Stereochemical course and structure of the products of the enzymic action of endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis.
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ABSTRACT:
SUBMITTER: Malet C
PROVIDER: S-EPMC1137759 | biostudies-other | 1993 Dec
REPOSITORIES: biostudies-other
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Project description:?-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single ?-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-?-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley ?-1,3:1,4-glucan. To find the minor structure on which the endo-?-1,3(4)-glucanase acts, we prepared oligosaccharides from barley ?-1,3:1,4-glucan by endo-?-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-?-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as ?-Glc-1,3-?-Glc-1,4-?-Glc-1,3-?-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-?-1,3(4)-glucanase acts on the cellobiosyl units of barley ?-1,3:1,4-glucan in an endo-manner.
| S-EPMC4673573 | biostudies-literature
Project description:The food enzyme glucan 1,4-?-maltotetraohydrolase (EC 3.2.1.8) is produced with the genetically modified Bacillus licheniformis strain DP-Dzf24 by Danisco US Inc. The production strain contains multiple copies of a known antimicrobial resistance gene. However, based on the absence of viable cells and DNA in the food enzyme, this is not considered to be a risk. The food enzyme is intended to be used in baking processes and starch processing for the production of glucose syrups. The residual amounts of the Total Organic Solids (TOS) in glucose syrups are removed by filtration and purification during starch processing. Consequently, dietary exposure was not calculated for this use. Based on the maximum use levels recommended for the baking processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-TOS was estimated to be up to 0.271 mg TOS/kg body weight per day in European populations. Toxicological tests with the food enzyme indicated that there was no concern with respect to genotoxicity or systemic toxicity. A no-observed-adverse-effect level (NOAEL) was identified in rats, which, compared with the dietary exposure, results in a margin of exposure of at least 347. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens; no match was found. The Panel considers that, under the intended conditions of use, the risk for allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered low. Based on the microbial source, genetic modifications, the manufacturing process, the compositional and biochemical data, the dietary exposure assessment and the findings in the toxicological studies, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.
| S-EPMC7009165 | biostudies-literature
Project description:The food enzyme glucan 1,4-?-maltotetraohydrolase (4-?-d-glucan maltotetraohydrolase, EC 3.2.1.60) is produced with a genetically modified Bacillus licheniformis strain DP-Dzr46 by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. The glucan 1,4-?-maltotetraohydrolase food enzyme is intended to be used in baking processes. Based on the maximum use levels, dietary exposure to the food enzyme-Total Organic Solids (TOS) was estimated to be up to 0.405 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with another glucan 1,4-?-maltotetraohydrolase from B. licheniformis (strain DP-Dzf24). The Panel considered this food enzyme as a suitable substitute to be used in the toxicological studies, because it derives from the same recipient strain as strain DP-Dzr46, the location of the inserts is comparable, no partial inserts were present and the production methods are comparable. Genotoxicity tests did not raise 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 (NOAEL) at the highest dose of 94 mg TOS/kg bw per day that, compared with the estimated dietary exposure, results in a sufficiently high margin of exposure of at least 232. Similarity of the amino acid sequence to those of known allergens was searched and none was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be 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.
| S-EPMC7009068 | biostudies-literature
Project description:The food enzyme endo-1,4-?-xylanase (4-?-d-xylan xylanohydrolase; EC 3.2.1.8) is produced with a genetically modified Bacillus licheniformis (strain NZYM-CE) by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. This xylanase is intended to be used in baking and cereal-based processes. Based on the maximum use levels recommended for the respective food processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.012 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a genotoxic concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of at least 1,020 mg TOS/kg bw per day, the highest dose tested. When the NOAEL value is compared to the estimated dietary exposure, this results in a margin of exposure (MoE) of at least 85,000. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered to be low. Overall, the Panel concluded that based on the data provided and the derived MoE, this food enzyme does not give rise to safety concerns under the intended conditions of use.
| S-EPMC7009109 | biostudies-literature
Project description:BackgroundMannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis.ResultsThe mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose.ConclusionOur study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.
| S-EPMC2868798 | biostudies-literature
Project description:The food enzyme glucan 1,4-α-maltohydrolase (4-α-d-glucan α-maltohydrolase; 3.2.1.133) is produced with the genetically modified Bacillus licheniformis strain NZYM-CY by Novozymes A/S. The genetic modifications did not give rise to safety concerns. The production strain has been shown to qualify for Qualified Presumption of Safety (QPS) status. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in three food manufacturing processes, namely baking and brewing processes and starch processing for glucose syrup production and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, dietary exposure was calculated only for the baking and brewing processes. Dietary exposure was estimated to be up to 0.45 mg TOS/kg body weight (bw) per day in European populations. Given the QPS status of the production strain and the lack of hazards resulting from the food enzyme manufacturing process, toxicological studies were not considered necessary. Similarity of the amino acid sequence to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood of such reactions to occur is considered to be low. Based on the data provided, the QPS status of the production strain and the absence of issues arising from the production process, the Panel concluded that the food enzyme glucan 1,4-α-maltohydrolase produced with the genetically modified B. licheniformis strain NZYM-CY does not give rise to safety concerns under the intended conditions of use.
