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Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis

Kahar UM, Ng CL, Chan KG, Goh KM

Appl Microbiol Biotechnol, 2016 Jul;100(14):6291-307.
PMID: 27000839 DOI: 10.1007/s00253-016-7451-6

Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0-6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60 °C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26-72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362-370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme.

Matched MeSH terms: Glucans/chemistry
Nutritional composition, antioxidant properties, and toxicology evaluation of the sclerotium of Tiger Milk Mushroom Lignosus tigris cultivar E

Kong BH, Tan NH, Fung SY, Pailoor J, Tan CS, Ng ST

Nutr Res, 2016 Feb;36(2):174-83.
PMID: 26598045 DOI: 10.1016/j.nutres.2015.10.004

The Tiger Milk Mushroom (Lignosus spp.) is an important medicinal mushroom in Southeast Asia and has been consumed frequently by the natives as a cure for a variety of illnesses. In this study, we hypothesized that Lignosus tigris (cultivar E) sclerotium may contain high nutritional value and antioxidant properties, is nontoxic and a potential candidate as a dietary supplement. The chemical and amino acid compositions of the sclerotium were evaluated and antioxidant activities of the sclerotial extracts were assessed using ferric reducing antioxidant power; 1,1-diphenyl-2-picrylhydrazyl; and superoxide anion radical scavenging assays. Acute toxicity of the L. tigris E sclerotium was assessed using a rat model study. The sclerotium was found to be rich in carbohydrate, protein, and dietary fibers with small amounts of fat, calories, and sugar. The amino acid composition of the protein contains all essential amino acids, with a protein score of 47. The sclerotial extracts contain phenolics, terpenoids, and glucan. The ferric reducing antioxidant power values of the various sclerotial extracts (hot water, cold water, and methanol) ranged from 0.008 to 0.015 mmol min(-1) g(-1) extract, while the 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radical scavenging activities ranged from 0.11 to 0.13, and -2.81 to 9.613 mmol Trolox equivalents g(-1) extract, respectively. Acute toxicity assessment indicated that L. tigris E sclerotial powder was not toxic at the dose of 2000 mg kg(-1). In conclusion, L. tigris E sclerotium has the potential to be developed into a functional food and nutraceutical.

Matched MeSH terms: Glucans/analysis
Fulltext Modulation of NKG2D, KIR2DL and Cytokine Production by Pleurotus ostreatus Glucan Enhances Natural Killer Cell Cytotoxicity Toward Cancer Cells

El-Deeb NM, El-Adawi HI, El-Wahab AEA, Haddad AM, El Enshasy HA, He YW, et al.

Front Cell Dev Biol, 2019;7:165.
PMID: 31457012 DOI: 10.3389/fcell.2019.00165

Medicinal mushrooms have been used for centuries against cancer and infectious diseases. These positive biological effects of mushrooms are due in part to the indirect action of stimulating immune cells. The objective of the current study is to investigate the possible immunomodulatory effects of mushroom polysaccharides on NK cells against different cancer cells. In this current study, fruiting bodies isolated from cultured Pleurotus ostreatus were extracted and partially purified using DEAE ion-exchange chromatography. The activation action of the collected fractions on Natural Killer cells was quantified against three different cancer cell lines in the presence or absence of human recombinant IL2 using three different activation and co-culture conditions. The possible modes of action of mushroom polysaccharides against cancer cells were evaluated at the cellular and molecular levels. Our results indicate that P. ostreatus polysaccharides induced NK-cells cytotoxic effects against lung and breast cancer cells with the largest effect being against breast cancer cells (81.2%). NK cells activation for cytokine secretion was associated with upregulation of KIR2DL genes while the cytotoxic activation effect of NK cells against cancer cells correlated with NKG2D upregulation and induction of IFNγ and NO production. These cytotoxic effects were enhanced in the presence of IL2. Analysis of the most active partially purified fraction indicates that it is predominantly composed of glucans. These results indicate bioactive 6-linked glucans present in P. ostreatus extracts activate NK-cell cytotoxicity via regulation of activation and induction of IFNγ and NO. These studies establish a positive role for bioactive P. ostreatus polysaccharides in NK-cells activation and induction of an innate immune response against breast and lung cancer cells.

Matched MeSH terms: Glucans
Fulltext Preparation and characterization of curdlan/polyvinyl alcohol/ thyme essential oil blending film and its application to chilled meat preservation

Zhang Y, Zhou L, Zhang C, Show PL, Du A, Fu J, et al.

Carbohydr Polym, 2020 Nov 01;247:116670.
PMID: 32829798 DOI: 10.1016/j.carbpol.2020.116670

With the growing interest in food safety and in environmental protection, it is more attractive to develop novel biodegradable packaging films. In this regard, one new blending film was prepared with curdlan (CD)/polyvinyl alcohol (PVA)/thyme essential oil. Our results demonstrated that the mechanical properties of the blending film were the best when the ratio of the CD and PVA was 4:1. Further, the barrier properties of the film were optimized by incorporating with thyme essential oil. It was proved that not only water vapor permeability was lower, but also the elongation at break was improved, when 2% (w/w) thyme essential oil used. The potential interactions of the film matrix were analyzed by FTIR, XRD and Cryo-scanning electron microscopy. Importantly, both the antioxidant activity and antibacterial activity were improved. Finally, the blending film was employed for the preservation of chilled meat, while the shelf life was extended up to 10 days.

