Digestive capacities allow the Mexican long-nosed bat (Leptonycteris nivalis) to live in cold environments

Digestive capabilities of nectar-feeding vertebrates to assimilate sugars affect their ability to acquire and store energy and could determine the minimal temperatures at which these animals can survive. Here, we described the sugar digestive capability of Leptonycteris nivalis and related it with its capacity to live in cold environments. We measured the enzymatic activity, food intake rate and changes in body mass of bats feeding at four different sucrose concentrations (from 5 to 35% wt./vol.). Additionally, we used a mathematical model to predict food intake and compared it with the food intake of bats. L. nivalis was able to obtain ~ 111.3 kJ of energy regardless of the sugar concentration of their food. Also, bats gained ~ 2.57 g of mass during the experimental trials and this gain was independent of sugar concentration. The affinity (1 / K_m) of sucrase (EC 3.2.1.48) was one order of magnitude higher relative to that reported for its sister species Leptonycteris yerbabuenae (0.250 and 0.0189 mmol^? 1 L, respectively), allowing this species to have a higher energy intake rate. We propose that the high ability to acquire energy conferred L. nivalis the faculty to invade cold environments, avoiding in this way the ecological competition with its sympatric species L. yerbabuenae.     

Effects of six sugars on the longevity,fecundity and nutrient reserves of Microplitis mediator

Parasitoid adults can directly feed on floral nectar and honeydew containing monosaccharides and disaccharides. Oligosaccharides such as maltose, melezitose and raffinose are also found in honeydew but are rare in floral nectar. The effects of six different sugar resources on the longevity, fecundity and nutrient reserves of Microplitis mediator, a larval endoparasitoid in the cotton bollworm Helicoverpa armigera (Hübner) were determined in our laboratory. The results showed that both food and sex affected longevity of this wasp. Females and males of M. mediator fed with 1 M sucrose solution survived longer than controls fed with water (5.7- and 3.7-fold longer, respectively). When provided with sucrose, glucose or fructose solutions, the parasitoid generated 3.6- to 3.7-fold more offspring than controls, and 60–75% of these progenies were produced during the first 5 days. When separately given fructose, sucrose or glucose, this wasp accumulated fructose and total sugar at the highest level, which means a high sugar levels might lead to prolonging longevity and more offspring in M. mediator. In addition, compared with organisms fed galactose or raffinose, M. mediator fed sucrose or fructose accumulated high glycogen levels. Furthermore, in M. mediator, the lipid content declined with the advancing age. Females showed the slowest lipid metabolic rates when fed with sucrose, glucose, fructose, mannose and galactose solutions versus when fed with raffinose and control. In addition, only sucrose had a significant effect on lipid levels in males nearing the end of life.     

The intake responses of three species of leaf-nosed Neotropical bats

Flower-visiting bats encounter nectars that vary in both sugar composition and concentration. Because in the new world, the nectars of bat-pollinated flowers tend to be dominated by hexoses, we predicted that at equicaloric concentrations, bats would ingest higher volumes of hexoses than sucrose-containing nectars. We investigated the intake response of three species of Neotropical bats, Leptonycteris curasoae, Glossophaga soricina and Artibeus jamaicensis, to sugar solutions of varying concentrations (292, 438, 584, 730, 876, and 1,022 mmol L⁻¹) consisting of either sucrose or 1:1 mixtures of glucose and fructose solutions. Bats did not show differences in their intake response to sucrose and 1:1 glucose–fructose solutions, indicating that digestion and absorption in bat intestines are designed under the principle of symmorphosis, in which no step is more limiting than the other. Our results also suggest that, on the basis of energy intake, bats should not prefer hexoses over sucrose. We used a mathematical model that uses the rate of sucrose hydrolysis measured in vitro and the small intestinal volume of bats to predict the rate of nectar intake as a function of sugar concentration. The model was a good predictor of the intake responses of L. curasoae and G. soricina, but not of A. jamaicensis.     