| S-EPMC9202445 | biostudies-literature
Project description:The food enzyme glucan 1,4-α-maltohydrolase (4-α-d-glucan α-maltohydrolase; 3.2.1.133) is produced with the genetically modified Bacillus licheniformis strain NZYM-SD by Novozymes A/S. The genetic modifications did not give rise to safety concerns. The production strain has been shown to qualify for Qualified Presumption of Safety (QPS) status. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in three food manufacturing processes, namely baking processes and brewing processes and starch processing for glucose syrup production and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, dietary exposure was calculated only for baking and brewing processes. Dietary exposure was estimated to be up to 0.57 mg TOS/kg body weight (bw) per day in European populations. Given the QPS status of the production strain and the lack of hazards resulting from the food enzyme manufacturing process, toxicological studies were not considered necessary. Similarity of the amino acid sequence to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood of such reactions to occur is considered to be low. Based on the data provided, the QPS status of the production strain and the absence of issues arising from the production process, the Panel concluded that the food enzyme glucan 1,4-α-maltohydrolase produced with the genetically modified B. licheniformis strain NZYM-SD does not give rise to safety concerns under the intended conditions of use.
| S-EPMC9200369 | biostudies-literature
Project description:The food enzyme glucan 1,4-α-maltohydrolase (4-α-d-glucan α-maltohydrolase; 3.2.1.133) is produced with the genetically modified Bacillus licheniformis strain NZYM-FR by Novozymes A/S. The genetic modifications did not give rise to safety concerns. The production strain has been shown to qualify for Qualified Presumption of Safety (QPS) status. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in three food manufacturing processes, namely baking and brewing processes and starch processing for glucose syrup production and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, dietary exposure was calculated only for the baking and brewing processes. Dietary exposure was estimated to be up to 0.30 mg TOS/kg body weight (bw) per day in European populations. Given the QPS status of the production strain and the lack of hazards resulting from the food enzyme manufacturing process, toxicological studies were not considered necessary. Similarity of the amino acid sequence to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood of such reactions to occur is considered to be low. Based on the data provided, the QPS status of the production strain and the absence of issues arising from the production process, the Panel concluded that the food enzyme glucan 1,4-α-maltohydrolase produced with the genetically modified B. licheniformis strain NZYM-FR does not give rise to safety concerns under the intended conditions of use.
| S-EPMC9199875 | biostudies-literature
Project description:In a previous paper, we reported the first stopped-flow experiments on a Bacillus licheniformis 1,3-1,4-beta-glucanase [Abel, Planas and Christensen (2001) Biochem. J. 357, 195-202]. It was shown that the pre-steady-state kinetics of the 1,3-1,4-beta-glucanase using the substrate 4-methylumbelliferyl 3-O-beta-cellobiosyl-beta-D-glucoside may be explained by a reaction scheme involving an induced fit and the binding of two substrates as well as a second enzymic conformational change, whereas the results definitely could not be explained in terms of the simple double-displacement scheme. In the present study, we report further stopped-flow kinetic results on the glucanase using a series of low-molecular-mass substrates with various leaving groups and varying chain length. The analysis of the resulting data leads to the conclusion that the free enzyme exists in two conformations, one of which binds the substrates rather strongly in a regulatory site, before any productive interactions can take place. This corresponds to an allosteric activation mechanism. With these substrates, however, the productive enzyme-substrate species are also able to change into less active or inactive forms. This may be seen as a feedback inhibitory mechanism.
| S-EPMC1223346 | biostudies-other
Project description:Bacillus licheniformis-fermented products (BLFP) are probiotics with antibacterial, antiviral, and anti-inflammatory properties that can improve growth performance. This study aimed to, firstly, compare the fecal microbiota of cats with chronic diarrhea (n = 8) with that of healthy cats (n = 4) from the same household using next-generation sequencing and, secondly, evaluate the effectiveness of oral administration of BLFP in relieving clinical signs and altering the intestinal microbiota in diarrheal cats. Six out of eight cats with diarrhea showed clinical improvement after BLFP administration for 7 days, and in two cats the stool condition was normal. A higher Firmicutes/Bacteroidetes ratio was noted in the feces of diarrheal cats without clinical improvement as compared with those in the healthy control group and in the diarrheal cats with clinical improvement after receiving BLFP. The phylum Bacteroidetes and class Bacteroidia decreased significantly in diarrheal cats regardless of BLFP administration. Blautia spp., Ruminococcus torques, and Ruminococcus gnavus, which belong to the Clostridium cluster XIVa and have been reported as beneficial to intestinal health, increased significantly in feces after BLFP treatment. Furthermore, a significant decrease in Clostridium perfringens was noted in diarrheal cats after BLFP administration. Overall, BLFP could be a potential probiotic to relieve gastrointestinal symptoms and improve fecal microbiota in cats with chronic diarrhea.
2022-08-24 | GSE203254 | GEO