Matched MeSH terms: beta-Glucans
Fulltext Pellet diameter of Ganoderma lucidum in a repeated-batch fermentation for the trio total production of biomass-exopolysaccharide-endopolysaccharide and its anti-oral cancer beta-glucan response

Abdullah NR, Sharif F, Azizan NH, Hafidz IFM, Supramani S, Usuldin SRA, et al.

AIMS Microbiol, 2020;6(4):379-400.
PMID: 33364534 DOI: 10.3934/microbiol.2020023

The pellet morphology and diameter range (DR) of Ganoderma lucidum were observed in a repeated-batch fermentation (RBF) for the trio total production of biomass, exopolysaccharide (EPS) and endopolysaccharide (ENS). Two factors were involved in RBF; broth replacement ratio (BRR: 60%, 75% and 90%) and broth replacement time point (BRTP: log, transition and stationary phase) in days. In RBF, 34.31 g/L of biomass favoured small-compact pellets with DR of 20.67 µm< d < 24.00 µm (75% BRR, day 11 of BRTP). EPS production of 4.34 g/L was prone to ovoid-starburst pellets with DR of 34.33 µm< d <35.67 µm (75% BRR, day 13 of BRTP). Meanwhile, the highest 2.43 g/L of ENS production favoured large-hollow pellets with DR of 34.00 µm< d < 38.67 µm (90% BRR, day 13 of BRTP). In addition, RBF successfully shortened the biomass-EPS-ENS fermentation period (31, 33 and 35 days) from batch to 5 days, in seven consecutive cycles of RBF. In a FTIR detection, β-glucan (BG) from EPS and ENS extracts were associated with β-glycosidic linkages (2925 cm-1, 1635 cm-1, 1077 cm-1, 920 cm-1 and 800 cm-1 wavelengths) with similar 1H NMR spectral behaviour (4.58, 3.87 and 3.81 ppm). Meanwhile, 4 mg/L of BG gave negative cytotoxic effects on normal gingival cell line (hGF) but induced antiproliferation (IC50 = 0.23 mg/mL) against cancerous oral Asian cellosaurus cell line (ORL-48). Together, this study proved that G. lucidum mycelial pellets could withstand seven cycles of long fermentation condition and possessed anti-oral cancer beta-glucan, which suits large-scale natural drug fermentation.

Matched MeSH terms: beta-Glucans
TRAIL-based tumor sensitizing galactoxyloglucan, a novel entity for targeting apoptotic machinery

Aravind SR, Joseph MM, George SK, Dileep KV, Varghese S, Rose-James A, et al.

Int J Biochem Cell Biol, 2015 Feb;59:153-66.
PMID: 25541375 DOI: 10.1016/j.biocel.2014.11.019

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells, without causing significant toxicity in normal tissues. We previously reported that galactoxyloglucan (PST001) possesses significant antitumor and immunomodulatory properties. However, the exact mechanism in mediating this anticancer effect is unknown. This study, for the first time, indicated that PST001 sensitizes non-small cell lung cancer (A549) and nasopharyngeal (KB) cells to TRAIL-mediated apoptosis. In vitro studies suggested that PST001 induced apoptosis primarily via death receptors and predominantly activated caspases belonging to the extrinsic apoptotic cascade. Microarray profiling of PST001 treated A549 and KB cells showed the suppression of survivin (BIRC5) and anti-apoptotic Bcl-2, as well as increased cytochrome C. TaqMan low density array analysis of A549 cells also confirmed that the induction of apoptosis by the polysaccharide occurred through the TRAIL-DR4/DR5 pathways. This was finally confirmed by in silico analysis, which revealed that PST001 binds to TRAIL-DR4/DR5 complexes more strongly than TNF and Fas ligand-receptor complexes. In summary, our results suggest the potential of PST001 to be developed as an anticancer agent that not only preserves innate biological activity of TRAIL, but also sensitizes cancer cells to TRAIL-mediated apoptosis.

Matched MeSH terms: Glucans/pharmacology*
Hericium erinaceus (Bull.: Fr.) Pers., a medicinal mushroom, activates peripheral nerve regeneration

Wong KH, Kanagasabapathy G, Naidu M, David P, Sabaratnam V

Chin J Integr Med, 2016 Oct;22(10):759-67.
PMID: 25159861 DOI: 10.1007/s11655-014-1624-2

OBJECTIVE: To study the ability of aqueous extract of Hericium erinaceus mushroom in the treatment of nerve injury following peroneal nerve crush in Sprague-Dawley rats.
METHODS: Aqueous extract of Hericium erinaceus was given by daily oral administration following peroneal nerve crush injury in Sprague-Dawley rats. The expression of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways; and c-Jun and c-Fos genes were studied in dorsal root ganglia (DRG) whereas the activity of protein synthesis was assessed in peroneal nerves by immunohistochemical method.
RESULTS: Peripheral nerve injury leads to changes at the axonal site of injury and remotely located DRG containing cell bodies of sensory afferent neurons. Immunofluorescence studies showed that DRG neurons ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for Akt, MAPK, c-Jun and c-Fos as compared with negative control group (P <0.05). The intensity of nuclear ribonucleoprotein in the distal segments of crushed nerves of treated groups was significantly higher than in the negative control group (P <0.05).
CONCLUSION: H. erinaceus is capable of promoting peripheral nerve regeneration after injury. Potential signaling pathways include Akt, MAPK, c-Jun, and c-Fos, and protein synthesis have been shown to be involved in its action.

Matched MeSH terms: Glucans/analysis