Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.

Flavonoids and proanthocyanidins-rich fractions from Eugenia dysenterica fruits and leaves inhibit the formation of advanced glycation end-products and the activities of α-amylase and α-glucosidase.

ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Eugenia dysenterica have been popularly used in Brazil for treating diabetes mellitus and its complications. The present study aimed to screen extracts from E. dysenterica fruit pulp, peel, seed and leaf for carbohydrate digestive enzymes inhibitors with antioxidant and anti-glycation capacities. MATERIALS AND METHODS: Ethanol extracts of E. dysenterica were subjected to a liquid-liquid fractionation and the fractions were used to evaluate their antioxidant properties and inhibitory potential against the formation of advanced glycation end-products (AGEs) and α-amylase and α-glucosidase. RESULTS: The ethyl acetate fraction (EtOAcF) from seed and the dichloromethane fraction (CH2Cl2F) and EtOAcF from leaf had high antioxidant capacities (ORAC >5500 µmol trolox eq g-1, FRAP >1500 µmol trolox eq g-1 and DPPH IC50 < 35 µg mL-1) and showed exceptional inhibitory activities against AGEs formation (glycation inhibition above 80% at 10 µg mL-1) and α-amylase and α-glucosidase (inhibition above 50% at 10 µg mL-1). The gallated B-types proanthocyanidins were the most active ingredients found in the leaf of E. dysenterica (CH2Cl2 and EtOAcF), being responsible for the notorious inhibitory effects against glycation and glycoside hydrolases due to their ortho-hydroxyl groups, which play role in scavenge and quench free radicals and glycated products, and may occupy the enzymes' substrate binding pocket. Furthermore, gallic acid, quercetin and its glycoside derivatives were detected by the first time in the E. dysenterica fruit seed (EtOAcF). CONCLUSIONS: The results strongly contribute to the understanding of the antidiabetic potential of seeds and leaves from E. dysenterica, a species from a global biodiversity hotspot, which appears to be linked to the prevention of oxidative stress, AGEs production and postprandial hyperglycemia.

Assessment of Chemopreventive Potential of the Plant Extracts against Liver Cancer Using HepG2 Cell Line.

The edible parts of the plants Camellia sinensis, Vitis vinifera and Withania somnifera were extensively used in ancient practices such as Ayurveda, owing to their potent biomedical significance. They are very rich in secondary metabolites such as polyphenols, which are very good antioxidants and exhibit anti-carcinogenic properties. This study aims to evaluate the anti-cancerous properties of these plant crude extracts on human liver cancer HepG2 cells. The leaves of Camellia sinensis, Withania somnifera and the seeds of Vitis vinifera were collected and methanolic extracts were prepared. Then, these extracts were subjected to DPPH, α- amylase assays to determine the antioxidant properties. A MTT assay was performed to investigate the viability of the extracts of HepG2 cells, and the mode of cell death was detected by Ao/EtBr staining and flow cytometry with PI Annexin- V FITC dual staining. Then, the protein expression of BAX and BCl2 was studied using fluorescent dye to determine the regulation of the BAX and BCl2 genes. We observed that all the three extracts showed the presence of bioactive compounds such as polyphenols or phytochemicals. The W. somnifera bioactive compounds were found to have the highest anti-proliferative activity on human liver cancer cells.

Optimization and scale-up of α-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes.

BACKGROUND: Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box-Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. RESULTS: The substrate optimization for α-amylase production by the Box-Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. CONCLUSIONS: The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

Regulation of gene expression encoding the digestive α-amylase in the larvae of Colorado potato beetle, Leptinotarsa decemlineata (Say) in response to plant protein extracts.

