AZCL-Amylose

New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1→4)-β-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1→4)-β-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.

Background: Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. Results: In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca²⁺ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. Conclusions: This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.

Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.

Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.

The purpose of this work was the observation of the differences between the microbial communities living in the gut of the termite Reticulitermes flavipes fed on different diets. The termites were fed on poplar wood (original diet) and artificial diets consisting of crystalline cellulose (with and without lignin), α-cellulose (with and without lignin) and xylan. The termites were then dissected and the protist communities were analyzed through microscopy, leading to the conclusion that protist species are strongly influenced by diets. BIOLOG ECO Microplates^® were used to assess the metabolic properties of the different types of consortia, highlighting strong differences on the basis of principal component analysis and calculation of similarity rates. The microorganisms were cultivated in liquid media corresponding to the artificial diets before being characterized through a metagenetic analysis of gut microbiota (16S ribosomal DNA). This analysis identified several phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Fibrobacteres, Firmicutes, Nitrospirae, OP9, Planctomycetes, Proteobacteria, Spirochaetes, TM6, Tenericutes, Verrucomicrobia and WS3. The OTUs were also determined and confirmed the abundance of Proteobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia. It was possible to isolate several strains from the liquid media, and one bacterium and several fungi were found to produce interesting enzymatic activities. The bacterium Chryseobacterium sp. XAvLW produced α-amylase, β-glucosidase, endo-1,4-β-D-glucanase, endo-1,4-β-D-xylanase and filter paper-cellulase, while the fungi Sarocladium kiliense CTGxxyl and Trichoderma virens CTGxAviL generated the same activities added with endo-1,3-β-D-glucanase.

A cold-active α-amylase, Amy_13C6, identified by a functional metagenomics approach was expressed in Escherichia coli and purified to homogeneity. Sequence analysis showed that the Amy_13C6 amylase was similar to α-amylases from the class Clostridia and revealed classical characteristics of cold-adapted enzymes, as did comparison of the kinetic parameters K _m and K _cat to a mesophilic α-amylase. Amy_13C6 was shown to be heat-labile. Temperature optimum was at 10–15°C, and more than 70 % of the relative activity was retained at 1°C. The pH optimum of Amy_13C6 was at pH 8–9, and the enzyme displayed activity in two commercial detergents tested, suggesting that the Amy_13C6 α-amylase may be useful as a detergent enzyme in environmentally friendly, low-temperature laundry processes.

Earlier harvest dates have become necessary for Canadian Christmas tree producers to meet international demand, though by harvesting these trees early they may experience poor needle retention. The objective of this study is to understand the effect of harvest date on needle retention and link those changes to cold acclimation. In one experiment, balsam fir trees with varied needle abscission resistance (NAR) were collected in October and in January and monitored for needle retention. This was repeated for 3 years. In a second experiment, 45 branches were collected each month from September to January and monitored for needle retention, xylem pressure, membrane injury, capacitance, and accumulation of galactose, raffinose, and abscisic acid. High-NAR trees had little improvement in needle retention from October to January, whereas low-NAR trees had significantly improved needle retention from October to January. Between September and January, there was an 85% increase in raffinose, 147% increase in galactose, 80% increase in abscisic acid, and 62% decrease in stem capacitance. Early harvest was not detrimental for all trees, and it appears that cold acclimation is linked to postharvest needle abscission, though cold acclimation does not adequately explain differences between NAR classes.

Corn and wheat starches as well as wheat and cassava flours were hydrolyzed using sorghum malt at 65°C for 6 h. During these reactions, dextrose equivalent (DE) values were followed under three concentrations of sorghum malt and calcium chloride. Wheat flour presented the highest DE values and cassava flour had the highest hydrolysis yield. Thus, different dextrins were produced in a pilot plant and were analyzed by HPSEC and HPAEC-PAD for their molecular weight distribution and oligosaccharides composition, respectively. The results indicated that oligosaccharides with broad molecular weight distributions were present in the dextrins produced and that the proportion of maltose was very high.

Maltogenic α-amylase from Bacillus stearothermophilus (BStA) is widely used as bread crumb anti-firming enzyme. A maltotetraose-forming α-amylase from Pseudomonas saccharophila (PSA) was recently proposed as alternative, hence the need to compare both exo-acting enzymes with some endo-action component. A purely exo-acting thermostable β-amylase from Clostridium thermosulfurogenes (CTB) was included for reference purposes. Under the experimental conditions used, temperature optima of the enzymes are rather similar (60–65°C), but temperature stability decreased in the order BStA, PSA and CTB. The action of the enzymes on different substrates and their impact on the rheological behaviour of maize starch suspensions demonstrated that, while CTB acts exclusively through an exo-action mechanism, BStA displayed limited endo-action which became more pronounced at higher temperatures. PSA has more substantial endo-action than BStA, which is rather temperature independent. This is important for their impact in processes such as breadmaking, where temperature is gradually increased.

A semi-qualitative screening based on protease and amylase activity evaluation in a basal agar medium supplemented with insoluble chromogenic substrates based on AZCL (Azurine-Crosslinked with amylose or casein) using a plate assay was used for selecting the polar streptomycetes able to produce cold actives and alkaline amylases and proteases. This technique provides a specific and rapid simultaneous detection of high active hydrolase producing strains based on the visible solubilization of small particles of AZCL and the formation of haloes on plates. It has a great potential in increasing the efficacy of screening streptomycetes able to produce hydrolytic enzymes. This study revealed the potential of the selected streptomycetes isolated from polar soils to biosynthesize amylases and proteases cold-adapted at low temperatures (from 5 to 20°C) and alkaline pH values (8 to 9).

