Artificial neural network-based modelling of optimized experimental study of xylanase production by Penicillium citrinum xym2

The industrial production of enzymes is generally optimized by one-factor-at-a-time (OFAT) approach. However, enzyme production by the method involves submerged or solid-state fermentation, which is laborious and time-consuming and it does not consider interactions among process variables. Artificial neural network (ANN) offers enormous potential for modelling biochemical processes and it allows rational prediction of process variables of enzyme production. In the present work, ANN has been used to predict the experimental values of xylanase production optimized by OFAT. This makes the reported ANN model to predict further optimal values for different input conditions. Both single hidden layered (6-3-1) and double hidden layered (6-12-12-1) were able to closely predict the actual values with MSE equals to 0.004566 and 0.002156, respectively. The study also uses multiple linear regression (MLR) analysis to calculate and compare the outcome with ANN predicted xylanase activity, and to establish a parametric sensitivity.

The Effects of Non-Fiber Carbohydrate Content and Forage Type on Rumen Microbiome of Dairy Cows

The main objective of our current study was evaluating the effects of NFC supplementation and forage type on rumen microbiota and metabolism, by comparing microbial structures and composition among samples collected from cows fed AH (alfalfa-based diet), H-NFC (CS-based diet with high NFC) and L-NFC (CS-based diet with low NFC) diets. Our results show that microbial communities were structurally different but functionally similar among groups. When compared with L-HFC, NFC increased the population of Treponema, Ruminobacter, Selenomonas and Succinimonas that were negatively correlated with ruminal NH3-N, and urea nitrogen in blood, milk and urine, as well as significantly increasing the number of genes involved in amino acid biosynthesis. However, when compared to the AH group, H-NFC showed a higher abundance of bacteria relating to starch degradation and lactate production, but a lower abundance of bacteria utilizing pectin and other soluble fibers. This may lead to a slower proliferation of lignocellulose bacteria, such as Ruminococcus, Marvinbryantia and Syntrophococcus. Lower fibrolytic capacity in the rumen may reduce rumen rotation rate and may limit dry matter intake and milk yield in cows fed H-NFC. The enzyme activity assays further confirmed that cellulase and xylanase activity in AH were significantly higher than H-NFC. In addition, the lower cobalt content in Gramineae plants compared to legumes, might have led to the significantly down-regulated microbial genes involved in vitamin B12 biosynthesis in H-NFC compared to AH. A lower dietary supply with vitamin B12 may restrict the synthesis of milk lactose, one of the key factors influencing milk yield. In conclusion, supplementation of a CS-based diet with additional NFC was beneficial for nitrogen conversion by increasing the activity of amino acid biosynthesis in rumen microbiota in dairy cattle. However, lower levels of fibrolytic capacity may limit dry matter intake of cows fed H-NFC and may prevent increased milk yield.

Coordinately express hemicellulolytic enzymes in Kluyveromyces marxianus to improve the saccharification and ethanol production from corncobs

Background Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K.marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs. Results Two polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K.marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular β-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and β-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of β-xylanase and β-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, β-mannanase, β-xylanase, and β-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. Conclusions The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

Xylanase Production By Thermobacillus Xylanilyticus is Impaired By Population Diversification But Can Be Mitigated Based On the Management of Cheating Behavior

Background: The microbial production of hemicellulasic cocktails is still a challenge for the sector of biorefineries and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, and notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneities impairs the production capability of the whole microbial population.Results: Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~75%) after 23.5 generations. Accordingly, the expression of the Tx-xyn11 gene decreased drastically and followed the same trend as the xylanase activity. Flow cytometry analyses pointed out that two subpopulations, differing at the level of their light scattering properties, were potentially involved in this progressive loss of enzymatic activities. Interestingly, upon successive cultivations on xylan, the subpopulation exhibiting low forward scatter (FSC) signal. Additionally, the evolution of the ratio between the two subpopulations was correlated to the decrease in xylanase activity. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. Serial cultivations on glucose, followed by the addition of a xylan pulse led to a ~1.5-fold to ~15-fold improvement of xylanase, depending on the moment for pulse addition, , suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. Conclusions: Taken altogether, the data from this study point out that a cheating behaviour is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation condition between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium.

