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 towards 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. U. 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.

Inhibition and kinetic studies of phytochemical constituents of Goniothalamus wynaadensis and their isoxazoline derivatives on α-glucosidase.

α-Glucosidase, an enzyme involved in post-prandial hyperglycaemia, was used as a target to study the effect of compound(s) isolated from Goniothalamus wynaadensis and its isoxazoline derivatives. Among thirteen compounds screened, compounds 1, 3a and 3j exhibited significant inhibition with IC50 values of 63.42, 61.36 and 58.89 µg/mL, respectively, outperforming acarbose (71.72 µg/mL). Kinetic studies revealed competitive binding for compound 1 and uncompetitive/non-competitive binding for 3a and 3j. Fluorescence quenching showed a linear relationship between I0/I at different inhibitor concentrations. The binding sites in α-glucosidase were ≤ 1. The binding constants 3a (0.7307) > 3j (0.6563) > 1 (0.5415) displayed strong interactions. Docking study revealed binding affinities; 3j (-8.9) > 3a (-7.7) > 1 (-7), and acarbose, 1, 3a and 3j had ARG-312, PHE-157 interactions in common to α-glucosidase. The toxicity profile showed compounds fell in classes IV and V. Overall, the results indicate that compounds 1, 3a and 3j are effective against α-glucosidase.

Structural insights into α-(1→6)-linkage preference of GH97 glucodextranase from Flavobacterium johnsoniae.

Glycoside hydrolase family 97 (GH97) comprises enzymes like anomer-inverting α-glucoside hydrolases (i.e., glucoamylase) and anomer-retaining α-galactosidases. In a soil bacterium, Flavobacterium johnsoniae, we previously identified a GH97 enzyme (FjGH97A) within the branched dextran utilization locus. It functions as an α-glucoside hydrolase, targeting α-(1→6)-glucosidic linkages in dextran and isomaltooligosaccharides (i.e., glucodextranase). FjGH97A exhibits a preference for α-(1→6)-glucoside linkages over α-(1→4)-linkages, while Bacteroides thetaiotaomicron glucoamylase SusB (with 69% sequence identity), which is involved in the starch utilization system, exhibits the highest specificity for α-(1→4)-glucosidic linkages. Here, we examined the crystal structures of FjGH97A in complexes with glucose, panose, or isomaltotriose, and analyzed the substrate preferences of its mutants to identify the amino acid residues that determine the substrate specificity for α-(1→4)- and α-(1→6)-glucosidic linkages. The overall structure of FjGH97A resembles other GH97 enzymes, with conserved catalytic residues similar to anomer-inverting GH97 enzymes. A comparison of active sites between FjGH97A and SusB revealed differences in amino acid residues at subsites +1 and +2 (specifically Ala195 and Ile378 in FjGH97A). Among the three mutants (A195S, I378F, and A195S-I378F), A195S and A195S-I378F exhibited increased activity toward α-(1→4)-glucoside bonds compared to α-(1→6)-glucoside bonds. This suggests that Ala195, located on the Gly184-Thr203 loop (named loop-N) conserved within the GH97 subgroup, including FjGH97A and SusB, holds significance in determining linkage specificity. The conservation of alanine in the active site of the GH97 enzymes, within the same gene cluster as the putative dextranase, indicates its crucial role in determining the specificity for α-(1→6)-glucoside linkage.

Understanding Antidiabetic Potential of Oligosaccharides from Red Alga Dulse Devaleraea inkyuleei Xylan by Investigating α-Amylase and α-Glucosidase Inhibition.

