Polygalacturonate Lyase Research Articles

Increased production of alkaline polygalacturonate lyase in the recombinant WB43CB by optimizing the medium

Alkaline polygalacturonate lyase (PGL), which was the central enzyme of green environmental enzyme treatment technology, has been widely used in textile, papermaking and beverage production. In the previous work, Bacillus subtilis was used as the expression host, and the PGL gene derived from B. subtilis WSHB04-02 was successfully expressed in the engineered strain WB43CB by regulating of molecular elements such as signal peptide, promoter and SD sequence. In this study, the fermentation medium was optimized from four aspects of carbon source, nitrogen source, liquid volume and inoculum volume, and the extracellular PGL enzyme activity in WB43CB increased from 264.5 U·mL-1 to 461.96 U·mL-1, which laid a solid foundation for the industrial production of PGL.

High PGL productivity in Bacillus subtilis by optimizing the SD sequence

Alkaline polygalacturonate lyase (PGL, EC 4.2.2.2) can catalyze the cleavage of a-1, 4-glycosidic bonds of polygalacturonate by a trans-elimination reaction and generate an unsaturated oligogalacturonates. As the critical enzyme of many environmental friendly processes, alkaline PGL has been widely used in many fields including paper, textile and beverage industries. At present, Bacillus subtilis is an ideal strain for producing PGL, but the yield is too low for industrial production. In this study, the effect of different SD sequences on the production of PGL was comparatively investigated, and the strong SD sequence (AGAGAACAAGGAGGG G) directed efficient PGL secretory expression and increased PGL yield to 264.5 U·mL-1 with a high productivity. As a result, the PGL yield in B. subtilis was effecively increased and laid the solid foundation for PGL industrial production.

An efficient expression tag library based on self-assembling amphipathic peptides

BackgroundSelf-assembling amphipathic peptides (SAPs) may improve protein production or induce the formation of inclusion bodies by fusing them to the N-terminus of proteins. However, they do not function uniformly well with all target enzymes and systematic research on how the composition of SAPs influence the production of fusion protein is still limited.ResultsTo improve the efficiency of SAPs, we studied factors that might be involved in SAP-mediated protein production using S1 (AEAEAKAK)2 as the original SAP and green fluorescent protein (GFP) as the reporter. The results indicate that hydrophobicity and net charges of SAPs play a key role in protein expression. As hydrophobicity regulation tend to cause the formation of insoluble inclusion bodies of protein, an expression tag library composed of SAPs, which varied in net charge (from + 1 to + 20), was constructed based on the random amplification of S1nv1 (ANANARAR)10. The efficiency of the library was validated by polygalacturonate lyase (PGL), lipoxygenase (LOX), l-asparaginase (ASN) and transglutaminase (MTG). To accelerate preliminary screening, each enzyme was fused at the C-terminus with GFP. Among the four enzyme fusions, the SAPs with + 2 – + 6 net charges were optimal for protein expression. Finally, application of the library improved the expression of PGL, LOX, ASN, and MTG by 8.3, 3.5, 2.64, and 3.68-fold relative to that of the corresponding wild-type enzyme, respectively.ConclusionsThis is the first report to study key factors of SAPs as an expression tag to enhance recombinant enzyme production. The SAP library could be used as a novel plug-and-play protein-engineering method to screen for enzymes or proteins with enhanced production.

Simultaneous production of industrially important alkaline xylanase-pectinase enzymes by a bacterium at low cost under solid-state fermentation conditions.

Simultaneous production of alkaline xylanase and all seven types of pectinases by a bacterial isolate, under solid-state fermentation was checked in this study. Under optimized conditions, high concurrent production of xylanase (22,800±578IU/g substrate) and pectinase (4,832±189IU/g substrate) was achieved. The different types of pectinases produced were exo-polymethylgalacturonase (782IU/g), endo-polymethylgalacturonase (6.42U/g), exo-polygalacturonase (2,250IU/g), endo-polygalacturonase (11.57U/g), polymethylgalacturonate lyase (53.99IU/g), polygalacturonate lyase (59.78IU/g), and pectin esterase (5.78IU/g). Wheat bran resulted in the highest titer of both enzymes. The maximum xylanase-pectinase yield was detected after 7 days of incubation with 2mM MgSO4 and 1.5g/L K2 HPO4 at wheat bran to moisture ratio 1:1.5 (w/v), media to flask volume ratio 1:25, pH 7.0, temperature 37°C, and inoculum size 15%. Xylanase was most stable at pH 8.0, retained more than 75% activity up to 24H, whereas pectinase was most stable at pH 9.0, having full activity even after 24H. At 45°C, the xylanase showed 82% residual activity after 6H of incubation. The pectinase was 97% and 61% stable up to 3H at 50 and 55°C, respectively. This is the first report showing the production of xylanase-pectinases by bacterium along with high titer of seven types of pectinases, suitable for industries.

Development of a purification tag to produce thermostable fused protein

Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.

Optimizing Culture Conditions by Statistical Approach to Enhance Production of Pectinase from Bacillus sp. Y1.

It was found that Bacillus sp. Y1 could secrete alkaline pectinase with suitable enzyme system for powerful and fast degumming of ramie fiber. In this study, the medium components and fermentation conditions were optimized by some statistical methods including mixture design, fractional factorial design, central composite design and response surface methodology, and single factor method for enhancing the alkaline pectinase production. The optimized conditions for pectinase production were that the culture was shaken at 34°C for 60 h in 50 mL of medium containing 10.5% (w/v) carbon source (consisting of 3.8% starch, 4.2% wheat bran, and 2.5% sucrose), 0.37% (NH4)2SO4, 0.3% MgSO4, and 0.1% Tween-80, with initial pH 8.2 and inoculation amount of 1.3 mL (with the OD600 of the seed medium about 5.77). Using the optimizing conditions, the activities of polygalacturonate lyase (PGL) and polygalacturonase (PG) in fermentation liquor were increased to 2.00-fold and 3.44-fold, respectively, and the fermentation time shortened 12 hours (from 72 h to 60 h), which showed good application potential in degumming of ramie.

