Supplementation of amylase combined with glucoamylase or protease changes intestinal microbiota diversity and benefits for broilers fed a diet of newly harvested corn

Background: The effect of amylases combined with exogenous carbohydrase and protease in a newly harvested corn diet on starch digestibility, intestine health and cecal microbiota was investigated in broiler chickens.Methods: Two hunderd and eighty-eight 5-day-old female chickens were randomly divided into six treatments: a newly harvested corn-soybean meal diet(control); control supplemented with 1,500 U/g α-amylase(Enzyme A);Enzyme A + 300 U/g amylopectase + 20,000 U/g glucoamylase(Enzyme B); Enzyme B + protease 10,000 U/g(Enzyme C); Enzyme C + xylanase 15,000 U/g(Enzyme D); and Enzyme D + cellulase 200 U/g + pectinase 1,000 U/g(Enzyme E). Growth performance, starch digestibility, digestive organ morphology, and intestinal microbiota were evaluated in the birds at 16 and 23 d of age.Results: Compared with the control diet, supplementation with Enzyme A significantly decreased ileum lesion scoring at 16 d of age(P < 0.05); supplementation with Enzyme B or Enzyme C showed positive effects on ileal amylopectin and total starch digestibility(P < 0.05); Broilers fed with a diet supplemented with Enzyme D had a tendency to decrease body weight gain at 23 d. Enzyme E supplementation improved lesion scoring of jejunum and ileum at 16 d(P < 0.05), and increased ileal amylopectin or total starch digestibility at 23 d(P < 0.05).Supplementation of enzymes changed cecal microbiota diversity. High numbers of Campylobacter, Helicobacter and Butyricicoccus, Anaerostipes and Bifidobacterium, Sutterella and Odoribacter were the main genera detected in supplementations with Enzymes B, C, D, and E respectively.Conclusions: Supplementation with amylase combined with glucoamylase or protease showed a beneficial effect on starch digestibility and intestinal microbiota diversity, and increased growth of broilers fed with newly harvested corn.

Immobilization of Glucoamylase onto Novel Porous Supports Containing Cyclic Carbonate

Glucoamylase was immobilized onto novel porous polymer supports containing cyclic carbonate. The relationship between activity of immobilized glucoamylase and the properties of porous polymer supports was investigated. The operation stability and storage stability of immobilized glucoamylase were studied.

Hydrolysis of Raw Corn Starch Granules by Glucoamylase and Product Inhibition During the Hydrolysis

Raw corn starch granules were hydrolysized by glucoamylase in a chemostat. The hydro- lysis of three different-sized granules shows that smaller granules undergo more hydrolyzation than larger ones. After 78 h, 9700 of the granules was hydrolysized with diameter between 0.15 mm and 0.3 mm at 50 ℃. When corn starch concentration increased from 100 g/L to 250 g/L, the amount of reducing sugar produced was proportional to the initial substrate concentration and no substrate inhibition phenomenon appeared. In order to study the product inhibition exactly, the product from hydrolysis reaction itself was added into the hydrolysis system at the beginning of starch hydrolysis. Product inhibition with different quantities of product added were studied in the initial several hours, during which period enzyme inactivation could be neglected and product inhibition could be studied separately. The experiments indicate that product inhibition happens when the additional quantity exceeds 9.56 g/L.

Fusion Expression of Glucoamylase and Xylanase in Pichia pastoris

Employing RT-PCR amplification method, the mature pepfide coding sequence of glucoamylase （amyA） gene was amplified from total RNA of Talaro- myces emersonii and the xylanase （xyrut） gene from genomic DNA ofPaenibacillus sp. H10-3. The result showed that the mature peptide sequence of amyA is 1857 bp long and encodes 618 amino acids; and the mature peptide sequence of xynA is 636 bp long and encodes 211 amino acids. Then these two genes were spliced by overlap extension PCR （ SOE-PCR）, yielding fusion gene amyA-l-xynA. The fusion gene amyA-l-xynA was then cloned into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid pPIC9-amyA-l-xynA. The recombinant plasmid pPICg-amyA-l-xynA was linearized and transformed into Pichia pastoris GSll5 via electric shock, yielding engineering strain ALX2. The maximum yields of glucoamylase and xylanase in ALX2 fermentation supernatant were determined as 10.7 and 51.8 U/ml, respectively.

