Mining for novel cyclomaltodextrin glucanotransferases unravels the carbohydrate metabolism pathway via cyclodextrins in Thermoanaerobacterales

Carbohydrate metabolism via cyclodextrins (CM-CD) is an uncommon starch-converting pathway that thoroughly depends on extracellular cyclomaltodextrin glucanotransferases (CGTases) to transform the surrounding starch substrate to α-(1,4)-linked oligosaccharides and cyclodextrins (CDs). The CM-CD pathway has emerged as a convenient microbial adaptation to thrive under extreme temperatures, as CDs are functional amphipathic toroids with higher heat-resistant values than linear dextrins. Nevertheless, although the CM-CD pathway has been described in a few mesophilic bacteria and archaea, it remains obscure in extremely thermophilic prokaryotes (Topt ≥ 70 °C). Here, a new monophyletic group of CGTases with an exceptional three-domain ABC architecture was detected by (meta)genome mining of extremely thermophilic Thermoanaerobacterales living in a wide variety of hot starch-poor environments on Earth. Functional studies of a representative member, CldA, showed a maximum activity in a thermoacidophilic range (pH 4.0 and 80 °C) with remarkable product diversification that yielded a mixture of α:β:γ-CDs (34:62:4) from soluble starch, as well as G3–G7 linear dextrins and fermentable sugars as the primary products. Together, comparative genomics and predictive functional analysis, combined with data of the functionally characterized key proteins of the gene clusters encoding CGTases, revealed the CM-CD pathway in Thermoanaerobacterales and showed that it is involved in the synthesis, transportation, degradation, and metabolic assimilation of CDs.

Changes of starch and sucrose content and related gene expression during the growth and development of Lanzhou lily bulb

As the main forms of carbohydrates, starch and sucrose play a vital role in the balance and coordination of various carbohydrates. Lanzhou lily is the most popular edible lily in China, mainly distributed in the central region of Gansu. To clarify the relationship between carbohydrate metabolism and bulb development of Lanzhou lily, so as to provide a basis for the promotion of the growth and development in Lanzhou lily and its important economic value, we studied lily bulbs in the squaring stage, flowering stage, half withering stage and withering stage. The plant height, fresh weight of mother and daughter bulbs continued to increase during the whole growth period and fresh weight of stem and leaf began to decrease in the half withering stage. The content of starch, sucrose and total soluble sugar in the lily mother bulb accumulated mostly in the flowering, withering and half withering stages, respectively. Starch, sucrose and total soluble sugar accumulated in the daughter bulb with the highest concentration during the withering stage. In the transcription level, sucrose synthase (SuSy1) and sucrose invertase (INV2) expressed the highest in squaring stage, and the expression was significantly higher in the mother bulb than in the daughter bulb. In flowering stage, the expression levels of soluble starch synthase (SSS1), starch-branching enzyme (SBE) and adenosine diphosphate-glucose pyrophosphorylase (AGP1) genes were higher in the mother bulb than in the daughter bulb. Altogether, our results indicate that starch and sucrose are important for the bulb growth and development of Lanzhou lily.

Physiological Biochemistry-Combined Transcriptomic Analysis Reveals Mechanism of Bacillus cereus G2 Improved Salt-Stress Tolerance of Glycyrrhiza uralensis Fisch. Seedlings by Balancing Carbohydrate Metabolism

Salt stress severely threatens the growth and productivity of Glycyrrhiza uralensis. Previous results found that Bacillus cereus G2 enhanced several carbohydrate contents in G. uralensis under salt stress. Here, we analyzed the changes in parameters related to growth, photosynthesis, carbohydrate transformation, and the glycolysis Embden-Meyerhof-Parnas (EMP) pathway-tricarboxylic acid (TCA) cycle by G2 in G. uralensis under salt stress. Results showed that G2 helped G. uralensis-accumulating photosynthetic pigments during photosynthesis, which could further increase starch, sucrose, and fructose contents during carbohydrate transformation. Specifically, increased soluble starch synthase (SSS) activity caused to higher starch content, which could induce α-amylase (AM) and β-amylase (BM) activities; increased sucrose content due to the increase of sucrose synthase (SS) activity through upregulating the gene-encoding SS, which decreased cell osmotic potential, and consequently, induced invertase and gene-encoding α-glucosidase that decomposed sucrose to fructose, ultimately avoided further water loss; increased fructose content-required highly hexokinase (HK) activity to phosphorylate in G. uralensis, thereby providing sufficient substrate for EMP. However, G2 decreased phosphofructokinase (PFK) and pyruvate kinase (PK) activities during EMP. For inducing the TCA cycle to produce more energy, G2 increased PDH activity that enhanced CA content, which further increased isocitrate dehydrogenase (ICDH) activity and provided intermediate products for the G. uralensis TCA cycle under salt stress. In sum, G2 could improve photosynthetic efficiency and carbohydrate transformation to enhance carbohydrate products, thereby releasing more chemical energy stored in carbohydrates through the EMP pathway-TCA cycle, finally maintain normal life activities, and promote the growth of G. uralensis under salt stress.

Improved Neomycin Sulfate Potency in Streptomyces fradiae Using Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Fermentation Medium Optimization

To improve the screening efficiency of high-yield neomycin sulfate (NM) Streptomyces fradiae strains after mutagenesis, a high-throughput screening method using streptomycin resistance prescreening (8 μg/mL) and a 24-deep well plates/microplate reader (trypan blue spectrophotometry) rescreening strategy was developed. Using this approach, we identified a high-producing NM mutant strain, Sf6-2, via six rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and screening. The mutant displayed a NM potency of 7780 ± 110 U/mL and remarkably stable genetic properties over six generations. Furthermore, the key components (soluble starch, peptone, and (NH4)2SO4) affecting NM potency in fermentation medium were selected using Plackett-Burman and optimized by Box-Behnken designs. Finally, the NM potency of Sf6-2 was increased to 10,849 ± 141 U/mL at the optimal concentration of each factor (73.98 g/L, 9.23 g/L, and 5.99 g/L, respectively), and it exhibited about a 40% and 100% enhancement when compared with before optimization conditions and the wild-type strain, respectively. In this study, we provide a new S. fradiae NM production strategy and generate valuable insights for the breeding and screening of other microorganisms.

