Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity

BACKGROUND: Manno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain. RESULTS: Galactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose. CONCLUSIONS: The effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.

Purification and physicochemical characterisation of Aspergillus niger USM F4 β-mannanase

Aims@#This present study focused on purification of fungal β-mannanase produced by Aspergillus niger USM F4 and also physicochemical characterisation of the purified enzyme.@*Methodology and results@#The purified β-mannanase with a molecular mass of ~47.4 kDa was demonstrated on SDSPAGE gel. The enzyme signified a purification degree of 4-fold, with final specific activity of 196.42 U/mg. It reached an optimum catalytic activity at pH 4.0 and 60 °C. The thermal stability of the enzyme was up to 70 °C and maintained the 50% activity after 30 min at 80 °C. Meanwhile, the pH stability was in the range of pH 3.0-9.0 and a 30 min half-life at pH 10.0. All chemical substances manifested an inhibitory effect on purified β-mannanase, with SDS (28.16 ± 0.05% residual activity) as the strongest inhibitor, followed by cupric ion (Cu2+) (49.51 ± 0.09% residual activity). As a whole, the enzyme displayed a substrate specificity in the order of locust bean gum (LBG) > carboxymethylcellulose > soluble starch > xylan from oat spelt > α-cellulose. Its preference for LBG has generated the Km and Vmax values of 0.20 mg/mL and 9.82 U/mL, respectively.@*Conclusion, significance and impact of study@#The outcomes of our study offer potential for use at industrial scales, particularly in the oligosaccharides production that involve acid-related activity, wide-ranging temperature and pH stability.

Screening and Identification of Mannanase- Producing Fungi Isolated from Selected Agricultural Wastes.

Aim: The study evaluated potential performance of different fungal isolates from agricultural by-products for mannanase production. Study Design: The first experiment, fungal isolates were screened for mannanase production on agar medium containing Locust Bean Gum (LBG) and total fungal count was conducted. In the second experiment, the fungal isolates were further screened for mannanase production in submerged state fermentation. Place and Duration of Study: Microbiology Research Laboratory Federal University of Technology, Akure and Postgraduate Research Laboratory, Obafemi Awolowo University Ile-Ife, Nigeria between September 2011 and March 2012. Methodology: The fungal isolates associated with some agricultural wastes were isolated on LBG containing agar medium by plate assay techniques and counted by standard microbiological methods. Mannanase production was conducted in submerged state fermentation (shaken & static) into which copra meal had been supplemented as the sole carbon source and enzyme activity was determined by dinitrosalicylic acid method. Results: In this study, 11 fungal isolates showed positive results with clear zone around their cultures. Fungal isolate 5A showed the highest activity ratio of 1.8, while the least was observed in isolate 9A12 with activity ratio of 0.64. The highest fungal counts were recorded in fermented coconut with 7.4×102 sfu/g, while cocoa pod and groundnut shell had no fungal growth. In terms of percentage occurrence of fungal isolates from selected agrowastes, it was revealed that Rhizopus japonicus had the highest occurrence of 66.67%, while the same value of 8.33% was observed for Aspergillus fumigatus, A. glaucus, R. stolonifer and Trichosporonoides oedocephalis. In fermentation broth, all the 11 isolates displayed mannanase activity ranging from 0.370 to 21.667 U/ml for static and 0.278 to 3.982 U/ml for shaken condition, with the highest mannanase activity observed with isolate 5A for both culture conditions. According to the cultural characters and microscopic morphology, the isolate 5A being the highest mannanase producer was identified as the Aspergillus fumigatus. Conclusion: In this study, fungal isolates screened and evaluated for mannanase production from agricultural by-products elaborated considerable mannanase activity and this could be exploited for prebiotic preparation.

Fermenation Products of β-Mannanase Producing Bacteria Inhibit Selected Poultry Borne Pathogens.

Aim: The study evaluated the inhibitory effect of fermentation products of β-mannanaseproducing bacteria on selected poultry borne pathogens. Study Design: The first experiment, bacterial isolates previously confirmed positive for mannanase by plate assay technique were further screened for mannanase production in submerged state fermentation. In the second experiment, inhibitory effect of fermentation products of mannanase-producing bacteria on selected poultry pathogens was evaluated. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between September 2011 and March 2012. Methodology: Bacterial isolates from agricultural wastes previously confirmed positive for mannanase activity by plate assay were further screened for their potential performance under submerged state fermentation and enzyme activity determined by dinitrosalicylic acid method. The inhibitory action of β-mannanase-producing bacteria was determined by supplementation of supernatant and plating method. Results: Isolate 1A showed highest mannanase activity (13.430 U/ml), displayed broad inhibition to selected poultry borne pathogens; Klebsiella oxytoca, Shigella alkalescens, Escherichia coli, Salmonella typhii, Staphylococcus aureus and Streptococcus sp. Apart from isolate 1A, fermentation products of other isolates generated from the mannolytic action of β-mannanase on mannan containing substrate displayed different percentage inhibition on selected poultry borne pathogens. Conclusion: The results suggested that fermentation products from β-mannanaseproducing bacteria might possess antibacterial properties which could be applied in poultry farms.