Kinetic Parameter of Alfa-amylase and Glucoamylase Enzymatic Reaction on the Glucose Yield from Hydrolyzed Processes of Tapioca Solid Waste

The increasing of tapioca production nowadays effected the production of waste. The waste of tapioca industries consists of two kinds, which were liquid waste and solid waste. Further more, tapioca solid waste treatment was ineffective. Weather solid waste produced from the extraction process still contains high concentration of starch that can be used to produce high quality product, for example, bio ethanol or other alternative energy sources. Objective of these experimental work was utilizing solid waste of tapioca industries and looking for the exactly composition of n-amylase and gluco-amylase enzymes on the hydrolysis processes of the solid waste of tapioca. The exact composition from both enzymes can be expected to increase the yield of glucose. Variables of cx-amylase enzyme for this research were 0.3% （w/w） and 0.5% （w/w） with liquefaction time were 1 hour and 1.5 hours, and variables of glucoamylase enzyme were 0.3% （w/w） and 0.5% （w/w）. To achieve these goals, the experimental work was held in laboratory scale with batch process. Firstly, tapioca solid waste was pretreated at 90 ~C and added u-amylase enzyme for 1 hour and 1.5 hours （variable of liquefaction time）. Then, substrate was cooled down to 60 ~C added with proposed variables of glucoamylase enzyme, and was analysed 24 hours after added. This experiment showed the best ratio between a-amylase and glucoamylase enzymes 0.5%：0.5% with 1 hour of liquefaction time. The highest glucose reaches 8.468% and yields 0.892 （g glucose/g starch） with starch conversion of 59.94%. KM = 0.0468 g/mL and rmax = 0.311 g/mL·h,

Sweet Potato Starch as a Carbon Source for Growth and Glucoamylase Production from <i>Aspergillus niger</i>

A mesophilic strain of Aspergillus niger isolated from cassava effluent samples produced extracellular glucoamylase in submerged culture containing 2% (w/v) soluble or sweet potato starch. On soluble starch medium the maximum glucoamylase activity in the culture filtrate was 9.40 U/mg compared to 8.24 U/mg on sweet potato starch culture filtrate. The mycelial dry weight for both media was 494 and 418 mg respectively. The maximum glucoamylase activity was obtained at a growth temperature of 40&deg;C and pH 4.5. The implication is that the bioprocess of utilizing sweet potato starch in the culture is attractive due to its relatively cheaper availability in Nigeria, making it even more favorable when economics is considered.

Study on the molecular basis of glucoamylase overproduction of a mutant strain Aspergillus niger T21

The molecular basis for increasing of the glucoamylase (GLA) production of an Aspergillus niger mutant T21 was investigated . Northern blot analysis showed that the amount of glaA specific mRNA of A . niger T21 was about 20 times higher than that of its start strain A . niger AS 3.795. The two glaA promoter fusions (PglaA)-uidAs were respectively introduced into A . niger. Analysis of GUS activity of the transformants revealed that the PglaA activity of the strain T21 is about 3 times stronger than that of the strain AS 3.795. It is considered to be one of the reasons for the increase of glaA transcriptional level in the strain T21. However, comparing with the 20 times increase in the amount of glaA mRNA the alteration of trans regulation should be the most important reason for that. The results of deletion analysis of 5′-cis region of A . niger T21 glaA gene indicated that the region from - 408 to - 513 bp upstream of ATG is responsible for the high level expression of glaA.

