Establishing an itaconic acid production process with Ustilago species on the low-cost substrate starch.

Ustilago maydis and Ustilago cynodontis are natural producers of a broad range of valuable molecules including itaconate, malate, glycolipids, and triacylglycerols. Both Ustilago species are insensitive toward medium impurities, and have previously been engineered for efficient itaconate production and stabilized yeast-like growth. Due to these features, these strains were already successfully used for the production of itaconate from different alternative feedstocks such as molasses, thick juice, and crude glycerol. Here, we analyzed the amylolytic capabilities of Ustilago species for metabolization of starch, a highly abundant and low-cost polymeric carbohydrate widely utilized as a substrate in several biotechnological processes. Ustilago cynodontis was found to utilize gelatinized potato starch for both growth and itaconate production, confirming the presence of extracellular amylolytic enzymes in Ustilago species. Starch was rapidly degraded by U. cynodontis, even though no α-amylase was detected. Further experiments indicate that starch hydrolysis is caused by the synergistic action of glucoamylase and α-glucosidase enzymes. The enzymes showed a maximum activity of around 0.5 U ml-1 at the fifth day after inoculation, and also released glucose from additional substrates, highlighting potential broader applications. In contrast to U. cynodontis, U. maydis showed no growth on starch accompanied with no detectable amylolytic activity.

Environmental factors drive microbial community succession in biofortified wheat Qu and its improvement on the quality of Chinese huangjiu.

Wheat Qu plays the role of saccharification fermentation, providing microorganisms and flavor in the fermentation of huangjiu, and the use of functional microorganisms to fortify wheat Qu is becoming increasingly popular. Yet, the mechanisms promoting microbial successions of wheat Qu remain unclear. In this study, we first correlated microbial community succession with physicochemical factors (moisture, temperature, acidity, glucoamylase and amylase) in inoculated raw wheat Qu (IRWQ) with Saccharopolyspora rosea. The Mantel test was performed to investigate the significance and found that temperature (r = 0.759, P = 0.001), moisture (r = 0.732, P = 0.006), and acidity (r = 0.712, P = 0.017) correlated significantly with the bacterial community in phase 1 (0-40 h). Meanwhile, temperature correlated significantly with the fungal community in phases 1 and 2 (40-120 h). To confirm the effect of temperature on microbial communities, the artificial reduction of bio-heat (37°C) in IRWQ also reduced the relative abundance of heat-resistant microorganisms including Bacillus and Saccharopolyspora. A higher abundance of Saccharopolyspora (87%) in IRWQ was observed following biofortified inoculation of S. rosea, in which glucoamylase activity increased by 40% compared to non-inoculated raw wheat Qu (NIRWQ) (1086 U/g vs 776 U/g). Finally, the IRWQ was employed to mechanized huangjiu fermentation and it was found to reduce the bitter amino acid and higher alcohol content by 27% and 8%, respectively, improving the drinking comfort and quality of huangjiu.

Multidimensional analysis of wheat original crucial endogenous enzymes driving microbial communities metabolism during high-temperature Daqu fermentation.

Knowledge of the metabolism of functional enzymes is the key to accelerate the transformation and utilization of raw materials during high temperature Daqu (HTD) manufacturing. However, the metabolic contribution of raw materials-wheat is always neglected. In this research, the relationship between the metabolism of wheat and microorganisms was investigated using physicochemical and sequencing analysis method. Results showed that the process of Daqu generation was divided into three stages based on temperature. In the early stage, a positive correlation was found between Monascus, Rhizopus and glucoamylase metabolism (r > 0.8, p < 0.05). Meanwhile, the glucoamylase metabolism in wheat occupied 63.8 % of the total matrix at the day 4. In the middle to later stages, the wheat metabolism of proteases, α-amylases and lipases in gradually reached their peak. Additionally, Lactobacillus and α-amylases presented a positive correlation (r > 0.7, p < 0.05), and the α-amylases metabolism in wheat occupied 22.18 % of the total matrix during the same time period. More importantly, the changes of enzyme activity metabolic pathway in wheat and microorganism were reflected by respiratory entropy (RQ). Overall, these results guide the choice of substrate during Daqu production.

Polarity-dependent expression and localization of secretory glucoamylase mRNA in filamentous fungal cells.