Transglucosylation reaction of amylomaltase for the synthesis of anticariogenic oligosaccharides

Synthesis of maltooligosylsucroses by the recombinant amylomaltase from Corynebacterium glutamicum as a N terminal (His)₆ chimera is reported. From the analysis of the products, by TLC and HPLC analysis on a Rezex RSO-Oligosaccharide column, the suitable glucosyl donor was found to be raw tapioca starch. The optimal condition was 2.0% (w/v) sucrose, 2.5% (w/v) raw tapioca starch and 9 U/ml of amylomaltase at 30 °C for 48 h, giving an overall 82% yield of maltooligosylsucrose products. After purified by Bio-Gel P-2 size exclusion column chromatography, the main products were determined by MS and NMR analysis to be maltosyl-, maltotriosyl-, maltotretraosyl- and maltopentaosyl-fructosides (G₂F, G₃F, G₄F and G₅F, respectively, where G = glucosyl unit and F = fructose) with an α-1,4 linkage between the added glucosyl unit and the sucrose. The low cariogenic property of these maltooligosylsucrose products was confirmed by analyzing the effect on the synthesis of water insoluble glucan, acid fermentation, plaque formation and cell aggregation of Streptococcus mutans when compared to those exerted by sucrose. Moreover, by adding sucrose to maltooligosylsucrose products at ratios of 1:1, 1:2 and 1:4, the inhibitory effects on glucosyltransferase activity of S. mutans by 7, 33 and 50%, respectively, were observed. These results suggest that the obtained maltooligosylsucrose products have an anticariogenic property and could be used to substitute for sucrose in food or related products.     

Do hummingbirds have a sweet-tooth? Gustatory sugar thresholds and sugar selection in the broad-billed hummingbird Cynanthus latirostris

Nectar is a solution of mainly three sugars: sucrose, glucose and fructose. Studies have demonstrated that pollinators have preferences according to the sugar composition presented in their diet, and these preferences may be caused by sugar assimilation capacities. However, sugar flavor could also play an important role for sugar preferences of nectar-feeding animals. We evaluated the sugar gustatory thresholds of the broad-billed hummingbird Cynanthus latirostris for sucrose, glucose, fructose and a 1:1 mixture of glucose-fructose. We presented eight C. latirostris to paired feeders containing either a sugar solution or pure water. Additionally, we conducted sugar preference tests at three different concentrations (146, 730 and 1022 mmol L^− 1), to relate sugar preferences with sugar gustatory thresholds. C. latirostris had different gustatory thresholds for the three different sugars tested. At low sugar concentrations (146 mmol L^− 1), sugar selection followed the gustatory thresholds. Hummingbird sugar preference patterns can be affected by different mechanisms, both pre- and post-ingestive. At low concentrations gustatory thresholds may play an important role to determine sugar selection. However, at intermediate and high concentrations, sugar assimilation rates, and velocity of food processing generated by osmotic constraints, can be the mechanisms that explain the sugar selection of these animals.     

Bio-based succinate from sucrose: High-resolution 13C metabolic flux analysis and metabolic engineering of the rumen bacterium Basfia succiniciproducens

Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium Basfia succiniciproducens DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using ¹³C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1 mol mol⁻¹ and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, B. succiniciproducens ΔfruA achieved a titer of 71 g L⁻¹ succinate and a yield of 2.5 mol mol⁻¹ from sucrose.     

Effect of pig faecal donor and of pig diet composition on in vitro fermentation of sugar beet pulp

