementation on the gut microbiota. Overall, this review updates the recent studies on β-glucans and gut microbiota and also inputs the demanding questions to be addressed in β-glucans–microbiota research in the future.     

beta-1,3-Endoglucanase from Soybean Releases Elicitor-Active Carbohydrates from Fungus Cell Walls

Two enzymes from soybean (Glycine max L. Merr. cv Harosoy 63) cotyledons released elicitor-active carbohydrates from cell walls of the phytopathogenic fungus Phytophthora megasperma f.sp. glycinea. They were identified as isoenzymes of β-1,3-endoglucanase (EC 3.2.1.39) with isoelectric points of pH 8.7 and 10.5. The pI 10.5 enzyme was extracted in the greatest amount and was isolated as a homogeneous protein of about 33,000 daltons as determined by gel filtration and sodium dodecyl sulfategel electrophoresis. The purified enzymes hydrolyzed several β-1,3-glucans in a strictly random manner, but degraded neither β-1,6- nor β-1,4-glucans.     

Single-reversal charge in the β10-β11 receptor-binding loop of Bacillus thuringiensis Cry4Aa and Cry4Ba toxins reflects their different toxicity against Culex spp. larvae

Bacillus thuringiensis Cry4Aa toxin was previously shown to be much more toxic to Culex mosquito-larvae than its closely related toxin – Cry4Ba, conceivably due to their sequence differences within the β10-β11 receptor-binding loop. Here, single-Ala substitutions of five residues (Pro⁵¹⁰, Thr⁵¹², Tyr⁵¹³, Lys⁵¹⁴ and Thr⁵¹⁵) within the Cry4Aa β10-β11 loop revealed that only Lys⁵¹⁴ corresponding to the relative position of Cry4Ba-Asp⁴⁵⁴ is crucial for toxicity against Culex quinquefasciatus larvae. Interestingly, charge-reversal mutations at Cry4Ba-Asp⁴⁵⁴ (D454R and D454K) revealed a marked increase in toxicity against such less-susceptible larvae. In situ binding analyses revealed that both Cry4Ba-D454R and D454K mutants exhibited a significant increase in binding to apical microvilli of Culex larval midguts, albeit at lower-binding activity when compared with Cry4Aa. Altogether, our present data suggest that a positively charged side-chain near the tip of the β10-β11 loop plays a critical role in determining target specificity of Cry4Aa against Culex spp., and hence a great increase in the Culex larval toxicity of Cry4Ba was obtained toward an opposite-charge conversion of the corresponding Asp⁴⁵⁴.     

Comparative biochemistry of α-glucan-utilization in pseudomonas amyloderamosa and Pseudomonas saccharophila

Growth patterns on and utilization of various α-glucans were investigated in Pseudomonas amyloderamosa and P. saccharophila. Maltose, maltodextrins (average chain length 7 glucosyl units) and glycogen supported excellent growth of both organisms and were extensively metabolized, although glycogen utilization in P. saccharophila was preceded by a prolonged lag phase. P. amyloderamosa produced limited growth on amylopectin and the carbohydrate was only partly degraded. It seemed likely that many of the unit chains liberated from amylopectin had a length exceeding the substrate range accepted by the maltodextrin permease (transport) system. A correlation was established between the pH of the medium and the utilization of glycogen and amylopectin for growth in P. amyloderamosa. The carbohydrates were at least partly utilizable at pH 6.0, whereas they could not support any growth at pH 6.5. Most likely, the lack of growth at the higher pH reflected the low activity of isoamylase at this pH. The enzyme patterns of maltodextrin catabolism in the two bacteria were established. Intracellularly, maltodextrin phosphorylase and 4-α-glucanotransferase occurred in both. Degradation of extracellular α-glucans was mediated by a mainly intracellular isoamylase in P. amyloderamosa, whereas P. saccharophila possessed an extracellular α-amylase and a firmly cell-bound pullulanase.