Preparation of glucosamine by hydrolysis of chitosan with commercial α-amylase and glucoamylase

Objective： In order to overcome the defects of chemical hydrolysis approach to prepare glucosamine, an enzymatic hydrolysis method was developed. Methods： Glucosamine was prepared by hydrolyzing chitosan, em- ploying e-amylase initially, and subsequently, glucoamylase. Results： The optimal hydrolyzing conditions were as follows： reaction time, 4 h; pH, 5.0; temperature, 50 ℃; and, α-amylase, 80 U/g for the initial reaction. Subsequently, glucoamylase was added in the presence of a-amylase. The optimal reaction conditions were found to be： reaction time, 8 h; pH, 4.5; temperature, 55 ℃; and, glucoamylase, 4000 U/g. The hydrolysates were subject to filtrating, concentrating to about 20% （w/w）, precipitating with five volumes of ethanol, and drying at 60 ℃ for 2 h. The content and the yield of glucosamine in the dried precipitate were 91.3% （w/w） and 86.2% （w/w）, respectively. Conclusions： The method developed in this study is a promising option in the preparation of glucosamine.

Purification and Characterization of Two Thermostable Glucoamylases Produced from Aspergillus niger B-30

Two thermostable glucoamylases were produced from Aspergillus niger B-30 by submerged fermentation. The two glueoamylases GAM-1 and GAM-2 were purified by ammonium sulfate precipitation, diethylaminoethyl- cellulose fast flow（DEAE FF） and Superdex G-75 gel filtration columns. The molecular weights of GAM-1 and GAM-2 were determined as 9.72x 104 and 7.83x104 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, while the molecular weights of GAM-1 and GAM-2 were determined to be 8.05x 104 and 7.04x 104 by matrix assisted laser desorption ionizationtime-of-flight（MALDI-TOF） mass spectrometry, respectively. Both the enzymes were glycosylated, with 10.4% and 11.4% carbohydrate content, respectively. The optimal pH and tempera- ture were 4.0--4.6 and 70 ℃ for both. The two glucoamylases were maintained 100% relative activity after incuba- tion at 60 ℃ for 120 min. After the hydrolysis of starch for 120 min, glucose was the only product, confirming that the two enzymes were of high efficiency towards starch. The GAM-2 exhibited higher catalytic activity towards oli- gosaccharides such as maltose than GAM-1, and the kinetic analysis shows that the affinity of GAM-2 to starch was lower than that of GAM-1. The high thermostability and effectiveness make the two glucoamylases potentially attrac- tive for biotechnological application.

Expression and secretion of barley α-amylase and A.niger glucoamylase in Saccharomyces cerevisiae

cDNAs of barley α amylase and A.niger glucoamylase were cloned in one E.coli yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed into S.cerevisiae GRF18 by protoplast transformation. The barley α amylase and A.niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18(pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used for the production of ethanol.

N-GIycosylation-induced Difference Between Multiple Forms of Glucoamylase

Glucoamylase from Monascus rubiginosus has been found to exist in multiple forms on PAGE pattern （E1-E5）. Both major molecular forms E3 and E4 are glycoproteins.Sugar content as mannose （%） is 7 for E3 and 9 for E4, respectively,

Expression and secretion of Aspergillus niger glucoamylase in Saccharomyces cerevisiae

Aspergillus niger glucoamylase GA 1 cDNA was inserted in between the yeast PGK promoter and terminator on plasmid pMA91. The resultant plasmid pMAG69 was introduced into Saccharomyces cerevisiae GRF18 by protoplast transformation. The A niger GA I cDNA was expressed efficiently under the contiol of PGK promoter and 99% of the gene products were secreted into the culture medium using its own signal sequence The recombmant yeast can digest 87% of starch in 2 d in the medium containing 10% starch. The recombinant plasmid pMAG69 can exist stably in 5. cerevisiae.

Cloning of the Aspergillus awamori glucoamylase gene and expression in Saccharomyces cerevisiae

The A. awamori glucoamylase I gene was obtained by RT_PCR and inserted into the plasmid pAC1. We constructed an expression vector pAC1_GA, which was used to transform the yeast S. cerevisiae AS 2 1364 without auxotrophic marker. The transformant secreted glucoamylase into the medium efficiently and degraded starch. The glucoamylase activity of the culture filtrate is up to 8 4 U/mL.