Considering the relevance of insect α-amylases and natural α-amylase inhibitors present in plants to protect against insect damage, we investigated the effect of white bean and rapeseed protein extracts on digestive α-amylase gene expression of the Colorado potato beetle, Leptinotarsa decemlineata (Say). For this purpose, in vitro and in vivo trials were performed to determine the inhibitory activity of seed proteins on the third and fourth instar larvae. In both trials, the significant inhibitory effect of each extracts on the third and fourth instar larval α-amylase activity and considerable mortality in treatments were observed compared to control trials. In the RT-qPCR, expression ratio demonstrated that the α-amylase gene of two different larval stages grown on both proteins treated leaves had significantly differentiated expression and was up-regulated in third instar larvae and down-regulated in fourth instar larvae compared to control. Results suggest that the hyper-production of α-amylase in third instar larvae is elicited to compensate for the enzyme activity inhibition at an earlier stage and also down-regulation suggests the existence of a negative feedback of plant proteins on the last instar larvae via impaired food intake and digestive α-amylase activity in Colorado potato beetle. Therefore, disruption of the insect's digestive physiology by plant defensive proteins can be considered in the development of innovative controlling methods of this crucial potato pest.

Effects of maternal protein supplementation and inclusion of rumen-protected fat in the finishing diet on nutrient digestibility and expression of intestinal genes in Nellore steers.

The study aimed to evaluate nutrient digestibility and intestine gene expression in the progeny from cows supplemented during gestation and fed diets with or without rumen-protected fat (RPF) in the feedlot. Forty-eight Nellore steers, averaging 340 kg, were housed in individual pens and allotted in a completely randomized design using a 2 × 2 factorial arrangement (dams nutrition × RPF). Cows' supplementation started after 124 ± 21 days of gestation. The feedlot lasted 135 days and diets had the inclusion of zero or 6% of RPF. Digestibility was evaluated by total feces collection. Steers were slaughtered using the concussion technique and samples of pancreas and small intestine were collected immediately after the slaughter to analyze α-amylase activity, and the expression of SLC5A1, CD36, and CCK and villi morphometry. Feeding RPF increased nutrients digestibility (p < 0.01). There was no effect of maternal nutrition on digestibility and α-amylase activity in steers (p > 0.05). Duodenal expression of SLC5A1, CD36, and CCK increased in the progeny from restricted cows. In conclusion, protein restriction during mid to late gestation of dams has long-term effects on small-intestine length and on expression of membrane transporters genes in the duodenum of the progeny. However, maternal nutrition does not affect digestibility in the feedlot.

Evaluation of in-vitro and in-vivo antidiabetic, antilipidemic and antioxidant potentials of aqueous root extract of Strophanthus hispidus DC (Apocynaceae).

Background Antidiabetic activity of aqueous root extract of Strophanthus hispidus (SHP) was evaluated based on its folklore used in traditional medicine for the treatment of diabetes. Objectives: This study aimed to investigate the in-vitro and in-vivo antidiabetic potential of the aqueous root extract of SHP. Methods SHP (50, 100 and 200 mg/kg p.o.), glibenclamide (5 mg/kg p.o.), normal saline (10 mL/kg; diabetic control) and distilled water (10 mL/kg; normal control) were administered once daily for 28 days, with the measurement of fasting blood glucose level at 7 days interval. Blood samples were collected on day 28 for serum biochemical (albumin, total protein [TP], creatinine, alanine transaminase [ALT], aspartate transaminase [AST], alkaline phosphatase [ALP], triglycerides [TG], total cholesterol [TC], high-density lipoprotein [HDL], low-density lipoprotein [LDL], bilirubin and urea) and hematological assays. The in-vitro antidiabetic activity was investigated using α-amylase and α-glucosidase enzymes inhibitory assays. Results SHP produced a day-dependent reduction in glucose level. Peak reduction (82.94 %; p < 0.05) was produced at the dose of 100 mg/kg. SHP significantly (p < 0.05) increased the level of HDL and TP but significantly (p < 0.05) reduced the levels of TG, LDL, TC, AST, ALT, ALP, bilirubin, creatinine and urea compared with diabetic control rats. Furthermore, SHP significantly (p < 0.05) increased the level of catalase, superoxide dismutase and reduced glutathione compared to diabetic control rats. SHP significantly (p < 0.05) inhibited α-amylase and α-glucosidase enzymes compared with acarbose. Conclusion The findings in this study showed that SHP possesses beneficial antidiabetic activity.