In this study, the α-glucosidase (maltase-glucoamylase: MGAM) and α-amylase inhibitory properties elicited by xylooligosaccharides (XOSs) prepared from dulse xylan were analysed as a potential mechanism to control postprandial hyperglycaemia for type-2 diabetes prevention and treatment. Xylan was purified from red alga dulse powder and used for enzymatic hydrolysis using Sucrase X to produce XOSs. Fractionation of XOSs produced xylobiose (X2), ß-(1→3)-xylosyl xylobiose (DX3), xylotriose (X3), ß-(1→3)-xylosyl-xylotriose (DX4), and a dulse XOS mixture with n ≥ 4 xylose units (DXM). The different fractions exhibited moderate MGAM (IC50 = 11.41-23.44 mg/mL) and α-amylase (IC50 = 18.07-53.04 mg/mL) inhibitory activity, which was lower than that of acarbose. Kinetics studies revealed that XOSs bound to the active site of carbohydrate digestive enzymes, limiting access to the substrate by competitive inhibition. A molecular docking analysis of XOSs with MGAM and α-amylase clearly showed moderate strength of interactions, both hydrogen bonds and non-bonded contacts, at the active site of the enzymes. Overall, XOSs from dulse could prevent postprandial hyperglycaemia as functional food by a usual and continuous consumption.

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.

Preparation of layer-assembled W/O/W-type microencapsulated beads and application in solid-state fermentation.

In this study, microcapsule beads-0-3-layers (M-0-3 indicates microencapsulated beads with 0, 1, 2, and 3 layers) were prepared, their properties were measured and characterized, and the effects of M-0-3 on solid-state fermentation were investigated. The results showed that in a liquid environment, the releasing glucoamylase activities of M-0-3 were 55.77%, 47.67%, 45.85%, and 42.87% in 360 h, respectively. In the solid environment, the reducing sugar production efficiency of M-0-3 was 29.84%, 27.72%, 19.16%, and 15.93% in 15 days, respectively. Adding M-0-3 improved the alcohol and reduced sugar content while decreasing the residual starch content of the Jiupei, indicating that adding M-0-3 was beneficial to the solid-state fermentation of Baijiu. Solid-state fermentation simulation experiments illustrated that microcapsule beads play a positive role in the production of Baijiu, enhancing raw material utilization and yield of Baijiu production.

Biochemical Characterization of a Novel Thermostable 1,4-α-Glucoamylase from Aspergillus brasiliensis Strain Isolated in the Brazilian Atlantic Forest.

Glucoamylases are exo-enzymes that cleave the ends of the starch chain, releasing glucose units. In the current work, we described a novel 1,4-α-glucoamylase from an A. brasiliensis strain isolated from an environmental sample. The purified glucoamylase, GlaAb, has a molecular mass of 69 kDa and showed a starch binding domain. GlaAb showed a similar sequence to other fungal glucoamylases, and the molecular 3D model analysis of GlaAb suggests an overall structure as described in the literature, except by elongation in the loop connecting the 4th and 5th α-helices. The enzyme showed activity over a wide range of pH and temperature, with maximum activity at pH 4.5 and 60 °C. GlaAb was stable at 50 °C for 7 h, maintaining 67% residual activity, and it was not inhibited by glucose up to 0.1 M. The glucoamylase was 65% more active in the presence of Mn2+ and showed a Km of 2.21 mg mL-1, Vmax of 155 U mg-1, Kcat 179 s-1, and Kcat/Km 81.06 mg mL-1 s-1 using potato starch as substrate. The results obtained are promising and provide the basis for the development of applications of GlaAb in the industrial process.

Enzyme-Mediated Exponential Glucose Release: A Model-Based Strategy for Continuous Defined Fed-Batch in Small-Scale Cultivations.