Two experiments were undertaken to investigate the influence of (1) pig bodyweight and (2) dietary fibre content of the diet on the in vitro gas production of sugar beet pulp fibre using faecal inoculum.In the first experiment, inocula prepared from young pigs (Y; 16–50 kg), growing pigs (G; 62–93 kg) and sows (S; 216–240 kg) were compared. Sugar beet pulp, hydrolysed in vitro with pepsin and then pancreatin, was used as the fermentation substrate. The cumulated gas productions over 144 h were modelled and the kinetics parameters compared. Lag times (Y: 4.6 h; G: 6.4 h; S: 9.2 h) and half-times to asymptote (Y: 14.7 h; G: 15.9 h; S: 20.8 h) increased with pig bodyweight (P<0.001) and the fractional degradation rates of the substrate differed between the pig categories (Y: 0.110 h⁻¹; G: 0.115 h⁻¹; S: 0.100 h⁻¹; P<0.001). The final gas production was not affected (P=0.10) by the inoculum source.In the second experiment hydrolysed sugar beet pulp was fermented with four inocula prepared from pigs fed diets differing in their total and soluble dietary fibre contents, i.e. low fibre diet rich in soluble fibre (LOW-S) or in insoluble fibre (LOW-I) or high fibre diet rich in soluble fibre (HIGH-S) or in insoluble fibre (HIGH-I). The total and the soluble dietary fibres influenced the kinetics of gas production. The presence of soluble fibres decreased the lag times, whatever the total dietary fibre content (2.7 h for LOW-S versus 3.5 h for LOW-I, 4.0 h for HIGH-S versus 4.4 h for HIGH-I; P<0.001). The half-times to asymptote were higher with the low fibre diets (P<0.001) and, for similar total dietary fibre contents, they were lower when the proportion of soluble fibres increased (LOW-S: 9.9 h; LOW-I: 11.4 h; HIGH-S: 8.9 h; HIGH-I: 10.1 h; P<0.001). The fractional degradation rates of the substrate were the highest with the fibre-rich diet containing a high proportion of soluble fibres (0.158 h⁻¹; P<0.001).In conclusion, the bodyweight of the faeces donors and the dietary fibre composition of the pig diet influence the in vitro fermentation kinetics of hydrolysed sugar beet pulp, but not the final gas production.     

Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures

Conidia of Trichoderma harzianum produced from either solid or liquid fermentation must be dried to prevent spoilage by microbial contamination, and to induce dormancy for formulation development and prolonged self-life. Drying conidia of Trichoderma spp. in large scale production remains the major constraint because conidia lose viability during the drying process at elevated temperatures. Moreover, caking must be avoided during drying because heat generated by milling conidial chunks will kill conidia. It is ideal to dry conidia into a flow-able powder for further formulation development. A method was developed for microencapsulation of Trichoderma conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10¹⁰ cfu in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31 °C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of T. harzianum was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations.     

Unmarked insertional inactivation in the gfo gene improves growth and ethanol production by Zymomonas mobilis ZM4 in sucrose without formation of sorbitol as a by-product,but yields opposite effects in high glucose

Sorbitol, one of the main by-products of growth on high sucrose concentrations, is catalyzed by glucose-fructose oxidoreductase (GFOR, EC 1.1.99.28) in Zymomonas mobilis, which decreases the ethanol yield. In this study, an unmarked gfo mutant from Z. mobilis ZM4 was constructed using a site-specific FLP recombinase, and growth and ethanol production were evaluated with or without the addition of sorbitol to the media. The inactivation of gfo had contrasting effects in different substrates, especially at high concentrations. The maximum specific growth rate (μ_m) and theoretical ethanol yield value (Y_m) increased from 0.065 h⁻¹ and 60.56% to 0.094 h⁻¹ and 83.87% in 342 g/L sucrose, respectively. Conversely, in 200 g/L glucose, gfo inactivation decreased μ_m and Y_m from 0.15 h⁻¹ and 89.85% to 0.10 h⁻¹ and 67.59%, respectively, and prolonged the lag period from 16 h to 40 h. The addition of sorbitol slightly accelerated growth and sucrose hydrolysis by the gfo mutant in 342 g/L sucrose; however, addition of sorbitol restored the μ_m and Y_m of the gfo mutant in 200 g/L glucose to 0.14 h⁻¹ and 82.50%, respectively. Inactivation of gfo had a small effect on fructose utilization, and a positive one on mixture of glucose and fructose similar to that on sucrose. These results provide further understanding of the osmoregulation mechanisms in Z. mobilis and may help to exploit the biotechnological applications of this industrially important bacterium.     