To gain insight into the distribution of arabinoxylans (AX), endoxylanases, and endoxylanase inhibitors in industrial wheat roller milling, all streams, that is, 54 flour fractions, 4 bran fractions, and the germ, were analyzed for ash, starch, and protein contents, α-amylase activity levels, total (TOT-AX) and water-extractable arabinoxylan (WE-AX) contents, endoxylanase activity levels, and endoxylanase inhibitor (TAXI and XIP) contents. In general, bran fractions were significantly richer in TOT-AX and WE-AX contents, endoxylanase activity levels, and endoxylanase inhibitor contents than germ and, even more so, than flour fractions. In the 54 different flour fractions, minimal and maximal values for TOT-AX and WE-AX contents differed by ca. 2-fold, whereas they differed by ca. 15-fold for endoxylanase activity levels. The latter were positively correlated with ash and negatively correlated with starch content, suggesting that the endoxylanase activity in flour is strongly influenced by the level of bran contamination. TAXI contents in the flour fractions varied ca. 4-fold and were strongly correlated with bran-related parameters such as ash content and enzyme activity levels, whereas XIP contents varied ca. 3-fold and were not correlated with any of the parameters measured in this study. The results can be valuable in blending and optimizing wheat flour fractions to obtain flours with specific technological and nutritional benefits.

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca⁺², Mn⁺², Mg⁺², Ba⁺², Li⁺², NaN₃, and isopropanol was observed. The K _m and V _max values determined for Xyl 1 with 4-O-methyl-D-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed α-1,6 glycosidic linkages of pullulan, producing maltotriose, and also α-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.

High performance anion-exchange chromatography with pulsed amperometric detection was applied for the rapid analysis of malto-oligosaccharides formed by extracellular enzyme preparations from 49 starch-degrading bacterial strains isolated from soil and compost samples. Malto-oligosaccharide-forming amylases, indicated by a predominant formation of maltohexaose from starch, were produced by enzyme preparations from four of the isolates growing at pH 7·0 and 10.

Among the most important rhizosphere bacteria are the pseudomonads, which are aggressive colonizers and utilize a wide range of substrates as carbon sources. The objective of this study was to determine if the taxonomic or metabolic diversity of pseudomonads differed among field-grown canola cultivars. Bacteria (n=2257) were isolated from the rhizosphere and root interior of six cultivars of field-grown canola, including three transgenic varieties. The bacteria were identified by fatty acid methyl ester (FAME) analysis, and about 35% were identified as Pseudomonas species. The most abundant species were Pseudomonas putida and Pseudomonas chlororaphis. Dendrograms based on FAME analysis revealed that many pseudomonad strains were found in all of the canola cultivars. Pseudomonads of the same strain were found in both the rhizosphere and the root interior of canola plants, suggesting that endophytic bacteria were a subset of the rhizosphere community. Because metabolic profiling provides more useful information than taxonomy, P. putida and P. chlororaphis isolates were characterized for their ability to utilize carbon substrates and produce several enzymes. Bacteria isolated from different plant cultivars had different carbon utilization profiles, but when only carbon substrates found in root exudates were analyzed, the cultivar effect was less pronounced. These characterizations also demonstrated that bacteria that were determined by FAME to be the same strain were metabolically different, suggesting functional redundancy among Pseudomonas isolates. The results of this study suggest that pseudomonads were functionally diverse. They differed in their metabolic potential among the canola cultivars from which they were isolated. Because bacteria capable of using many substrates can effectively adapt to new environments, these results have implications for the use of pseudomonads as biofertilizers, biological control agents and plant growth-promoting bacteria in canola.

The conversion of soluble starch to cyclomaltohexaose (α-CD), cyclomaltoheptaose (β-CD), cyclomaltooctaose (γ-CD) and cyclomaltononaose (δ-CD) by cyclodextrin glycosyltransferases (E.C. 2.4.1.19) from Bacillus spp. and bacterial isolates was studied. The results show that δ-CD was formed by all the enzymes investigated in the range of 5%–11.5% of the total amount of α-, β-, γ-, and δ-CD produced.

Bacillus licheniformis α-amylase, a thermostable starch-degrading enzyme, has been assessed as a candidate enzyme for the genetic transformation of malting barley. The temperature optimum, pH optimum and thermostability of B. licheniformis α-amylase were compared with those of barley α-amylase. The bacterial enzyme has a higher pH optimum (~9), a higher temperature optimum (~90°C) and much higher thermostability at elevated temperatures than the barley enzyme. The specific activity of the bacterial enzyme under conditions of pH and temperature relevant to the brewing process (pH 5.5, 65°C) is ~1.5-fold higher than that of the barley enzyme. Measurements of α-amylase activity during a micro-mash showed that the bacterial enzyme is at least as stable as the barley enzyme under these conditions, and that a level of expression for the bacterial enzyme corresponding to ~0.5% of total malt protein would approximately double the α-amylase activity in the mash. B. licheniformis α-amylase activity was rapidly eliminated by boiling following mashing as would occur during brewing. The combined results suggest that barley expressing the bacterial enzyme may be useful in the brewing process.