Miniaturized cultivation systems offer the potential to enhance experimental throughput in bioprocess development. However, they usually lack the miniaturized pumps necessary for fed-batch mode, which is commonly employed in industrial bioprocesses. An alternative are enzyme-mediated glucose release systems from starch-derived polymers, facilitating continuous glucose supply. Nevertheless, while the glucose release, and thus the feed rate, is controlled by the enzyme concentration, it also strongly depends on the type of starch derivative, and the culture conditions as well as pH and temperature. So far it was not possible to implement controlled feeding strategies (e.g., exponential feeding). In this context, we propose a model-based approach to achieve precise control over enzyme-mediated glucose release in cultivations. To this aim, an existing mathematical model was integrated into a computational framework to calculate setpoints for enzyme additions. We demonstrate the ability of the tool to maintain different pre-defined exponential growth rates during Escherichia coli cultivations in parallel mini-bioreactors integrated into a robotic facility. Although in this case study, the intermittent additions of enzyme and dextrin were performed by a liquid handler, the approach is adaptable to manual applications. Thus, we present a straightforward and robust approach for implementing defined continuous fed-batch processes in small-scale systems, where continuous feeding was only possible with low accuracy or high technical efforts until now.

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.

Mixed food waste valorization using a thermostable glucoamylase enzyme produced by a newly isolated filamentous fungus: A sustainable biorefinery approach.

Food waste is a lucrative source of complex nutrients, which can be transformed into a multitude of bioproducts by the aid of microbial cell factories. The current study emphasizes isolating Glucoamylase enzyme (GA) producing strains that can effectively break down mixed food waste (MW), which serves as a substrate for biomanufacturing. The screening procedure relied heavily on the growth of isolated fungi on starch agar media, to specifically identify the microbes with the highest starch hydrolysis potential. A strain displayed the highest GA activity of 2.9 ± 0.14 U/ml which was selected and identified as Aspergillus fumigatus via molecular methods of identification. Exposure of the A. fumigatus with 200 mM Ethyl methanesulphonate (EMS) led to a 23.79% increase compared to the wild-type GA. The growth conditions like cultivation temperature or the number of spores in the inoculum were investigated. Further, maximum GA activity was exhibited at pH 5, 55 °C, and at 5 mM Ca2+ concentration. The GA showed thermostability, retaining activity even after long periods of exposure to temperatures as high as 95 °C. The improvement of hydrolysis of MW was achieved by Taguchi design where a maximum yield of 0.57 g g-1 glucose was obtained in the hydrolysate. This study puts forth the possibility that mixed food waste, despite containing spices and other microbial growth-inhibitory substances, can be efficiently hydrolyzed to release glucose units, by robust fungal cell factories. The glucose released can then be utilized as a carbon source for the production of value-added products.

Biochemical characterization of an acid-thermostable glucoamylase from Aspergillus japonicus with potential application in the paper bio-deinking.

Aspergillus species have been highlighted in enzyme production looking for industrial applications, notably, amylases are one of the most interesting enzymes. They are capable of hydrolyzing α-glycosidic linkages of starch and widely used in industrial processes to produce ethanol, glucose, and fructose syrup as well as in the textiles, detergents, and paper industries applications. In this context, this work aimed at the biochemical characterization of the glucoamylase from Aspergillus japonicus and its application in the bio-bleaching process of recycled paper. The optimum temperature and pH for the glucoamylase assay were standardized as 50°C and 5.5. After 1 h of incubation, glucoamylase retained 90% of its activity at 30-50°C. It also kept 70% of its activity in the pH range of 4.0-6.5 after an hour of incubation. The enzyme led to an increase of 30% in the relative whiteness of 10 dry grams of sulfite paper and magazine paper when applied along with commercial cellulase and 10 mM MnCl2 . In addition, after the treatments, the glucoamylase recovered activity was 30%-32%, which indicates a prolonged availability of the enzyme and can considerably curtail the redundant downstream process of the recycled paper bio-bleaching. Thus, the glucoamylase from A. japonicus has a significant role in bio-bleaching recycled paper, reducing the necessity of hard chemicals, and improving the industrial process in an interesting economic and ecological mode.

Preparation of glucoamylase microcapsule beads and application in solid-state fermentation.