In multinuclear and multicellular filamentous fungi little is known about how mRNAs encoding secreted enzymes are transcribed and localized spatiotemporally. To better understand this process we analyzed mRNA encoding GlaA, a glucoamylase secreted in large amounts by the industrial filamentous fungus Aspergillus oryzae, by the MS2 system, in which mRNA can be visualized in living cells. We found that glaA mRNA was significantly transcribed and localized near the hyphal tip and septum, which are the sites of protein secretion, in polarity-dependent expression and localization manners. We also revealed that glaA mRNA exhibits long-range dynamics in the vicinity of the endoplasmic reticulum (ER) in a manner that is dependent on the microtubule motor proteins kinesin-1 and kinesin-3, but independent of early endosomes. Moreover, we elucidated that although glaA mRNA localized to stress granules (SGs) and processing bodies (PBs) under high temperature, glaA mRNA was not seen under ER stress, suggesting that there are different regulatory mechanisms of glaA mRNA by SG and PB under high temperature and ER stress. Collectively, this study uncovers a dynamic regulatory mechanism of mRNA encoding a secretory enzyme in filamentous fungi.

Evaluation of extruded starch foam for glucose-supplying biomaterials.

The survival rate of mesenchymal stem cells (MSC), a crucial factor in tissue engineering, is highly dependent on glucose supply. The purpose of this paper is to study the potential of starch foams as glucose suppliers. It is investigated through in vitro hydrolysis by amyloglucosidase in conditions that respect physiological constraints (37 °C and pH 7.4), including a duration of 21 days, and no stirring. Nine extruded starch foams with amylose contents ranging from 0 to 74 %, with various cell wall thicknesses (50 to 300 µm), and different crystallinities (0-30 %) were hydrolysed. These kinetics were fitted by a model which shows that the maximum rate of hydrolysis varies from 7 to 100 %, and which allows the rate of hydrolysis at 21 days to be calculated precisely. The results reveal the major role of amylose in glucose delivery kinetics, and the secondary roles of crystallinity and cell wall thickness of the foams. Additional hydrolysis of starch films revealed that thickness positively influences the amylose chain reorganisation during hydrolysis, which, in slows down and limits glucose delivery. A simple glucose delivery kinetics analysis procedure is proposed to select samples for testing as MSC glucose suppliers.

Mammalian maltase-glucoamylase and sucrase-isomaltase inhibitory effects of Artocarpus heterophyllus: An in vitro and in silico approach.

Alpha-glucosidase (maltase, sucrase, isomaltase and glucoamylase) activities which are involved in carbohydrate metabolism are present in human intestinal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). Hence, these proteins are important targets to identify drugs against postprandial hyperglycemia thereby for diabetes. To find natural-based drugs against MGAM and SI, Artocarpus heterophyllus leaf was explored for MGAM and SI inhibition in in vitro and in silico. A. heterophyllus leaf aqueous active fraction (AHL-AAF) was prepared using Soxhlet extraction followed by silica column chromatography. The phytoconstituents of AHL-AAF were determined using LC-ESI-MS/MS. AHL-AAF showed dose-dependent and mixed inhibition against maltase (IC50 = 460 µg/ml; Ki = 300 µg/ml), glucoamylase (IC50 = 780 µg/ml; Ki = 480 µg/ml), sucrase (IC50 = 900 µg/ml, Ki = 504 µg/ml) and isomaltase (IC50 = 860 µg/ml, Ki = 400 µg/ml). AHL-AAF phytoconstituents interaction with N-terminal (Nt) and C-terminal (Ct) subunits of human MGAM and SI was analyzed using induced-fit docking, molecular dynamics (MD), and binding free energy calculation. In docking studies, rhamnosyl hexosyl methyl quercetin (RHMQ), P-coumaryl-O-16-hydroxy palmitic acid (PCHP), and spirostanol interacted with active site amino acids of human MGAM and SI. Among these RHMQ stably interacted with all the subunits (Nt-MGAM, Ct-MGAM, Nt-SI and Ct-SI) whereas PCHP with Ct-MGAM and Nt-SI during MD analysis. In molecular docking, the docking score of RHMQ with NtMGAM, CtMGAM, NtSI and CtSI was -8.48, -12.88, -11.98 and -11.37 kcal/mol. The docking score of PCHP for CtMGAM and NtSI was -8.59 and -8.4 kcal/mol, respectively. After MD simulation, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values further confirmed the stable protein-ligand interaction. The RMSD value of all the complexes were around 2.5 Šand the corresponding RMSF values were also quite low. In MM/GBSA analysis, the involvement of Van der Waals and lipophilic energy in the protein/ligand interactions are understood. Further binding free energy for Nt-MGAM-PCHP, Nt-MGAM-RHMQ, Nt-SI-PCHP, Nt-SI-RHMQ, Ct-MGAM-PCHP, Ct-MGAM-RHMQ and Ct-SI-RHMQ complexes was found to be -24.94, -46.60, -46.56, -44.48, -40.3, -41.86 and -19.39 kcal/mol, respectively. Altogether, AHL-AAF showed inhibition of α-glucosidase activities of MGAM and SI. AHL-AAF could be further studied for its effect on diabetes in in vivo.

