Oligosaccharide biotechnology: an approach of prebiotic revolution on the industry.

Oligosaccharides are polymers with two to ten monosaccharide residues which have sweetener functions and sensory characteristics, in addition to exerting physiological effects on human health. The ones called nondigestible exhibit a prebiotic behavior being fermented by colonic microflora or stimulating the growth of beneficial bacteria, playing roles in the immune system, protecting against cancer, and preventing cardiovascular and metabolic issues. The global prebiotics market is expected to grow around 12.7% in the next 8 years, so manufacturers are developing new alternatives to obtain sustainable and efficient processes for application on a large scale. Most studied examples of biotechnological processes involve the development of new strategies for fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, and mannanooligosaccharide synthesis. Among these, the use of whole cells in fermentation, synthesis of microbial enzymes (ß-fructofuranosidases, ß-galactosidases, xylanases, and ß-mannanases), and enzymatic process development (permeabilization, immobilization, gene expression) can be highlighted, especially if the production costs are reduced by the use of agro-industrial residues or by-products such as molasses, milk whey, cotton stalks, corncobs, wheat straw, poplar wood, sugarcane bagasse, and copra meal. This review comprises recent studies to demonstrate the potential for biotechnological production of oligosaccharides, and also aspects that need more investigation for future applications in a large scale.

Crude glycerol from biodiesel as a carbon source for production of a recombinant highly thermostable ß-mannanase by Pichia pastoris.

OBJECTIVE: To explore an efficient use of crude glycerol for the production of a highly thermostable ß-mannanase (ReTMan26) by Pichia pastoris X33. RESULTS: Cell growth was significantly inhibited by 4 and 6% (w/v) crude glycerol in 250 ml shake-flasks and in 5 l bioreactor batch cultures, respectively, but not affected by pure glycerol at the same concentrations. For further study, the impact of various impurities in crude glycerol on the cell growth of, and ReTMan26 production by, Pichia pastoris was investigated. Salts and methanol did not exert an inhibitory effect, but ≥ 0.2% and 0.3% (w/v) soap in shake-flask and bioreactor cultures, respectively, inhibited fermentation. Under identical conditions, the biomass and ReTMan26 activity produced by high-cell-density fermentation using 5% crude glycerol (glycerol at 80%, w/w) were slightly higher than those using 4% (w/v) pure glycerol. CONCLUSIONS: Non-pretreated ≤ 5% (w/v) crude glycerol could be effectively utilized for industrial production of ReTMan26, and the total production costs using crude glycerol were ~ 4.2% lower than those using pure glycerol.

Purification, characterization, and overexpression of an endo-1,4-ß-mannanase from thermotolerant Bacillus sp. SWU60.

Endo-ß-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the ß-1,4-linkage in the mannan backbone and release short ß-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300 ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38 kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60 °C, respectively. The enzyme was stable up to 60 °C for 1 h and at pH 5-9 at 4 °C for 16 h. Its enzyme activity was inhibited by Hg2+. The full-length mans2 gene was 1,008 bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.

Production of high activity Aspergillus niger BCC4525 ß-mannanase in Pichia pastoris and its application for mannooligosaccharides production from biomass hydrolysis.

A cDNA encoding ß-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pastoris KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high kcat/Km (461 mg-1 s-1 mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-ß-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An in vitro digestibility test simulating the gastrointestinal tract system of broilers suggested that the recombinant ß-mannanase could effectively liberate reducing sugars from PKM-containing diet. These characteristics render this enzyme suitable for utilization as a feed additive to improve animal performance.

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.