Beta-1,3-glucomannanase assisted lipid extraction from Rhodosporidium toruloides / 生物工程学报

To evaluate the effectiveness of enzymatic assisted extraction (EAE) of lipid from the oleaginous yeast Rhodosporidium toruloides in the presence of beta-1,3-glucomannanase at a larger scale, we investigated the effects of enzymatic treatment and extraction conditions on lipid extraction yields at 10-L scale by using the broth of R. toruloides Y4 as the feed and ethyl acetate as the solvent. When it was treated for 0.5 h, the lipid extraction yield reached 71.1%, indicating that the enzymatic treatment process reached similar efficiency to that obtained at 10-mL scale. The inhibitory effect of emulsification was greatly reduced by repeated extraction. After extracted for three times, yields of lipid extraction, solvent recovery and total material recovery reached 92.9%, 87.0% and 94.2% respectively. As it can use the lipid production slurry with good extraction efficiency, EAE technology is promising for industrial production of microbial lipids.

Directed evolution by error-prone PCR of Armillariella tabescens MAN47 beta-mannanase gene toward enhanced thermal resistance / 生物工程学报

Firstly, We used error-prone PCR to induce mutations on Armillariella tabescens MAN47 beta-mannanase gene, Secondly, we cloned the mutated fragments into secreted expression vector pYCalpha, Then the recombinant plasmids were transformed into Saccharomyces cerevisiae BJ5465 after amplified and extracted in DH5alpha cells. Through three cycles of error-prone PCR we built a mutant database, Then we screened one optimum (named M262) from about 104 mutants. The evoluted MAN47 beta-mannanase displayed both higher thermal stability and activity than wide type. The evoluted enzyme M262 retained high activity after treatment at 80 degrees C for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of M262 can reach to 25 U/mL, which is 4.3 times as wide type under optimum temperature. In addition, pH stability and pH range of evoluted enzyme M262 were both improved compared with wild-type enzyme. The optimum pH was estimated to be similar to that of wild-type enzyme. The sequence comparison illustrated that there were three nucleotide substitutions (T343A/C827T/T1139C) which carried corresponding amino acid changes (Ser115Thr/Thr276Met/Val380Ala). According to homologous modeling by SWISS-MODEL Repository, three mutated amino acids located at the sixth amino acid of the fourth beta-sheet, the first amino acid of the sixth alpha-helix, the turn between the tenth and eleventh beta-sheet, respectively.

Cloning, expression and characterization of mannanase from Armillariella tabescens EJLY2098 in Pichia pastoris / 生物工程学报

We used reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) techniques to obtain the full-length cDNA of beta-mannanase (EC 3.2.1.78) from Armillariella tabescens EJLY2098 (an edible fungus). Sequence analysis of the 1481 bp full-length cDNA encoding 445 amino acid residues indicated that the gene contained two structural domains, cellulose-binding domains (CBD) and glycoside hydrolase family 5 (GHF5) domains, other than the conserved beta-mannanase domain. Thus, we classified this gene as a member of glycoside hydrolase family 5. Next, we cloned a 1308 bp fragment encoding the beta-mannanase mature peptide (re-atMAN47) into the expression vector pPICZalphaA and expressed it in Pichia pastoris. The yield was 440 mg/L. Enzyme activity reached a maximum of 1.067 IU/mL after 72 h of methanol induction. The re-atMAN47 had an optimal temperature of 60 degrees C and an optimal pH of 5.5. It manifested broad thermostability from 30 degrees C-65 degrees C, and was stable between pH 4.5-7.0. This study represents the first record of a beta-mannanase from Armillariella tabescens EJLY2098 and provides a new source of carbohydrate hydrolysis enzyme with good biosafety, thermostability and wide pH stability. It is a good approach for the industrial needs of feed, food and pharmaceutical manufacturers.