BACKGROUND: Baijiu brewing adopts the solid-state fermentation method, using starchy raw materials, Jiuqu as saccharifying fermenting agent, and distilled spirits made by digestion, saccharification, fermentation and distillation. In the late stages of solid-state fermentation of Baijiu, the reduced activity of glucoamylase leads to higher residual starch content in the Jiupei, which affects the liquor yield. The direct addition of exogenous glucoamylase leads to problems such as the temperature of the fermentation environment rising too quickly, seriously affecting the growth of microorganisms. RESULTS: To solve the problem of reduced activity of glucoamylase in the late stage of solid-state fermentation of Baijiu, microcapsule beads (M-B) based on microcapsule emulsion were prepared and the effect of M-B on solid-state fermentation of Baijiu was investigated. The results showed that the release of M-B before and after drying was 53.27% and 25.77% in the liquid state (120 h) and 29.84% and 22.62% in the solid state (15 days), respectively. Adding M-B improved the alcohol by 0.33 %vol and reducing sugar content by 0.51%, reduced the residual starch content by 1.21% of the Jiupei, and had an insignificant effect on the moisture and acidity of the Jiupei. CONCLUSION: M-B have excellent sustained-release properties. The addition of M-B in solid-state fermentation significantly increased the alcohol content, reduced the residual starch content of Jiupei, ultimately improving the starch utilization rate and liquor yield of Baijiu brewing. The preparation of M-B provides methods and approaches for applying other active substances and microorganisms in the brewing of Baijiu. © 2023 Society of Chemical Industry.

A Coculture of Photoautotrophs and Hydrolytic Heterotrophs Enables Efficient Upcycling of Starch from Wastewater toward Biomass-Derived Products: Synergistic Interactions Impacting Metabolism of the Consortium.

Even with particular interest in sustainable development, due to the limited types of bioavailable carbon sources that could support heterotrophic/mixotrophic growth, microalgae-derived products still suffer from inconsistent yield and high costs. This study demonstrates a successful cocultivation of the photoautotroph Chlorella vulgaris with a hydrolytic-enzyme-abundant heterotroph, Saccharomycopsis fibuligera, enabling efficient starch upcycling from water/wastewater toward enhancing microalgae-dominant biomass and lipid production. The enzymatic activities of S. fibuligera contributed to the hydrolysis of starch into glucose, generating a 7-fold higher biomass through mixotrophic/heterotrophic growth of C. vulgaris. Further, scanning transmission electron microscopy (STEM) and quantitative analysis suggested a significantly induced accumulation of lipids in C. vulgaris. Results of meta-transcriptomics revealed the critical regulatory role of illumination in interaction shifting. Gene expression for glycolysis and lipid biosynthesis of C. vulgaris were highly activated during dark periods. Meanwhile, during illumination periods, genes coding for glucoamylase and the sulfur-related activities in S. fibuligera were significantly upregulated, leading to induced starch hydrolysis and potential increased competition for sulfur utilization, respectively. This study indicates that hydrolytic organisms could collaborate to make starch bioavailable for nonhydrolytic microalgae, thus broadening the substrate spectrum and making starch a novel biotechnological feedstock for microalgae-derived products, e.g., biofuels or single-cell protein.

Additional glucoamylase genes increase ethanol productivity on rice and potato waste streams by a recombinant amylolytic yeast.

The implementation of consolidated bioprocessing for converting starch to ethanol relies on a robust yeast that produces enough amylases for rapid starch hydrolysis. Furthermore, using low-cost substrates will assist with competitive ethanol prices and support a bioeconomy, especially in developing countries. This paper addresses both challenges with the expression of additional glucoamylase gene copies in an efficient amylolytic strain (Saccharomyces cerevisiae ER T12) derived from the industrial yeast, Ethanol Red™. Recombinant ER T12 was used as a host to increase ethanol productivity during raw starch fermentation; the ER T12.7 variant, selected from various transformants, displayed enhanced raw starch conversion and a 36% higher ethanol concentration than the parental strain after 120 h. Unripe rice, rice bran, potato waste and potato peels were evaluated as alternative starchy substrates to test ER T12.7's fermenting ability. ER T12.7 produced high ethanol yields at significantly improved ethanol productivity, key criteria for its industrial application.