Regioselective synthesis of neo-erlose by the β-fructofuranosidase from Xanthophyllomyces dendrorhous

The β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous (Xd-INV) catalyzes the synthesis of neo-fructooligosaccharides (neo-FOS of the ⁶G-series), which contain a β(2 → 6) linkage between a fructose and the glucosyl moiety of sucrose. In this work we demonstrate that the enzyme is also able to fructosylate other carbohydrates that contain glucose, in particular disaccharides (maltose, isomaltulose, isomaltose, trehalose) and higher oligosaccharides (maltotriose, raffinose, maltotetraose), but not monosaccharides (glucose, fructose, galactose). With maltose as acceptor, the reaction in the presence of Xd-INV proceeded with high regioselectivity; the product was purified and chemically characterized, and turned out to be 6′-O-β-fructosylmaltose (neo-erlose). Using 100 g/L sucrose as fructosyl donor and 300 g/L maltose as acceptor, the maximum concentration of neo-erlose was 38.3 g/L. Thus, novel hetero-fructooligosaccharides with potential applications in the functional food and pharmaceutical industries can be obtained with Xd-INV.     

Fruit nutritional composition and non-nutritive traits of indigenous South African tree species

Frugivorous animals play a major role in dispersing tropical, and to a lesser extent, temperate tree species. In order to attract potential seed dispersers, plants generally offer a reward of fleshy fruit pulp. Criteria for fruit choice by avian frugivores are influenced by a number of non-nutritive (e.g. fruit size and colour) factors; and nutritional composition of the fruit. There is a paucity of nutritional composition and other fruit trait data of indigenous South African fruit. This information is necessary in order to determine which frugivores are likely to ingest which fruits and consequently act as potential seed dispersal agents. This information would provide us with an understanding of the inter-relationships between indigenous fruit and frugivores in South Africa. Consequently nutritional composition was investigated in various indigenous fruit species that avian frugivores feed on. Fruits were collected from 38 indigenous tree species found in KwaZulu-Natal Afromontane and coastal forests. Pulp was freeze-dried to constant mass and then analysed for sugar, lipid and protein content; and for water content determination. Fruit width in this study ranged from 4 mm (Searsia rehmanniana and Trema orientalis) to 40 mm (Annona senegalensis, Ficus sur and Xylotheca kraussiana). Of the fruits examined in this study 29% were black and 43% were red when ripe. Most (84%) fruit species analysed for sugar content were hexose dominant with 50% being fructose and 34% being glucose dominant. Only 16% of the fruit species analysed were sucrose dominant. Fruits in this study were generally observed to be high (mean: 68.1 ± 3.3%; n = 30) in water content; and low in protein and lipid content respectively (mean: 8.2 ± 0.5%; 9.3 ± 2.2%; n = 30) indicating that these fruit species could be considered as nutrient-dilute. Future studies need to determine the nutritional composition of the remaining indigenous South African fruit in order to develop a comprehensive database as well as examining non-nutritive factors.     

Effects of feeding high- and low-fibre fractions of air-classified,solvent-extracted canola meal on diet nutrient digestibility and growth performance of weaned pigs

The dietary energy value of solvent-extracted canola meal (CM) is limited by its relative high fibre content. The fibre-rich hull of canola is denser than the oil-free cotyledons, so these seed components partially fractionate in a stream of air. Air classification thus separates CM into a low-fibre, light-particle fraction and a high-fibre, heavy-particle fraction of interest for feeding monogastric and ruminant species, respectively. Crude fibre (CF), acid detergent fibre (ADF) and neutral detergent fibre (NDF) in light-particle fraction were reduced by 96, 34 and 28% compared with CM (83 CF, 165 ADF, 238 NDF g/kg, as-is). Brassica (B) napus, Brassica juncea, or their fractions were evaluated feeding 288 weaned pigs (7.1 kg) for 37 d as a 2 × 3 factorial with 12 replicate pens per treatment. Wheat-based diets including 200 g of test feedstuff/kg provided 10.5 and 10.0 MJ net energy (NE)/kg and 1.27 and 1.15 g standardised ileal digestible lysine/MJ NE and were fed for 9 and 28 d, respectively. Pen feed added, orts, and individual pig body weight were measured weekly to calculate average daily feed intake (ADFI), average daily gain (ADG), and feed efficiency (G:F). Pen faecal samples were collected on d 16 and 17 to calculate diet apparent total tract digestibility coefficients (CATTD) of dry matter (DM), gross energy (GE), crude protein (CP) and digestible energy (DE) value. Pigs fed B. juncea had 3 and 2% higher (P<0.001) CATTD of DM (0.82 vs. 0.79) and GE (0.84 vs. 0.82) than pigs fed B. napus. Feeding the light-particle fraction increased (P<0.001) CATTD of DM (0.82 vs. 0.79), GE (0.84 vs. 0.82), and CP (0.79 vs. 0.77) by 4, 3 and 3% compared with CM, respectively. For the entire trial, pigs fed B. juncea consumed 33 g/d less (P<0.001) feed (723 vs. 756 g/d), had 0.02 higher (P<0.05) G:F (0.735 vs. 0.718 g:g), but ADG (503 vs. 514 g/d) was not different (P>0.05) compared to pigs fed B. napus. Feeding pigs the light-particle fractions did not affect (P>0.05) ADFI (741 vs. 736 g/d), increased (P<0.05) G:F 0.02 (0.739 vs. 0.721 g:g) and tended to increase (P=0.07) ADG (519 vs. 501 g/d) by 18 g/d compared to CM. In conclusion, air classification of canola meal increased diet nutrient digestibility, but only modestly increased G:F of weaned pigs due to dietary fibre reduction.     