Physiochemical characterization and ameliorative effect of rice resistant starch modified by heat-stable α-amylase and glucoamylase on the gut microbial community in T2DM mice.

In the presented study, natural rice containing high resistant starch content was used as a raw material to produce rice resistant starch (RRS) through enzymatic hydrolysis with heat-stable α-amylase and glucoamylase. The chemical composition, structural characteristics and in vitro glycemic index (GI) of RRS were evaluated. The effects of RRS at different doses on the body weight, serum biochemical levels, pathological indexes, production of short-chain fatty acids (SCFAs) in the gut and the intestinal microbial composition in T2DM mice were investigated. The results of physiochemical characterization indicated that, relative to rice flour, RRS mainly comprising resistant starch had higher crystallinity (25.85%) and a more stable structure, which contributed to its lower digestibility and decreased GI in vitro. Compared with the model control group, 1 g per kg BW and 2 g per kg BW oral gavage dosages of RRS effectively enhanced the SCFA productivity in the T2DM mouse gut, as well as alleviating T2DM symptoms, involving an increase in body weight, reduction in fasting blood glucose, total cholesterol, triglyceride, low-density lipoprotein cholesterol, alanine transaminase and aspartate aminotransferase, and an increase in serum insulin and high-density lipoprotein cholesterol. Besides, 1 g per kg BW and 2 g per kg BW dosages of RRS mitigated T2DM-induced pancreas damage. Furthermore, up-regulation in the abundance of probiotics (Lactobacillus, Ruminococcus, etc.) and down-regulation in the number of harmful bacteria (Desulfovibrio, Prevotella, etc.) were observed in all RRS-treated groups. In summary, this work suggested that RRS prepared using heat-stable α-amylase and glucoamylase could be a potential functional component for amelioration of T2DM applied in the fields of food and pharmaceutics.

Genetic study of auxotrophic and resistant mutants of Aspergillus niger and their glucoamylase production

Mutantes auxotróficos resistentes foram isolados de uma linhagem de A. niger produtora de glucoamilase. Somente um mutante resitente, mgrA2 (verde malaquita) e os auxotróficos, metA1 (metionina) e myA1 (metionina ou cisteína) näo mostraram diminuiçäo significativa de produçäo comparados com a linhagem parental. A produçäo dos diplóides heterozigotos indicou que a baixa produçäo observada nos outros mutantes poderia ser devido a efeito pleiotrópico. Quatro grupos de ligaçäo foram identificados através de segregaçäo de diplóides heterozigotos. O gene mgrA2 se mostrou recessivo e o etbA5 (resistência a brometo de etídio), semi-diminante. O segundo poderia ser usado para determinaçäo da ordem dos genes no grupo de ligaçäo I

Proceso descontínuo de síntese de amiloglicosidade por Aspergilus awamori: influência do pH e da concentraçäo inicial de polissacarídeo

Estudou-se a influência da concentraçäo inicial de polissacarídeo (So) e do pH na produçäo de amiloglicosidase por Aspergillus awaamori NRRL 3112. Foram efetuados ensaios descontínuos, a pH 4,0 e 5,0 em fermentador de 10 litros, empregando-se meio de cultura a base de farinha de mandioca. Através do estudo cinético do processo, verificou-se a partir de So = 20g/l, para ambos os valores de pH, um comportamento acentuadamente crescente da atividade enzimática final no caldo, até se atingir um valor máximo para um determinado So. Verificou-se que esta atividade máxima é significativamente maior a pH 5,0, chegando-se a aproximadamente 7000U/1 para So = 93g/1, enquanto que a pH 4,0, obteve-se 2500U/1