Expression patterns of alpha-amylase and beta-amylase genes provide insights into the molecular mechanisms underlying the responses of tea plants (Camellia sinensis) to stress and postharvest processing treatments.

MAIN CONCLUSION: The alpha-amylase and beta-amylase genes have been identified from tea plants, and their bioinformatic characteristics and expression patterns provide a foundation for further studies to elucidate their biological functions. Alpha-amylase (AMY)- and beta-amylase (BAM)-mediated starch degradation plays central roles in carbohydrate metabolism and participates extensively in the regulation of a wide range of biological processes, including growth, development and stress response. However, the AMY and BAM genes in tea plants (Camellia sinensis) are poorly understood, and the biological functions of these genes remain to be elucidated. In this study, three CsAMY and nine CsBAM genes from tea plants were identified based on genomic and transcriptomic database analyses, and the genes were subjected to comprehensive bioinformatic characterization. Phylogenetic analysis showed that the CsAMY proteins could be clustered into three different subfamilies, and nine CsBAM proteins could be classified into four groups. Putative catalytically active proteins were identified based on multiple sequence alignments, and the tertiary structures of these proteins were analyzed. Cis-element analysis indicated that CsAMY and CsBAM were extensively involved in tea plant growth, development and stress response. In addition, the CsAMY and CsBAM genes were differentially expressed in various tissues and were regulated by stress treatments (e.g., ABA, cold, drought and salt stress), and the expression patterns of these genes were associated with the postharvest withering and rotation processes. Taken together, our results will enhance the understanding of the roles of the CsAMY and CsBAM gene families in the growth, development and stress response of tea plants and of the potential functions of these genes in determining tea quality during the postharvest processing of tea leaves.

Silencing of α-amylase StAmy23 in potato tuber leads to delayed sprouting.

Potato tuber dormancy is critical for the postharvest quality. The supply of carbohydrates is considered as one of the important factors controlling the rate of potato tuber sprouting. Starch is the major carbohydrate reserve in potato tuber, but very little is known about the specific starch degrading enzymes responsible for controlling tuber dormancy and sprouting. In this study, we demonstrate that an α-amylase gene StAmy23 is involved in starch breakdown and regulation of tuber dormancy. Silencing of StAmy23 delayed tuber sprouting by one to two weeks compared with the control. This phenotype is accompanied by reduced levels of reducing sugars and elevated levels of malto-oligosaccharides in tuber cortex and pith tissue below the bud eye of StAmy23-deficient potato tubers. Changes in soluble sugars is accompanied by a slight variation of phytoglycogen structure and starch granule size. Our results suggest that StAmy23 may stimulate sprouting by hydrolyzing soluble phytoglycogen to ensure supply of sugars during tuber dormancy.

Biotic elicitation as a tool to improve strawberry and raspberry extract potential on metabolic syndrome-related enzymes in vitro.

BACKGROUND: Raspberry and strawberry are high value-added food products that can contribute to human health due to the abundance of polyphenols that they contain. Polyphenols are secondary metabolites and therefore devoted to improve plant adaptation, these polyphenol profile can be induced applying different stimuli, such as certain bacteria. The aim of this study was twofold: (i) to evaluate the ability of two bacterial strains to modulate secondary metabolisms in strawberry and raspberry, and (ii) to explore the ability of plant extracts to modify enzyme activities related to metabolic syndrome. RESULTS: Total phenolic and anthocyanin content was higher in strawberries than in raspberries, despite similar antioxidant capacities. Strawberry extracts performed better on the tested enzymes, except on α-glucosidase inhibition capacity. Bacillus amyloliquefaciens stabilized the effects of extracts at different points in time, and Pseudomonas fluorescens modified plant metabolism after more inoculations (spring) in both species, improving the effects of raspberry extracts on α-glucosidase, COX1, and COX2, and of strawberry on α-amylase and COX1. CONCLUSION: It is good to include these two fruits in the diet because they improve the activity of metabolic syndrome-related enzymes. Applying either strain during plant growth modifies the bioactive profile of the plants, improving the effects of the fruit extracts on human health. © 2018 Society of Chemical Industry.