Multi-enzymes Production Studies in Single Tray Solid State Fermentation with Opened and Closed System

The robustness ofA. awamori and A. oryzae as enzyme producers is exploited in fungal fermentation on agricultural solid waste. High-level production of extracellular glucoamylase, protease, cellulase and xylanase has been achieved. Three different types of ＇waste＇ solids （wheat bran, soybean hulls and rapeseed meal） have been used in studies of solid state fermentation （SSF）. The enzymes could be produced in significant levels by continuously supplying oxygen （02） through the tray system known as ＂closed＂ and ＂opened＂ tray systems. A perforated tray system was developed in this study that permits direct access to 02. Testing the tray system with different perforated mesh aperture sizes in this study did not yield different results in growth performance of A. awamori and A. oryzae. A. awamori and A. oryzae can be very versatile in producing various enzymes with different substrates with different starch, protein, hemiceilulose and cellulose contents. These studies indicate that A. awamori is more suitable for the efficient production of multiple enzymes in the closed system including xylanase and cellulase, while the production of glucoamylase and protease is superior in the opened system. A. oryzae is more suitable for the efficient production of protease and cellulase in the closed system, while the production of protease is more favourable the opened system. A. awamori efficiently consumed starch in wheat bran medium and produced very high glucoamylase activity, and after that, the fungus efficiently produced other enzymes to degrade other complex nutrients such as protein, hemicellulose and cellulose. Meanwhile, A. oryzae efficiently consumed protein in rapeseed meal and produced very high protease activity. The ability of both filamentous fungi, to convert biomass through SSF bioconversion will have a great impact on food and agro-industry in every aspect of life from food and medicine to fuel.

A multi-enzymatic cascade reaction for the synthesis of bioactive C-oligosaccharides

C-Oligosaccharides are rare in nature and possess diverse bioactivities.However,their chemical synthesis faces many challenges.In this work,enzymatic introduction of C-linked sugar chains to target aglycones was successfully achieved by multi-enzymatic cascade reactions.A C-glycosyltransferase from Aloe barbadensis was employed to introduce the first C-linked glucose and then a cyclomaltodextrin glucanotransferase from Bacillus licheniformis was used to extend the sugar chain.A total of twenty C-oligosaccharides with 2-6 sugars were synthesized from scale-up reactions and exhibited good water solubility and sodium-dependent glucose transporter 2(SGLT2)inhibitory activity.Furthermore,a glucoamylase was used to control the length of the sugar chain and the C-maltosides were efficiently synthesized.These findings not only expanded the structural diversity of C-oligosaccharides,but also provided a strategy for the modification of C-glycoside drugs to improve the druggability.

The synergetic effects of two CCAAT boxes in Aspergillus niger glaA gene promoter on activation of PglaA transcription

EMSA and footprinting analyses have revealed that the -489—-414 bp and the -390—-345 bp (designated DC and PC respectively) upstream of the Aspergillus niger T21 glaA gene were bound by one protein factor in the A. niger T21 whole cell extract. Both DC and PC contained CCAAT pentanucleotides. The functions of DC and PC in regulation of expression of glucoamylase (GLA) were studied. CCAAT pentanucleotides were replaced with CGTAA and the mutated DNA fragments DCm and PCm lost the binding activities of protein factors in vitro. In vivo when either DC or PC was mutated or the relative orientations between them were changed on the PglaA, the transcriptional activity of PglaA decreased to a basal level. Introduction of multi-copies of DC into the original site at the PglaA in A. niger T21 decreased the expression of endogenous GLA expression and the exogenous reporter E. coli uidA gene introduced under the PglaA promoter, while having no effect on the uidA gene under the control of PgpdA. EMSA re-vealed that the levels of the specific DNA-binding protein factors in the transformants maintained the same meaning that introduction of multi-copies of DC caused the titration effect. AnghapC gene was cloned from A. niger T21 cDNA and introduced into the DC multi-copied strains. The expression of AnghapC improved the expression of the endogenous GLA and the exogenous gene controlled by PglaA. These results showed that both the CCAAT pentanucleotides were necessary for DC and PC binding to the protein factors, and the simultaneous binding of DC and PC to the protein was necessary for promoting the transcriptional activity of PglaA. AngHapC was the specific positive trans-acting protein factor binding to DC.