Permeabilized whole-cell biocatalyst containing co-expressed two enzymes facilitates the synthesis of maltoheptaose (G7) from starch.

Maltoheptaose (G7) is one of the mixtures of maltodextrin widely used in the food, pharmaceutical, and cosmetics industries. A genetically engineered strain, which simultaneously expressed cyclodextrin glucanotransferase (CGTase) from Gracilibacillus alcaliphilus SK51.001 and cyclomaltodextrinase (CDase) from Bacillus sphaericus E-244, two enzymes, was constructed by cloning the above two genes into a plasmid and transformed into the host Escherichia coli BL21(DE3) (E. coli) strain, resulted in recombinant cells harboring the vector pETDuet-GaCGT/BsCD (pGaBs). These cells were used as whole-cell catalysts for the biotransformation of G7 from the inexpensive substrate (starch). Due to the high molecular weight of starch, the cell membrane prevents the entry of starch into the cellular system. Therefore, the pGaBs cell wall was permeabilized by lysozyme, EDTA, and heat treatment. After reaching the optimized conditions of permeabilized pGaBs cell amount, lysozyme amount, reaction temperature, and metal ion concentration, approximately 4.1 g/L of G7 was produced from 30 g/L starch in 1 h with the addition of Ca2+. This co-expression system offers a one-pot synthesis approach to the production of G7 using an inexpensive substrate, avoiding enzyme purification steps.

One-pot production of maltoheptaose (DP7) from starch by sequential addition of cyclodextrin glucotransferase and cyclomaltodextrinase.

Maltodextrins (dextrins) are glucose chains normally produced by starch hydrolysis. Maltodextrins are characterized by their degree of polymerization (DP), which indicates the average number of glucose units per chain. Maltoheptaose (DP7), also known as amyloheptaose, is one of the maltodextrin mixtures widely used in foods, cosmetics, and pharmaceutical industries. Recently, the enzymatic synthesis of DP7 has attracted considerable attention, owing to its considerable advantages over chemical methods. In this work, we have designed a one-pot cascade reaction bio-synthesis starting from soluble starch to produce a specific degree of polymerization (DP7). The reaction system was catalyzed by cyclodextrin glucotransferase (GaCGT) from Gracilibacillus alcaliphilus SK51.001CGTase (transglycosylation/cyclization reaction) and cyclomaltodextrinase (BsCD) from Bacillus sphaericus E-244CDase (ring-opening reaction). The one-pot cascade reaction exhibited an optimum temperature of 30 °C and pH 7.0, and the addition of Ca2+ enhanced the maltoheptaose production. The optimum enzyme units for the one-pot cascade reaction were 80 U/g of GaCGT and 1 U/g of BsCD. However, the sequential addition of the enzymes exhibited a 5-fold higher conversion rate over simultaneous addition. The one-pot cascade reaction converted 30 g/L of soluble starch to 5.4 g/L of maltoheptaose in 1 h reaction time with a conversion rate of 16 %.

Characterization of a recombinant l-ribose isomerase from Mycetocola miduiensis and its application for the production of l-ribulose.

An enzyme, l-ribose isomerase (l-RI), mostly catalyzes the isomerization of l-ribose and l-ribulose. These so-called rare sugars are essential for the treatment of cancer and other viral diseases. In the present study, l-ribose isomerase produced from a bacterium, Mycetocola miduiensis (Mm-LRIse), by using l-ribose as a carbon source. The recombinant l-ribose isomerase gene was cloned and overexpressed from M. miduiensis and purified with an exclusive band of 32 kDa by nickel-affinity chromatography. This gene possessed 267 amino acids protein having an estimated molecular weight of 29,568.17 Da. The native molecular weight of Mm-LRIse estimated by HPLC was 134.84 kDa. The recombinant l-ribose isomerase was highly active in sodium phosphate (50 mM) buffer at 40 °C and pH 7.5, showing the specific activity up to 47.40 U mg-1. Mm-LRIse showed no significant enhancement in activity with metallic ions except Mn2+ and Co2+. The values of Km, Kcat, Kcat/Km and Vmax of Mm-LRIse against l-ribose substrate were 42.48 mM, 9259.26 min-1, 217.43 min-1 mM-1, and 277.78 U mg-1 respectively. At equilibrium, the l-ribulose transformation rate was nearly 32 % (6.34 g L-1) using 20 g L-1 of l-ribose. The results revealed that the Mm-LRIse enzyme has a potential for L-ribulose production from l-ribose.

Biochemical characterization of recombinant L-fucose isomerase from Caldanaerobius polysaccharolyticus for L-fuculose production.

L-fuculose is a rare sugar that is useful for the agriculture and medicine industries. L-fucose isomerase (E.C.5.3.1.25), which is an aldose-ketose isomerase, plays a significant role in producing rare sugars. A recommended L-fucose isomerase gene was cloned from Caldanaerobius polysaccharolyticus and purified with a single band of 65 kDa using nickel-affinity chromatography, with a specific activity of 108.23 U mg-1. The native molecular mass existed with 214 kDa was a trimer. The purified enzyme showed a maximum activity in 1 mM Mn2+ at 55 °C and pH 6.5 with a melting temperature (Tm) of 80.3 °C in the presence of one molecule per monomer. L-fucose isomerase from C. polysaccharolyticus (Capo-LfIase) exhibited the highest activity of L-fucose with Km, kcat and Kcat/km values of 94.2 mM, 23854 min-1 and 253.3 min-1 mM-1, respectively. Capo-LfIase showed more than 50% thermostability after 20 h of incubation at 45, 55, 65, 75 and 85 °C. The 9 putative active site residues of the L-fucose substrate were described using a homology model, and the results showed that Tyr440, Met185, Trp499 and Asn527 are the candidates of metal-binding residues, while Ser393, Glu337, Glu302, His528 and Asp361 would be involved in substrate binding. The conversion rate of L-fuculose from L-fucose was almost 28.2%, with 80 g L-1 L-fucose, and no byproduct was found. To the best of our knowledge, Capo-LfIase produces high yield of L-fuculose from L-fucose by enzymatic methods.