Effects of residual phenolic compounds on xylanase-assisted ClO2 bleaching of hardwood kraft pulp

In both pulping and bleaching processes, lignin in the pulp fiber is degraded into smaller molecules that need to be rinsed away. However, despite the installation of automatic washing equipment, the small phenolic compounds among other lignin degradation products can hardly be completely removed from the brownstock. Among the myriad of small phenolic compounds degrading from lignin, some are water-soluble and highly reactive with bleaching reagents. To understand the impact of residual phenolic compounds from black liquor on pulp bleaching, six monomeric phenolic model compounds were tested in this study. Catechol and vanillin showed inhibitory effects on xylanase activity, while catechol, vanillin, and guaiacol interfered with the delignification reaction in the chlorine dioxide (D) and alkaline extraction (E) stages of the bleaching sequence, thereby preserving the integrity of cellulose in the pulp. Because the efficiency of xylanase and bleaching reagents is hindered by the presence of these phenolic compounds, higher operational cost and more bleaching reagents are needed, which are incompatible with modern environmental policies in the world. Nonetheless, the presence of remaining soluble phenolic compounds in the brownstock can improve the bleaching selectivity important for the production of high-quality pulp with less-degraded cellulose chains.

XYLANASE ACTIVITY OF ASPERGILLUS NIGER AT DIFFERENT ECOLOGICAL CONDITIONS

Maximization of xylanase activity at different media, temperature, pH and salt concentration has been presented in this paper. YpSs, Czapek dox and Malt extract medium were taken for evaluation of optimum growth and activity. Amongst all tested media, YpSs showed the highest growth. Three different natural carbon substitute i.e., wheat husk, rice husk and sugarcane baggase were used for xylanase activity. Maximum enzyme activity was observed in test fungus at rice husk. Production and maximum xylanase activity at rice husk has been observed at different temperatures, pH and Salt concentrations. The highest xylanase activity has been observed on day 5 at temperature 32° C, pH 6.5 and salt concentration of 2%.

Probe-mining of endo-1,4-beta-xylanase from goats-rumen bacterial metagenomic DNA data

Endo-1,4-beta-xylanases (xylanases) are classified into 9 glycoside hydrolase families, GH5, 8, 10, 11, 30, 43, 51, 98, and 141 based on the CAZy database. The probe sequences representing the enzymes were constructed from published sequences of actual experimental studies with xylan decomposition activity. From online databases, we found one sequence belonging to the GH5 family, 6 sequences belonging to the GH8 family and 5 sequences belonging to the GH30 family exhibiting xylanase activity. Thus specific probes for xylanase GH8 and GH30 families were designed with the length of 351 and 425 amino acids respectively. The reference values for the probe of the GH8 family were defined as the sequences with maximum score greater than 168, the lowest coverage was 84%, the lowest similarity was 36%; for the probe GH30, the maximum score was greater than 316, the coverage was greater than 98%, the similarity was greater than 41%. Using the built probes, including the probe of the two GH10 and GH11 families, we found 41 xylanase-encoding sequences from the metagenomic DNA data of bacteria in Vietnamese goats’rumen. Of the 41 exploited sequences, 19 were identical to the BGI company's annotation result based on KEGG database, whereas there were 16 sequences that are not annotated by the BGI company. Total 28 of 41 exploited sequences were complete open reading frames, of which the predicted ternary structure was highly similar to the published structures of xylanase.

One-step fermentation for producing xylo-oligosaccharides from wheat bran by recombinant Escherichia coli containing an alkaline xylanase