Effect of grazing and homeopathy on milk production and immunity of Merino derived ewes

The effect of grazing and homeopathic therapy on sheep immune response and milk production was investigated on 40 multiparous Merino derived ewes. Twenty animals were housed in an indoor-bedded pen (P), whereas 20 others were allowed to graze on pasture for 9 h/d (G). P and G animals were fed an equivalent diet in terms of dry matter intake, crude protein percentage and energy concentration. In each group, 10 animals were subjected to unicistic homeopathic treatments (H), while 10 ewes were kept as a control and treated with conventional medicine when necessary (C). The grazing rearing system had a marked positive effect on in vivo cellular immune response (delayed-type hypersensivity to PHA, P < 0.001). Grazing animals produced more milk than the penned ones (1048.00 ± 75.61 kg versus 853.04 ± 67.78 kg, P < 0.05), with increased content of milk fat (7.69 ± 0.15% versus 7.25 ± 0.14%, P < 0.05). Accordingly, blood levels of triglycerides (P < 0.01), urea (P < 0.001) and alanine aminotransferase (ALT) (P < 0.001) were significantly higher in group G. The homeopathic treatments produced limited effects on the milk production and immune response. However, such treatments reduced the risk of contamination of the products with medicinal traces, as H group received no allopathic treatment.     

Production,characterization and antioxidant activities in vitro of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

The production, characterization and antioxidant activities in vitro of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3 were investigated. For EPS production, the preferable culture conditions were 24 °C and pH 8 for 60 h with sucrose and yeast extract as the carbon and nitrogen sources, respectively. Notably, sucrose concentration was the prominent factor, and the maximum yield of EPS (22.82 g/L) was obtained at a sucrose concentration of 160 g/L. The crude EPS was purified by chromatography of DEAE-52 and Sephadex G-100, affording EPS-1 and EPS-2 with molecular weights of 1.22 × 10⁶ and 8.69 × 10⁵ Da, respectively. They were composed of mannose, fructose and glucose in a molar ratio of 2.59:29.83:1 and 4.23:36.59:1, respectively. In addition, both crude and purified EPS showed strong scavenging activities on superoxide and hydroxyl radicals, and their antioxidant activities decreased in the order of crude EPS > EPS-2 > EPS-1.     

Presence of Mycoplasma species and somatic cell counts in bulk-tank goat milk

This cross-sectional study performed on dairy goat herds was designed to establish the relationship between the presence of Mycoplasma species in bulk-tank milk samples from different farms and the bulk-tank milk somatic cell count (BTMSCC) in an area where contagious agalactia (CA) is endemic. Three BTMSCC thresholds, used in payment schemes or as legal requirements for milk quality in Europe and the USA, were considered: (1) 2,000,000 cells/ml; (2) 1,500,000 cells/ml and (3) 1,000,000 cells/ml. Of the 1068 milk samples tested, 7.9% (n = 84) showed the presence of Mycoplasma spp. (Mycoplasma agalactiae 82% and Mycoplasma mycoides subsp. mycoides large colony 17%). Somatic cell counts for bulk-tank samples containing mycoplasmas were higher than those recorded for negative samples (1,176,000 cells/ml vs. 875,000 cells/ml; P < 0.001). Two-by-two table analyses revealed that the presence of mycoplasmas in bulk-tank milk increased the risk of surpassing all SCC thresholds considered, with the highest risk for Mycoplasma positive bulk-tank milk samples exceeding the threshold of 1500 × 10³ cells/ml (odds ratio = 2.42 (1.49 < OR < 3.91). Our results indicate that the presence of mycoplasmas in goat milk had yet another economic consequence and a further incentive to encourage the implementation of specific programs for disease control.     