Efficient Hydrolysis of Raw Microalgae Starch by an α-Amylase (AmyP) of Glycoside Hydrolase Subfamily GH13_37.

Microalgae starch is receiving increasing attention as a renewable feedstock for biofuel production. Raw microalgae starch from Tetraselmis subcordiformis was proven to be very efficiently hydrolyzed by an α-amylase (AmyP) of glycoside hydrolase subfamily GH13_37 below the temperature of gelatinization (40 °C). The hydrolysis degree reached 74.4 ± 2.2% for 4% raw microalgae starch and 53.2 ± 1.7% for 8% raw microalgae starch after only 2 h. The hydrolysis efficiency was significantly stimulated by calcium ions. The enzyme catalysis of AmyP and its mutants (Q306A and E347A) suggested that calcium ions contributed to the hydrolysis of cyclic structures in raw microalgae starch by a distinctive calcium-binding site Ca2 of AmyP. The study explored raw microalgae starch as a new resource for cold enzymatic hydrolysis and extended our knowledge on the function of calcium in amylolytic enzyme.

Novel carbohydrate binding modules in the surface anchored α-amylase of Eubacterium rectale provide a molecular rationale for the range of starches used by this organism in the human gut.

Gut bacteria recognize accessible glycan substrates within a complex environment. Carbohydrate binding modules (CBMs) of cell surface glycoside hydrolases often drive binding to the target substrate. Eubacterium rectale, an important butyrate-producing organism in the gut, consumes a limited range of substrates, including starch. Host consumption of resistant starch increases the abundance of E. rectale in the intestine, likely because it successfully captures the products of resistant starch degradation by other bacteria. Here, we demonstrate that the cell wall anchored starch-degrading α-amylase, Amy13K of E. rectale harbors five CBMs that all target starch with differing specificities. Intriguingly these CBMs efficiently bind to both regular and high amylose corn starch (a type of resistant starch), but have almost no affinity for potato starch (another type of resistant starch). Removal of these CBMs from Amy13K reduces the activity level of the enzyme toward corn starches by ∼40-fold, down to the level of activity toward potato starch, suggesting that the CBMs facilitate activity on corn starch and allow its utilization in vivo. The specificity of the Amy13K CBMs provides a molecular rationale for why E. rectale is able to only use certain starch types without the aid of other organisms.

A tandem CBM25 domain of α-amylase from Microbacterium aurum as potential tool for targeting proteins to starch granules during starch biosynthesis.

BACKGROUND: Starch-binding domains from carbohydrate binding module family 20 have been used as a tool for starch engineering. Previous studies showed that expression of starch binding domain fusion proteins in planta resulted in modified starch granule structures and physicochemical properties. However, although 13 carbohydrate binding module families have been reported to contain starch-binding domains, only starch-binding domains from carbohydrate binding module family 20 have been well studied and introduced into plants successfully. In this study, two fragments, the tandem CBM25 domain and the tandem CBM25 with multiple fibronectin type III (FN3) domains of the α-amylase enzyme from Microbacterium aurum, were expressed in the tubers of a wild type potato cultivar (cv. Kardal) and an amylose-free (amf) potato mutant. RESULTS: The (CBM25)2 and FN3 protein were successfully accumulated in the starch granules of both Kardal and amf transformants. The accumulation of (CBM25)2 protein did not result in starch morphological alterations in Kardal but gave rise to rough starch granules in amf, while the FN3 resulted in morphological changes of starch granules (helical starch granules in Kardal and rough surface granules in amf) but only at a very low frequency. The starches of the different transformants did not show significant differences in starch size distribution, apparent amylose content, and physico-chemical properties in comparison to that of untransformed controls. CONCLUSION: These results suggest that the starch-binding domains from carbohydrate binding module family 25 can be used as a novel tool for targeting proteins to starch granules during starch biosynthesis without side-effects on starch morphology, composition and properties.