One-step fermentation is a cheap way to produce xylo-oligosaccharides (XOS), where production of xylanases and XOS is integrated into a single process. In spite of having cost advantage, one-step fermentation is still short in yield so far due to the limited exploration. To cope with this issue, production of XOS from wheat bran by recombinant Escherichia coli through one-step fermentation was investigated here. A xylanase gene belonging to glycoside hydrolase family 11 of Bacillus agaradhaerens was employed to construct recombinant E. coli. This xylanase showed maximal activity at 60°C and pH 8.0. Its activity retained more than 60% after incubation at 70°C for 4 hours, showing a good stability. The recombinant E. coli successfully secreted xylanases that directly hydrolyzed wheat bran to XOS in fermentation medium. The generated XOS consisted of xylose, xylobiose and xylotriose accounting for 23.1%, 37.3% and 39.6%, respectively. Wheat bran concentration was found to be the most crucial factor affecting XOS production. The yield reached 5.3 mg/mL at 10% of wheat bran, which is higher than previous reports employing one-step fermentation. Nitrogen source type could also affect XOS yield by changing extracellular xylanase activity, and glycine was found to be the best one for fermentation. Optimal fermentation conditions were finally studied by response surface optimization. The maximal yield emerged at 44.3°C, pH 7.98, which is affected by characteristics of the xylanase and growth conditions of E. coli. This work indicates that the integrated fermentation using recombinant E. coli is highly competitive in cost and yield for production of XOS.

Brewer’s spent grain as substrate for enzyme and reducing sugar production using Penicillium sp. HC1

Brewer’s spent grain (BSG) is the main solid waste from the brewing process. It is recognized as a valuable resource for biobased industries because of its composition, high availability, and low cost. The objective of this study was to employ BSG as a substrate to produce the enzymes endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase, as well as reducing sugars using Penicillium sp. HC1. For enzyme production, we evaluated BSG submerged fermentation at different concentrations (1%, 3%, and 5%, w/v) and two sources of nitrogen (yeast extract and ammonium sulfate) on different days (6, 10, and 12) in a 100 mL Erlenmeyer flask. The highest enzyme activity was obtained after 10 days. The enzyme extract obtained using 3% BSG (w/v) and 5 g L-1 of ammonium sulfate showed the highest xylanase activity (25013 ± 1075 U L-1). Using BSG 5% (w/v) without nitrogen supplementation, the endoglucanase activity was 909.7±14.2 U L-1 while underthe same conditions but using BSG 3% (w/v), the β-glucosidase and cellobiohydrolase activity was 3268.6 ±229.9 U L-1 and 103.15±8.1 U L-1, respectively. Maximum reducing sugar concentrations using an enzyme dosage of 1000 U g-1 of xylanase were: 2.7 g L-1 xylose, 1.7 g L-1 arabinose, and 3.3 g L-1 glucose after 6 h of hydrolysis. Result s demonstrated it is possible to produce enzymes and reducing sugars using Penicillium sp. HC1 and BSG as substrate and BSG grinding only as pretreatment.

Citrus Segment Degradation Potential, Enzyme Safety Evaluation, and Whole Genome Sequence of Aspergillus aculeatus Strain ZC-1005

Aspergillus aculeatus ZC-1005 (ZC-1005 was used as the abbreviation of this strain) is a hemicellulase-producing strain isolated from rotten citrus rind buried in the soil. Our previous study has shown its biochemical properties including high xylanase activity, mannanase activity, and degradation reaction with citrus mesocarp. In this study, we focused more on the enzyme safety evaluation and the genome sequencing via PacBio and Illumina platforms. High biological safety of the crude enzymes of ZC-1005 has been proven by the acute oral toxicity test, sub-chronic toxicity test, micronucleus test, and sperm malformation test. The genome of ZC-1005 had a GC content of 52.53%, with a size of 35,458,484 bp, and encoded 10,147 genes. Strain ZC-1005 harbored 269 glycosyl hydrolase (GH) genes of 64 families. The fungus produces cellulose-acting (GH3, GH5, GH12, and GH1) and hemicellulose-acting enzymes (GH16, GH31, GH2, and GH92). In genome annotation, we paid more attention to the genes encoding xylanase, such as gene 01512, gene 05833, gene 05469, gene 07781, gene 08432, gene 09042, gene 08008, and gene 09694. The collaboration between complete genome information and the degradation test confirmed that ZC-1005 could degrade cellulose and xylan. Our results showed that the citrus enzymatic decapsulation technology was efficacious and safe for canned citrus product processing, which may also solve the industrial waste problem. Therefore, ZC-1005 and the crude enzyme secreted from the strain were very promising to be used in the citrus processing industry.