Alterations in kidney enzyme pattern in acute hypervitaminosis A

The relation of excessive doses of vitamin A with various kidney pathologies is well known however, information concerning the relation of kidney enzyme activity with acute hypervitaminosis A is rather scarce. In this study we describe the kidney enzymatic alterations observed in rats that received daily intramuscular injectiosn of 10.000, 30.000, 50.000 and 100.000IU of vitamin A palmitate (VA) during seven days (TREATED GROUPS). A comparison is made with the enzyme activity in healthy rats pairfed and treated with sodium palmitte by intramuscular injection (CONTROL GROUP). The treated rats showed a proportional increase (<0.05) in activity of acid maltase, transaminases or aminotransferase (GOT and GPT), alkaline phosphatase (ALP) and acid protease with all doses of VA administered, Amylase, lipase and arginase tend to decrease (p<0.05) in activity only with doses of 50.000 and 100.000 I.U. of VA. Several factors are responsible for these findings, such as kidney necrosis due to release of lysosomal acid hydrolases produced by hypervitaminosis A.

Enzymatic determination of soluble and insoluble dietary fiber in rice and wheat bran

The information about dietary fiber presents controversies in many research areas such as in nomenclature, related illnesses, recommended quantities and terminology, mainly because of lack of analytical data. Different needs and interests for the dietary fiber composition of foods and forages have led to a proliferation of methods for its analysis. This research, a further adaptation of the enzymatic method of Asp et al. (1983) for its appplication is proposed for rice and wheat bran, byproducts of agroindustries in the southern region of Rio Grande do Sul (Brazil). The inclusion of Amyloglucosidase in the proposed methodology contributed to the decrease in the content of residual starch at the end of the experiment, like Prosky et al (1992). To increase the efficiency of the enzyme system inthis type of samples, other changes were made with respect to incubation time and proteolytic enzyme concentration. In the final adaptation, a decrease of 51.33 per cent of the starch content was observed in rice bran (RB) and of 52.93 per cent in wheat bran (WB). This decrease was also verified in the model system (MS) (52.08 per cent), which demonstrates the adequacy of the proposed adaptation. With respect to the residual protein, it was verified that the measures adopted provoked a reduction of 42.15 per cent (RB), 52.19 per cent (WB) and 42.11 per cent (MS) as compared to the original method. Then the proposed conditions has been shown to be efficient in decreasing the level of interference (indigestible starch and protein) in the quantification of dietary fiber in rice and wheat bran.

Influência da glicose na determinaçäo da atividade da amiloglicosidase / Influence of glucose on the determination of amyloglucosidase activity

Há inibiçäo da amiloglicosidase, quando se atinge 2g glicose/1 no volume de reaçäo. Este valor, além de sua importância no fenômeno de hidrólise, indica o cuidado a ser tomado na determinaçäo da atividade. Para permitir uma estimativa mais real, foi proposta a correçäo de valores, caso o limite de 2g glicose/1 seja ultrapassado

Produçäo e propriedades da amilase de Trichoderma pseudokoningüi / Production and proprieties of amylases of Trichoderma pseudokoningüi

Trichoderma pseudokoningii. Rifai foi isolado como contaminante em experimentos realizados sob nitrogênio líquido. Apresentou atividade amilolítica superior a de outros fungos isolados do solo ou do ambiente. Verificou-se que exuste relaçäo direta entre o crescimento do fundo e a produçäo de amilase. A maior produçäo de amilase ocorreu quando meio mineral mínimo foi suplementado com amido a 2%, farelo de trigo a 1% e líquido resultante do processamento do milho (corn steep liquor) a 1%. A amilase foi parcialmente purificada (4,9 vêzes) através de precipitaçäo com sulfato de amônio e cromatografia em Sephacryl S-200. Apresentou somente uma banda no gel corado para atividade, mas o gel para proteínas mostrou várias bandas, indicando que a preparaçäo näo está totalmente pura. A análise cromatográfica dos produtos de hidrólise, mostrou unicamente a presença de glucose, confirmando que a amilase em estudo é uma glicoamilase. O pH ótimo de atividade foi determinado ser entre 4,0 a 5,0 com máximo em 4,2. A enzima é estável na faixa de pH de 4,0 a 6,0. A temperatura ótima encontrada foi 60ªC, retendo 90% de atividade por um periodo de duas horas de pré incubaçäo a esta temperatura a esta temperatura. Ensaios de especificidade mostraram atividade superior sobre amido de milho que de batata ou amido solúvel, e que a preparaçäo atuava com menor velocidade, mas significativamente sobre amido cru. Mostrou açäo sobre o pululano, e näo sobre o ciclo dextrina ou maltose, indicando, neste caso a ausência de atividade de transglicosilase. O Km para o amido foi da ordem de 2,7 g/l