Study on release mechanism of berberine hydrochloride-loaded carboxymethyl konjac glucomannan pellets for colonic delivery / 中国中药杂志

<p><b>OBJECTIVE</b>To study release mechanism of berberine hydrochloride (BH) from carboxymethyl konjac glucomannan pellets for colonic delivery.</p><p><b>METHOD</b>The pellets were prepared by ionotropic gelation technique. The effects of the kinds of enzyme and enzyme concentration of dissolution media on the release of BH and the erosion properties of the pellets were studied.</p><p><b>RESULT</b>Compared with the dissolution media without enzymes, the release of BH and the erosion of the pellets were increased obviously in the media with rat cecal and colonic content or beta-mannase, the degradation of the carrier material of pellets by enzymes was the main factor which result in the erosion of the pellets. With the increased of beta-mannase concentration, the release of BH and the erosion of the pellets increased, the amount relationships of the release of BH and the erosion of the pellets were approximately 1:1. The release of BH exhibit Peppas equation, the n value was more than 1.</p><p><b>CONCLUSION</b>The release mechanism of BH from the pellets was enzymatic erosion-controlled, which indicates the potential of the pellets to serve as a colon-specific drug delivery system.</p>

Study on in vitro colon-specific enzymatic degradation performance of carboxymethyl konjac glucomannan / 中国中药杂志

<p><b>OBJECTIVE</b>In vitro enzymatic degradation of carboxymethy konjac glucomannan (CMKGM) were studied to evaluate the feasibility of CMKGM used as carrier materials to prepare colon-specific drug delivery systems.</p><p><b>METHOD</b>The solutions with rat gastrointestinal tract (GIT) contents or with commercial enzymes were chosen to stimulate in vivo GIT environment, respectively. Enzymatic degradation of CMKGM were studied by viscometic procedure. Degradation kinetics of CMKGM and konjac glucomannan (KGM) by enzymes, the effects of the degree of substitution (DS) of CMKGM and the pH of solution on its susceptibility to degradation were investigated.</p><p><b>RESULT</b>CMKGM were degraded mainly in the simulated cecal and colonic media, but not in the simulated gastric and enteric media. Degradation of KGM and CMKGM by enzymes obeyed Michaelis-Menton kinetics. CMKGM with lower DS were more susceptible substrates. CMKGM were more susceptible substrates in solution with pH 6. 0-6. 8.</p><p><b>CONCLUSION</b>CMKGM had colon-specific enzymatic degradation characteristics and could be used as carrier materials to prepare colon-specific drug delivery systems.</p>

Physiological, morphological, and mannanase production studies on Aspergillus niger uam-gs1 mutants

Mutant strains from Aspergillus niger UAM-GS1 were produced by UV radiation to increase their hemicellulolytic and cellulolytic activity production. The mutant strains showing more enzymatic activity were those labelled GS1-S059 and GS1-S067. These strains also showed the largest relationship between diameter of hydrolysis zone and colony diameter. The mutant GS1-S067 showed a colony radial extension rate and a biomass growth rate g biomass/(cm² h), 1.17 times higher than that achieved by strain UAM-GS1. The high invasive capacity makes this mutant strain a promising alternative for its use in solid substrate fermentation (SSF). The morphological properties of the two mutant strains were evaluated by using scanning electron microscopy. The diameter of the sporangium of the mutant strains GS1-S059 and GS1-S067 was significantly larger (P < 0.05) than that found for the parental strain. The hypha length and diameter of the mutant strains significantly changed (P < 0.05) compared to the parental strain. A Pearson correlation analysis on hypha length, sporangium diameter, and cellulase and xylanase activities indicated that there was a strong relationship among these variables in relation to mannanase activity. Mutant strains GS1-S059 and GS1-S067 significantly increased their level of mannanase, xylanase and cellulase production, compared to the parental strain, improving their potential industrial applications.

Expression of endo-beta-mannanase gene from Trichoderma reesei in Pichia pastoris / 生物工程学报

Complete mannanase gene with two introns was cloned from Trichoderrna reesei by PCR. The two introns were then removed by overlap extension PCR. The gene encoding the mature mannanase protein was inserted into the expression vector pPIC9K, downstream of a alpha-factor signal peptide sequence. The resultant recombinant vector was named pM242. After linearized with Sac I , pM242 was transformed to Pichia pastoris GS115 by electroporation. After screening, the recombinant strain Gpmf25 that expresses the secretory protein at high level was obtained. The activity of the recombinant mannanase reached 12.5 IU/mL. Optimum pH and temperature for the recombinant enzyme were 5.0 and 80 degrees C, respectively. The enzyme was stable at pH 5.0-6.0 and maintained over 50% of original activity after incubation at 70 degrees C for 30 min.