Bagasse minority pathway expression: Real time study of GH2 β-mannosidases from bacteroidetes

After being isolated from a sugarcane pile, the bacterium Chitinophaga sp. CB10 demonstrated to be a rich source of carbohydrases, with 350 predicted CAZyme domains. CB10 was able to grow on carbohydrates of different structural complexities: glucose, carboxymethylcellulose, corn starch, galactomannan, Aloe vera gum and sugarcane bagasse. The sugarcane bagasse is a rich source of complex polymers, and the diversity of metabolites released by its enzymatic hydrolysis has an important role for green chemistry, including minority pathways such as the degradation of mannan conjugates. In this sense, CB10 demonstrated considerable levels of gene expression for mannanases, and was stable for a period of 96–144 hours in the presence of sugarcane bagasse as sole carbon source. The bacterium showed respectively 4.8x and 5.6x expression levels for two genes predicted for GH2 β-mannosidase: one located within a gene cluster identified as “polysaccharide utilization loci” (PUL), and another a classic β-mannosidase. These enzymes shared less than 45% of identity with enzymes characterized from the genus Chitinophaga belonging to the phylum Bacteroidetes. The degree of novelty—as demonstrated by the low identity with previously characterized enzymes; the remarkable capability to grow in different substrates; mannanase activity, evidenced by the release of residual oligosaccharides in the cultivation with galactomannan (HPLC-RID, 12.3 mMol); associated to the ability of mannanases expression in a low concentration of inductor conditions (sugarcane bagasse, 0.2%) indicate the high potential for the application of CB10 as a source of enzymes in the production of oligosaccharides from biomass. This capacity might prove to be very valuable for the biorefinery process of pre-biotic precursors and other functional oligosaccharides focused on the food and pharmaceutical industries.

Man/Cel5B, a Bifunctional Enzyme Having the Highest Mannanase Activity in the Hyperthermic Environment

Thermotoga maritima (Tma) contains genes encoding various hyperthermophilic enzymes with great potential for industrial applications. The gene TM1752 in Tma genome has been annotated as cellulase gene encoding protein Cel5B. In this work, the gene TM1752 was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Interestingly, the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxy methyl cellulose, which is the highest among thermophilic mannanases. However, the putative enzyme did not show sequence homology with any of the previously reported mannanases; therefore, the enzyme Cel5B was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B. Man/Cel5B exhibited maximum activity at 85°C and pH 5.5. This enzyme retained more than 50% activity after 5 h of incubation at 85°C, and retained up to 80% activity after incubated for 1 h at pH 5–8. The Km and Vmax of Man/Cel5B were observed to be 4.5 mg/mL galactomannan and 769 U/mg, respectively. Thin layer chromatography depicted that locust bean gum could be efficiently degraded to mannobiose, mannotriose, and mannooligosaccharides by Man/Cel5B. These characteristics suggest that Man/Cel5B has attractive applications for future food, feed, and biofuel industries.

Enhancement of β-Mannanase Production by Bacillus subtilis ATCC11774 through Optimization of Medium Composition

Palm kernel cake (PKC) has been largely produced in Malaysia as one of the cheap and abundant agro-waste by-products from the palm oil industry and it contains high fiber (mannan) content. The present study aimed to produce β-mannanase by Bacillus subtilis ATCC11774 via optimization of the medium composition using palm kernel cake as substrate in semi-solid fermentation. The fermentation nutrients such as PKC, peptone, yeast extract, sodium chloride, magnesium sulphate (MgSO2), initial culture pH and temperature were screened using a Plackett-Burman design. The three most significant factors identified, PKC, peptone and NaCl, were further optimized using central composite design (CCD), a response surface methodology (RSM) approach, where yeast extract and MgSO2 were fixed as a constant factor. The maximum β-mannanase activity predicted by CCD under the optimum medium composition of 16.50 g/L PKC, 19.59 g/L peptone, 3.00 g/L yeast extract, 2.72 g/L NaCl and 0.2 g/L MgSO2 was 799 U/mL. The validated β-mannanase activity was 805.12 U/mL, which was close to the predicted β-mannanas activity. As a comparison, commercial media such as nutrient broth, M9 and Luria bertani were used for the production of β-mannanase with activities achieved at 204.16 ± 9.21 U/mL, 50.32 U/mL and 88.90 U/mL, respectively. The optimized PKC fermentation medium was four times higher than nutrient broth. Hence, it could be a potential fermentation substrate for the production of β-mannanase activity by Bacillus subtilis ATCC11774.