Total Starch Enzymatic Digestion v1

Enzymatic digestion of total soluble starch to glucose in plant tissue extracts for preparation for quantification via the GOD-POD Method (NZYtech).

Selection of Optimal Conditions for Cultivation of Bacillus megaterium UCM B-5710 – Producer of Keratinase

Every year the volume of production of poultry products all over the world is growing steadily. This contributes to a constant increase in the amount of by-products of poultry processing in the form of down and feather waste, which are dangerous for the environment due to the hard-to-degrade keratin protein and a large number of microbial pathogens. Therefore, the use of environmentally friendly methods for the destruction of keratin substrates due to keratinases of microorganisms is an urgent area of research. The aim of this work was to select the optimal cultivation conditions for the Bacillus megaterium strain UCM B-5710 to increase the activity of the keratinase synthesized by it. Methods. The culture was grown at 28°C, 201 rpm for 7 days on a basic nutrient medium containing defatted chicken feathers as the only source of carbon and nitrogen. The selection of optimal cultivation conditions was carried out according to the following parameters: temperature (21°C, 28°C, 42°C), stirring speed (201 rpm, 212 rpm), amount of inoculum (5%, 10%, 15% , 20%, 25%), the initial pH value of the nutrient medium (4.0–11.0), concentration of keratin-containing substrate (0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%), additional carbon source (glucose, galactose, lactose, maltose, sucrose, mannitol, potato and corn starch, soluble starch, soybean meal) and nitrogen (NH4Cl, NH4NO3, (NH4)2SO4, NaNO3, urea, peptone, tryptone, yeast extract and soybean meal) at a concentration of 1%. Keratinase activity was assessed by the UV absorption at 280 nm of the hydrolysis products of keratin-containing raw materials. Protein was determined by the Lowry method. Results. The dynamics of the enzyme synthesis showed that the culture of B. megaterium UCM B-5710 exhibited the highest keratinase activity on the 3rd day, and complete splitting of feathers was observed on the 4–5th days. The selection of the concentration of the keratin-containing substrate showed that 0.5% is the optimal concentration. The study of the influence of the initial pH value of the nutrient medium indicates that the culture grew well at pH 6.0–7.0 and pH 9.0–11.0, but at pH 8.0 its growth was very weak. The culture exhibited the maximum keratinase activity at pH 10.0. In addition, at this pH value, complete splitting of feathers was visually observed. The influence of such a key factor as temperature on the growth and synthesis of the enzyme by B. megaterium UCM B-5710 culture demonstrated complete splitting of feathers already on the 2nd day of cultivation at 42°C, at 21°C the culture split feathers very poorly. The introduction of the inoculum into the composition of the nutrient medium in an amount of 15% of the volume of the medium and the mixing intensity of 212 rpm turned out to be optimal. Besides, it was shown that the introduction of an additional source of carbon or nitrogen had an ambiguous effect on the level of keratinase activity of B. megaterium UCM B-5710. Complete inhibition of enzyme synthesis was observed when ammonium sulfate was added to the nutrient medium, and partial inhibition was observed in the case of glucose, lactose, and maltose. Potato, corn, and soluble starch stimulated keratinase synthesis. The majority of inorganic nitrogen sources (ammonium chloride and nitrate) did not affect the synthesis of B. megaterium UCM B-5710 keratinase, while organic sources (urea, peptone, tryptone, yeast extract) increased the level of keratinase activity by 20–50%. However, the most effective result was obtained using soybean meal, the addition of which to the nutrient medium increased the keratinase activity by 2.5 times. Conclusions. As a result of the studies, the optimal conditions for cultivation of the B. megaterium UCM B-5710 strain were selected: the optimum temperature for the growth and development of the culture is 42°C, the initial pH value is 10.0, the stirring speed is 212 rpm and the amount of inoculum introduced is 15%, an additional source of carbon and nitrogen in the form of soybean meal at a concentration of 0.5%. This made it possible to increase the activity of keratinase by 4 times.

High Amylase Production by a Novel Strain of Bacillus amyloliquefaciens M37 Isolated from Can Gio Mangrove Forest, Vietnam

Amylases are one of the most important industrial enzymes and find applications in many areas such as textiles, chemicals, food, and pharmaceuticals. Most of the amylases are derived from microbes. The objective of the present study was to evaluate amylase production by a bacterium isolated from the Can Gio mangrove forest. The bacterium was identified as a species of genus Bacillus based on morphological and biochemical characteristics. The analysis of 16S rRNA sequences was then confirmed that this strain belonged to Bacillus amyloliquefaciens species (100% similarity). The effect of culture conditions such as temperature, pH, and carbon sources on amylase production through shake-flask culture was investigated. Maximum amylase activity of 904 IU/mL was obtained after 24 h of cultivation in LB medium containing 1% soluble starch at 35oC and pH 7.0. The highest enzyme activity of 1279 IU/mL was achieved in the bioreactor after 30 h of cultivation at optimum conditions. In addition, B. amyloliquefaciens M37 can grow on soybean meal medium. The high bacterial cell number of 456 × 109 CFU/g and amylase activity of 1039 IU/g were obtained after 36 h of cultivation. This newly isolated B. amyloliquefaciens M37 could be a potential producer for industrial amylase production and probiotics with commercial implications.