OPTIMIZATION OF ALKALINE Α-AMYLASE PRODUCTION BY THERMOPHILIC BACILL US SUBTILIS.

BACKGROUND: Starch-degrading amylase enzyme is important in biotechnological applications as food, fermentation, textile, paper and pharmaceutical purposes. The aim of current study to isolate alkaline thermostable α-amylase bacteria and then study the composition of medium and culture conditions to optimize cells growth and a-amylase production. MATERIALS AND METHODS: Thermophilic amylase producing bacterium was isolated from local hot water-springs in Gazan city Saudi Arabia. RESULTS: Phylogenetic analysis of 16 S rRNA sequence for the strain revealed that the strain have the same sequence of Bacillus subtilis. Maximum amylase production was observed, when B. subtilis cultured in medium containing starch at concentration 0.5%, and 10 g/L peptones as nitrogen source at pH 8.5 in when it was incubated for 48 h at 45°C. CONCLUSION: An amylase-producing bacterium were isolated from hot-spring water and was identified as B. subtilis. Amylase produced from B.subtilis had optimum temperature 45°C and pH 8.5 in shaking media.

Natural Prenylchalconaringenins and Prenylnaringenins as Antidiabetic Agents: α-Glucosidase and α-Amylase Inhibition and in Vivo Antihyperglycemic and Antihyperlipidemic Effects.

Inhibition of α-glucosidase and α-amylase decreases postprandial blood glucose levels and delays glucose absorption, making it a treatment strategy for type 2 diabetes. This study examined in vivo and in vitro antidiabetic activities of natural prenylchalconaringenins 1 and 2 and prenylnaringenins 3 and 4, found in hops and beer. 3'-Geranylchalconaringenin (2) competitively and irreversibly inhibited α-glucosidase (IC50 = 1.08 µM) with activity 50-fold higher than that of acarbose (IC50 = 51.30 µM) and showed moderate inhibitory activity against α-amylase (IC50 = 20.46 µM). Docking analysis substantiated these findings. In addition, compound 2 suppressed the increase in postprandial blood glucose levels and serum levels of total cholesterol and triglycerides in streptozotocin-induced diabetic mice. Taken together, these results suggest that 2 has dual inhibitory activity against α-glucosidase and α-amylase and alleviates diabetic hyperglycemia and hyperlipidemia, making it a potential functional food ingredient and drug candidate for management of type 2 diabetes.

Carbohydrate-binding module 74 is a novel starch-binding domain associated with large and multidomain α-amylase enzymes.

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract.

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains.

The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.

Direct bioethanol production by amylolytic yeast Candida albicans.