Obtaining and characterization of modified mannan from the coffee sludge

Mannans are polysaccharides of natural origin. Their main chain consists of residues of D-mannose. They have an immunomodulatory effect, able to induce macrophage activation, inhibit tumor growth and virus development, normalize blood cholesterol, etc. Manooligosaccharides are effective prebiotics The main way to obtain mannan is to extract their alkaline solutions. This makes it impossible to use this polysaccharide in food technologe. This study proposes a biotechnological method of producing water-soluble mannan from coffee sludge. As a source of mannan used a by-product of obtaining instant coffee at the Odessa Combine Food Concentrates which is formed in the processing of coffee beans Arabica. The chemical composition of this coffee sludge was investigated. It is dominated by water-insoluble carbohydrates which are represented by hemicellulose polysaccharides and cellulose. Analysis of the monosaccharide composition of hemicelluloses showed that the hydrolyzate contains mannose, glucose and galactose residues in a ratio of 6: 0,5: 3., which may indicate the presence of galactomannan in their composition. The developed method involves the treatment of the coffee sludge with enzyme preparation with beta-mannanase activity. The process was carried out in aqueous medium at temperature 50 °C, varying the hydromodule in the range of 30… 50, the ratio of enzyme: substrate (1:25, 1:50 and 1:100) for 24…72 hours. This study presents the characteristics of the monomer composition and molecular weight distribution of polysaccharide samples obtained in this way. Only mannose was found in the hydrolyzate of water-soluble mannan. The rational conditions of enzymatic processing of raw materials are determined. The modified mannan is soluble in water. It contains the highest number of target fragments with a molecular weight of about 20 kDa which are considered to be the most physiologically active. Subsequently the modified mannan can be used in nanotechnology functional food ingredients and dietary supplements.

Enzyme activity in the micropylar region of Melanoxylon brauna Schott seeds during germination under heat stress conditions

Recent studies indicate that global temperatures will rise substantially in the 21st century, leading to the extinction of several plant species, as plant metabolism and germination are greatly affected by temperature. Melanoxylon brauna, a tree species native to the Atlantic Forest that occurs from northeastern to southeastern Brazil, is one of the many species threatened by global warming. Despite the economic and ecological importance of M. brauna, studies investigating the influence of heat stress on seed germination and biochemical responses are still incipient. This study aimed to evaluate enzyme activity in the micropylar region of M. brauna seeds during germination under heat stress conditions. Endo-β-mannanase, α-galactosidase, polygalacturonase, pectin methylesterase, pectin lyase, total cellulase, 1,3-β-glucosidase, and 1,4-β-glucosidase activities were determined in micropyles of seeds imbibed for 24, 48 and 72 h at 25, 35 and 45 °C. Seed germination was highest at 25 °C. Endo-β-mannanase activity was not detected under any of the experimental conditions, but imbibition temperature had a significant effect on the activity of all other enzymes.

Optimization of BS4 Enzyme production with Different Substrate Thickness and Type of Trays

BS4 enzyme that is produced from solid substrate fermentation (SSF) on coconut cake with Eupenicillium javanicum BS4 in tray bioreactor has been applied as a feed additive. It increases the nutritional value of animal feedstuff. The BS4 production on SSF may be influenced by the better aeration through the perforated trays or by the thinner substrate. The aim of this research is to optimize the production of BS4 with different substrate thicknesses and types of trays. The trial was carried out using a factorial randomized design (2x2x3) with 6 replicates. The first factor was the type of trays: i.e., non-perforated and perforated tray. The second factor was the thickness of the substrate: i.e., 1.5 and 3.0 cm, while the third factor was the duration of fermentation: i.e. 5, and 7 days. The variables observed were moisture content, dry matter loss (DML), mannanase and saccharification activities, soluble protein content, their specific activities, and yield. Statistical analysis showed no interactions between the three factors, but there were interactions between types of trays and substrate thicknesses, as well as type of trays and incubation times on the mannanase activity and yield of mannanase. The results showed that DML was observed on day 7 were around 31.43- 36.89. The highest mannanase activity was observed on the non-perforated tray with 3 cm thickness on day 7. The saccharification activity towards palm kernel meal was better in the non-perforated tray on day 7 but not influenced by The yield value of mannanase and saccharification activities on a non-perforated tray with 3.0 cm thickness on day 7 was also the highest. Based on energy efficiency and the cost of production, it can be concluded that the optimum condition to produce the BS4 enzyme was observed in the non-perforated tray with 3 cm thickness and fermented for 7 days.