Characterization of a novel d-arabinose isomerase from Thermanaeromonas toyohensis and its application for the production of d-ribulose and l-fuculose.

d-Ribulose and l-fuculose are potentially valuable rare sugars useful for anticancer and antiviral drugs in the agriculture and medicine industries. These rare sugars are usually produced by chemical methods, which are generally expensive, complicated and do not meet the increasing demands. Furthermore, the isomerization of d-arabinose and l-fucose byDd-arabinose and l-fucose by d-arabinose isomerase from bacterial sources for the production of d-ribulose and l-fuculose have not yet become industrial due to the shortage of biocatalysts, resulting in poor yield and high cost of production. In this study, a thermostable d-ribulose- and l-fuculose producing d-arabinose isomerase from the bacterium Thermanaeromonas toyohensis was characterized. The recombinant d-arabinose isomerase from T. toyohensis (Thto-DaIase) was purified with a single band at 66 kDa using His-trap affinity chromatography. The native enzyme existed as a homotetramer with a molecular weight of 310 kDa, and the specific activities for both d-arabinose and l-fucose were observed to be 98.08 and 85.52 U mg-1, respectively. The thermostable recombinant Thto-DaIase was activated when 1 mM Mn2+ was added to the reactions at an optimum pH of 9.0 at 75 °C and showed approximately 50% activity for both d-arabinose and l-fucose at 75 °C after 10 h. The Michaelis-Menten constant (Km), the turnover number (kcat) and catalytic efficiency (kcat/Km) for d-arabinose/l-fucose were 111/81.24 mM, 18,466/10,688 min-1, and 166/132 mM-1  min-1, respectively. When the reaction reached to equilibrium, the conversion rates of d-ribulose from d-arabinose and l-fuculose from l-fucose were almost 27% (21 g L-1) and 24.88% (19.92 g L-1) from 80 g L-1 of d-arabinose and l-fucose, respectively.

In vitro survival of Bifidobacterium bifidum microencapsulated in zein-coated alginate hydrogel microbeads.

The Bifidobacterium bifidum susceptibility in gastrointestinal conditions and storage stability limit its use as potential probiotics. The current study was design to encapsulate B. bifidum using sodium alginate (SA, 1.4% w/v) and different concentration of zein as coating material, that is, Z1 (1% w/v), Z2 (3% w/v), Z3 (5% w/v), Z4 (7% w/v), Z5 (9% w/v). The resultant microbeads were further investigated for encapsulation efficiency, survival in gastrointestinal conditions, release profile in intestinal fluid, storage stability and morphological characteristics. The highest encapsulation efficiency (94.56%) and viable count (>107 log CFU/g) was observed in Z4 (7% w/v). Viable cell count of B. bifidum was >106 log CFU/g in all the zein-coated microbeads as compare to free cells (103 log CFU/g) and SA (105 log CFU/g) at 4 °C after 32 days of storage. Therefore, B. bifidum encapsulated in zein-coated alginate microbeads present improved survival during gastric transit and storage.

Preparation of Doum fruit (Hyphaene thebaica) dietary fiber supplemented biscuits: influence on dough characteristics, biscuits quality, nutritional profile and antioxidant properties.

The increasing demand for functional foods has boosted up the food industry to produce fiber-enriched products. In this study, dietary fiber (DF) was isolated from Doum fruit by exploiting the combination of microwave reactor technique and superfine grinding technology. The isolated Doum dietary fiber (DDF) possessed a high content of total dietary fiber, essential minerals and total polyphenols with good antioxidant activity. Biscuits were prepared by substituting wheat flour with DDF at different levels (0, 2.5, 5, 7.5 and 10%) and assessed for dough mixing properties and biscuit quality. The results showed that an increase of DDF in the flour affected physical parameters of biscuits by increasing the biscuits hardness and reducing the diameter, thickness and spread ratio. Supplementation of biscuits with DDF improved the nutritional value in terms of DF contents and essential minerals. Improvement in total phenolic contents (TPC) and antioxidant activities of the biscuits were also noted as a result of DDF supplementation. Biscuits supplemented with 7.5% DDF showed overall better sensorial characteristics. Conclusively, this study has shown that supplementation of wheat flour with DDF improved nutritional profile, antioxidant properties and overall consumer acceptability of biscuits. The present findings will be helpful regarding the development of functional foods enriched with DDF.

Detarium microcarpum: A novel source of nutrition and medicine: A review.

Detarium microcarpum is a plant indigenous to Africa, which occurs naturally in many African countries, particularly in savannah regions. Its leaves and fruits are used mainly as food and as folk medicine. It has anti-diabetic, antioxidant, and hepatitis C inhibitor properties and has been traditionally utilised in cancer treatment. This review examines published work on the nutritional, pharmacological, and traditional uses of Detarium microcarpum. This plant may become valuable if the fruit, stems, roots, and leaves are extracted for nutraceutical purposes.