UNLABELLED: An attempt was made to produce bioethanol using optimized fermentation parameters and mutationally improved strain of Candida albicans. The mutant strain OMC3E6 obtained by UV irradiation followed by ethidium bromide successive mutations showed 2.6 times more glucoamylase secretion and 1.5 times more bioethanol production via direct conversion of starch. Enhanced hydrolysis of insoluble starch (72%) and potato starch (70%) was achieved with glucoamylase enzyme preparation from mutant C. albicans. In fermentation medium, the use of maltose, corn steep liquor, NaH2 PO4 , NaCl + MgSO4 and Triton X-100 has increased the glucoamylase production by the microbe. Under optimized conditions, C. albicans eventually produced 437 g ethanol kg(-1) potatoes. Earlier reports mentioned the use of thrice the quantity of starch as reported by us followed by more fermentation period (3-4 days) and demanded pretreatment of starch sources with alpha-amylase as well. Here, we simplified these three steps and obtained 73% conversion of insoluble starch into ethanol via direct conversion method in a period of 2 days without the involvement of cell immobilizations or enzyme pretreatment steps. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to fast depletion of fossil fuels in the modern world, bioethanol usage as an alternate energy source is the need of the hour. For the first time, we report bioethanol production by Candida albicans via direct conversion of starchy biomass into ethanol along with enhanced starch-hydrolysing capacity and ethanol conversion ratio. So far, C. albicans was dealt in the field of clinical pathology, but here we successfully employed this organism to produce bioethanol from starchy agri-substrates. Optimizing fermentation parameters and improving the microbial strains through successive mutagenesis can improve the end product yield.

Ectopic expression of human acidic fibroblast growth factor 1 in the medicinal plant, Salvia miltiorrhiza, accelerates the healing of burn wounds.

BACKGROUND: Healing of burns is a complex process and very few effective treatments exist to facilitate the burn recovery process. Human acidic fibroblast growth factor 1 (FGF-1) plays an important role in a variety of biological processes, including angiogenesis, and tissue repair. Salvia miltiorrhiza is widely used in traditional Chinese medicine as an herb for the treatment of various diseases, including cardiovascular and cerebrovascular diseases, and traumatic injuries. We present that expression of FGF-1 in S. miltiorrhiza significantly accelerates the healing of burn wounds. RESULTS: The human fgf-1 gene was fused with a barley α-amylase signal peptide DNA sequence and driven by a 35S promoter for constitutive expression in transgenic S. miltiorrhiza plants. The highest yield of recombinant FGF-1 obtained from leaves of transgenic S. miltiorrhiza lines was 272 ng/fresh weight. Aqueous extracts from transgenic S. miltiorrhiza exhibited FGF-1 activity approximately 19.2-fold greater than that of the standard FGF-1. Compared to the standard FGF-1 or the extracts obtained from non-transgenic plants, it stimulated proliferation of Balb/c 3 T3 mouse fibroblast cells assessed with the standard MTT assay and promoted angiogenesis in the chicken embryo chorioallantoic membrane (CAM) assay. Topical application of the extract significantly accelerated the burn wound healing process. CONCLUSIONS: The product appears to retain the biological activity of both FGF-1 as well as the medicinal properties of the plant. The extracts from transgenic S. miltiorrhiza combines the therapeutic functions of FGF-1 and the medicinal plant, S. miltiorrhiza. Topical application of the product can reduce the costs associated with extraction, purification, and recovery.

2,3-butanediol production from starch by engineered Klebsiella pneumoniae G31-A.

2,3-Butanediol (2,3-BD) is an organic compound, which is widely used as a fuel and fuel additive and applied in chemical, food, and pharmaceutical industries. Contemporary strategies for its economic synthesis include the development of microbial technologies that use starch as cheap and renewable feedstock. The present work encompasses the metabolic engineering of the excellent 2,3-BD producer Klebsiella pneumoniae G31. In order to perform direct starch conversion into 2,3-BD, the amyL gene encoding quite active, liquefying α-amylase in Bacillus licheniformis was cloned under lac promoter control in the recombinant K. pneumoniae G31-A. The enhanced extracellular over-expression of amyL led to the highest extracellular amylase activity (68 U/ml) ever detected in Klebsiella. The recombinant strain was capable of simultaneous saccharification and fermentation (SSF) of potato starch to 2,3-BD. In SSF batch process by the use of 200 g/l starch, the amount of total diols produced was 60.9 g/l (53.8 g/l 2,3-BD and 7.1 g/l acetoin), corresponding to 0.31 g/g conversion rate. The presented results are the first to show successful starch conversion to 2,3-BD by K. pneumoniae in a one-step process.