Postruminal Casein Infusion and Exogenous Glucagon-Like Peptide 2 Administration Differentially Stimulate Pancreatic α-Amylase and Small Intestinal α-Glucosidase Activity in Cattle.

BACKGROUND: Increasing luminal carbohydrate flow decreases pancreatic α-amylase activity but can increase jejunal maltase activity, suggesting that regulation of carbohydrase activity is perhaps uncoordinated in response to luminal carbohydrate flow. Increasing luminal casein flow increases pancreatic α-amylase activity in cattle, and exogenous glucagon-like peptide 2 (GLP-2) has been shown to increase small intestinal α-glucosidase activity in nonruminants. OBJECTIVES: The objective was to evaluate the effects of postruminal casein infusion, exogenous GLP-2, or their combination on endogenous pancreatic and small intestinal carbohydrase activity in cattle postruminally infused with starch. METHODS: Holstein steers [n = 24; 250 ± 23 kg body weight (BW)] received a continuous abomasal infusion of 3.94 g raw corn starch/kg of BW combined with either 0 or 1.30 g casein/kg of BW. Steers received subcutaneous injections in 2 equal portions daily of excipient (0.5% bovine serum albumin) or 100 µg GLP-2/kg of BW per day. At the end of the 7-d treatment period, steers were slaughtered for tissue collection. Data were analyzed using the MIXED procedure of SAS version 9.4 (SAS Institute Inc.). RESULTS: Postruminal casein infusion increased (P ≤ 0.03) pancreatic mass by 12.6%, total pancreatic α-amylase activity by 50%, and postruminal starch disappearance from 96.7% to 99.3%. Exogenous GLP-2 increased (P < 0.01) total small intestinal and mucosal mass by 1.2 kg and 896 g, respectively. Relative to control, GLP-2 and casein + GLP-2 increased (P = 0.04) total small intestinal α-glucosidase activity by 83.5%. Total small intestinal maltase, isomaltase, and glucoamylase activity was 90%, 100%, and 66.7% greater for GLP-2 and casein + GLP-2 steers compared with control. CONCLUSIONS: Casein increased pancreatic α-amylase activity, GLP-2 increased small intestinal α-glucosidase activity, and the combination of casein and GLP-2 increased both pancreatic α-amylase activity and small intestinal α-glucosidase activity. This novel approach provides an in vivo model to evaluate effects of increasing endogenous carbohydrase activity on small intestinal starch digestion.

Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants.

BACKGROUND: Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as Aspergillus and Rhizopus species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus Myceliophthora thermophila is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the M. thermophila platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering. RESULTS: In this study, to increase the genome targeting range, we upgraded the CRISPR-Cas12a-mediated technique by engineering two AsCas12a variants carrying the mutations S542R/K607R and S542R/K548V/N552R. Using the engineered AsCas12a variants, we deleted identified key factors involved in the glucoamylase expression and secretion in M. thermophila, including Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2. Deletion of four targets led to more than 1.87- and 1.85-fold higher levels of secretion and glucoamylases activity compared to wild-type strain MtWT. Transcript level of the major amylolytic genes showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2 and overexpressing Mtamy1 and Mtpga3. Total secreted protein and activities of amylolytic enzymes in the MtGM12 were about 35.6-fold and 51.9‒55.5-fold higher than in MtWT. Transcriptional profiling analyses revealed that the amylolytic gene expression levels were significantly up-regulated in the MtGM12 than in MtWT. More interestingly, the MtGM12 showed predominantly short and highly bulging hyphae with proliferation of rough ER and abundant mitochondria, secretion vesicles and vacuoles when culturing on starch. CONCLUSIONS: Our results showed that these AsCas12a variants worked well for gene deletions in M. thermophila. We successfully constructed the glucoamylase hyper-production strain of M. thermophila by the rational redesigning and engineering the transcriptional regulatory and secretion pathway. This targeted engineering strategy will be very helpful to improve industrial fungal strains and promote the morphology engineering for enhanced enzyme production.