Mitochondrial distribution patterns in canine oocytes as related to the reproductive cycle stage

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n = 2), follicular phase (F, n = 4), ovulation (O, n = 2), early luteal (EL, n = 7) and mid/late luteal phase (MLL, n = 2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (I) small aggregates diffused throughout the cytoplasm; (II) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II + III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P < 0.05; O vs. A: P < 0.001), in EL (61%; O vs. EL: P < 0.01), or in MLL (0%; F vs. MLL: P < 0.05; O vs. MLL: P < 0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P < 0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes.     

Effect of deglycosylation on the properties of thermophilic invertase purified from the yeast Candida guilliermondii MpIIIa

Invertase from Candida guilliermondii MpIIIa was purified and biochemically characterized. The purified enzyme (INV3a-N) is a glycoprotein with a carbohydrate composition comprising nearly 74% of its total molecular weight (MW) and specific activity of 82,027 U/mg of protein. The enzyme displayed optimal activity at pH 5.0 and 65 ˚C. The Km and V_max values for INV3a-N were 0.104 mM and 10.9 μmol/min/mg of protein, respectively, using sucrose as the substrate. The enzyme retained 50% and 20% of its maximal activity after 168 h and 30 days, respectively, at 50 ˚C. INV3a-N was fully active at sucrose concentrations of 400 mM and the activity of the enzyme dropped slowly at higher substrate concentration. Interestingly, the deglycosylated form of INV3a-N (INV3a-D) displayed 76–92% lower thermostability than that of INV3a-N at all temperatures assayed (50–70 ˚C), and was inhibited at sucrose concentrations of 200 mM. Findings here indicate glycosylation plays an important role, not only in the thermostability of INV3a-N, but also in the inhibition of the enzyme by sucrose. Since the enzyme is active at high sucrose concentrations, INV3a-N may be considered a suitable candidate for numerous industrial applications involving substrates with high sugar content or for improvement of ethanol production from cane molasses.     

Hexokinase—A limiting factor in lipid production from fructose in Yarrowia lipolytica

Microbial biolipid production has become an important part of making biofuel production economically feasible. Genetic engineering has been used to improve the ability of Yarrowia lipolytica, an oleaginous yeast, to produce lipids using glucose-based media. However, few studies have examined lipid accumulation by Y. lipolytica׳s ability to utilize other hexose sugars, and as of yet, the rate-limiting steps in this process are unidentified. In this study, we investigated the de novo accumulation of lipids by Y. lipolytica when grown in glucose, fructose, and sucrose. Three Y. lipolytica wild-type (WT) strains of varied origin differed significantly in their lipid production, growth, and fructose utilization. Hexokinase (ylHXK1p) activity partially explained these differences. Overexpression of the ylHXK1 gene led to increased hexokinase activity (6.5–12 times higher) in the mutants versus the WT strains; a pronounced reduction in cell filamentation in mutants grown in fructose-based media; and improved biomass production, particularly in the mutant whose parent had shown the lowest growth capacity in fructose (French strain W29). All mutants showed improved lipid yield and production when grown on fructose, although the effect was strain dependent (23–55% improvement). Finally, we overexpressed ylHXK1 in a highly modified strain of Y. lipolytica W29 engineered to optimize oil production. This modification was combined with Saccharomyces cerevisiae invertase gene expression to evaluate the resulting mutant׳s ability to produce lipids using cheap industrial substrates, namely sucrose (a major component of molasses). Sucrose turned out to be a better substrate than either of its building blocks, glucose or fructose. Over its 96 h of growth in the bioreactors, this highly modified strain produced 9.15 g L⁻¹ of lipids, yielding 0.262 g g⁻¹ of biomass.