Aspergillus clavatus UEM 04: An efficient producer of glucoamylase and α-amylase able to hydrolyze gelatinized and raw starch.

The best amylolytic activity production by Aspergillus clavatus UEM 04 occurred in submersed culture, with starch, for 72 h, at 25 °C, and 100 rpm. Exclusion chromatography partially purified two enzymes, which ran as unique bands in SDS-PAGE with approximately 84 kDa. LC-MS/MS identified a glucoamylase (GH15) and an α-amylase (GH13_1) as the predominant proteins and other co-purified proteins. Zn2+, Cu2+, and Mn2+ activated the glucoamylase, and SDS, Zn2+, Fe3+, and Cu2+ inhibited the α-amylase. The α-amylase optimum pH was 6.5. The optimal temperatures for the glucoamylase and α-amylase were 50 °C and 40 °C, and the Tm was 53.1 °C and 56.3 °C, respectively. Both enzymes remained almost fully active for 28-32 h at 40 °C, but the α-amylase thermal stability was calcium-dependent. Furthermore, the glucoamylase and α-amylase KM for starch were 2.95 and 1.0 mg/mL, respectively. Still, the Vmax was 0.28 µmol/min of released glucose for glucoamylase and 0.1 mg/min of consumed starch for α-amylase. Moreover, the glucoamylase showed greater affinity for amylopectin and α-amylase for maltodextrin. Additionally, both enzymes efficiently degraded raw starch. At last, glucose was the main product of glucoamylase, and α-amylase produced mainly maltose from gelatinized soluble starch hydrolysis.

A CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger.

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 µg·mL-1 PatE protein in the culture medium with a purity just under 90%.

Immobilized glucoamylase based on ZIF-8: Preparation, response surface optimization, characterization.

The glucoamylase@ZIF-8 was prepared using ZIF-8 material as the carrier in this study. The preparation process was optimized by response surface methodology, and the stability of glucoamylase@ZIF-8 was determined. The material was characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The results showed that the optimum preparation process of glucoamylase@ZIF-8 was 1.65 mol 2-methylimidazole, 5.85 mL glucoamylase, 33°C stirring temperature, 90 min stirring time, and 84.0230% ± 0.6006% embedding rate. At 100°C, the free glucoamylase completely lost its activity, whereas the glucoamylase@ZIF-8 still had a retained enzyme activity of 12.0123% ± 0.86158%; at pH 3-6, the highest activity of glucoamylase@ZIF-8 was 95.9531% ± 0.96181%, and about 80% of glucoamylase activity could be retained under alkaline conditions. When the ethanol concentration was 13%, the retained enzyme activity was 7.9316% ± 0.19805%, significantly higher than free enzymes. The Km of glucoamylase@ZIF-8 and free enzyme were 1235.6825 and 80.317 mg/mL, respectively. Vmax was 0.2453 and 0.149 mg/(mL min), respectively. The appearance, crystal strength, and thermal stability of glucoamylase@ZIF-8 were improved after optimization, and they had high reusability.

Safety evaluation of the food enzyme glucan 1,4-α-glucosidase from the non-genetically modified Aspergillus niger strain NZYM-BO.

The food enzyme glucan 1,4-α-glucosidase (4-α-d-glucan α-glucohydrolase; EC 3.2.1.3) is produced with the non-genetically modified Aspergillus niger strain NZYM-BO by Novozymes A/S. It was considered free from viable cells of the production organism. It is intended to be used in seven food manufacturing processes: baking processes, brewing processes, cereal-based processes, distilled alcohol production, fruit and vegetable processing for juice production, production of dairy analogues and starch processing for the production of glucose syrups and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by distillation and during starch processing, dietary exposure was not calculated for these two food manufacturing processes. For the remaining five food manufacturing processes, dietary exposure to the food enzyme-TOS was estimated to be up to 2.97 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,920 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 646. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure to this food enzyme cannot be excluded (except for distilled alcohol production), but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.