Development of strategy for simultaneous enhanced production of alkaline xylanase-pectinase enzymes by a bacterial isolate in short submerged fermentation cycle

The aim of this study is to enhance the production of industrially valuable xylanase and pectinase enzymes in short duration, using agrowaste extracted substrates. Conventional cum statistical multifactor analysis approaches were used in order to evaluate the effect of crude extracted substrates, supplemented for the production of xylanase-pectinase enzymes. Incorporation of crude extracted xylan (1.2 mg/ml of inoculum) and pectin (4.8 mg/ml of inoculum) substrates in inoculum resulted in maximal xylanase (320 ± 15) and pectinase titre (90 ± 8) after 48 h, using 2% wheat bran and 2% citrus peel in production medium with 48 h of fermentation time, with one variable factor at a time approach. The best condition obtained after performing statistical multifactor interaction analysis includes 5.50 mg/ml of pectin in inoculum,1.50 mg/ml of xylan in inoculum, wheat bran 3%, temperature 37.5 °C, time 48 h, 7 mg/ml of pectin in production medium, peptone 1.05%, inoculum size 2% and inoculum age of 20 h, with alkaline xylanase activity of 415.22 ± 18.50 IU/ml and alkaline pectinase activity of 109.10 ± 8.80 IU/ml. Activity of different pectinolytic enzymes per ml was also calculated, with 18.98 IU of exo-polymethylgalacturonase, 0.14 IU of endo-polymethylgalacturonase, 80 IU of exo-polygalacturonase, 0.28 IU of endo-polygalacturonase, 1.42 IU of polymethylgalacturonate lyase, 1.47 IU of polygalacturonate lyase, 0.15 IU of pectin esterase. This is the first report mentioning the utilization of crude extracted xylan and extracted pectin in inoculum to get the increment in the activity of both alkaline xylanase-pectinase enzymes simultaneously under short submerged fermentation cycle.

An efficient thermostabilization strategy based on self-assembling amphipathic peptides for fusion tags

Self-assembling amphipathic peptides (SAPs) have been used as stabilization tags to improve enzyme stability but do not function uniformly well with all target enzymes. Here, the key factors involved in SAPs stabilization were identified as the SAP length and linker length and flexibility, using S1 (AEAEAKAK)2 as an originated SAP and polygalacturonate lyase (PGL) as model protein. Biochemical analysis demonstrated that SAPs could induce loose protein oligomerization via intermolecular hydrophobic interactions. Based on this mechanism, a comprehensive protein stabilization strategy was proposed, in which a library of stabilizing tags through random combination of different SAPs and linker peptides was developed to design the fusion composition while the sodium chloride (NaCl) was used to enhance the intermolecular hydrophobic interactions. By using the strategy, the PGL, lipoxygenase (LOX) and L-asparaginase exhibited 33.25-, 17.55- and 15.6-fold increases, respectively, in the t1/2 value relative to that of the corresponding wild-type enzyme. The SAP library therefore shows great application potential in stability enhancement of enzymes/proteins.

A multifunctional tag with the ability to benefit the expression, purification, thermostability and activity of recombinant proteins

In this study, a novel multifunctional tag, S1v1 (AEAEAHAH)2, was generated from a self-assembling amphipathic peptide in the Zuotin protein sequence by replacing lysine residues with histidine residues. After fusing S1v1 at the N-terminus through a PT-linker, the expressions of polygalacturonate lyase (PGL), lipoxygenase (LOX) and green fluorescent protein (GFP) were enhanced by 3.8, 0.2 and 1.52-fold, respectively,compared to the wild-type proteins. However, the frequently used His-tag with a PT-linker had negligible effects on expression. Moreover, the three S1v1 fusions were purified with high purities and acceptable recovery rates due to their affinity to the nickel column. In contrast, PGL and LOX fused with His-tag were unable to be adsorbed by the nickel column, and His-tag fusion only achieved 8.23% of GFP recovery in the same purification process.In addition, S1v1 fusions induced the enhancement of thermostabilties and/or activities of PGL, LOX and GFP. These results indicated that S1v1 was much more effective than the frequently used His-tag during protein expression and purification in these cases, and will be especially suitable for those proteins requiring the simultaneous enhancement of expression, production and catalytic properties.

Characterization of bacterial community structure during in-vessel composting of agricultural waste by 16S rRNA sequencing.

Bacterial diversity during in-vessel (rotary drum) composting of agricultural waste was characterized using NGS-based 16S rRNA sequencing for microbial identification. The activity of the bacteria was observed to vary with the composting materials and degradation pattern. Taxonomic hits distribution at domain level revealed that 89.5% sequences belonged to bacteria, 9% to eukaryota followed by 1.4% archaea during drum composting. The lowest common ancestor (LCA) classification plot showed the high abundance of the phylum proteobacteria followed by actinobacteria in compost sample. Taxonomic hit distribution at family level showed that compost sample was enriched with Thermomonosporaceae. Thermomonospora curvata is an aerobic, cellulolytic, thermophilic Gram-positive bacterium which produces a number of industrially important compounds, i.e., cellulase, alpha-amylase, and polygalacturonate lyase. Thermomonospora family of bacteria play a major role in organic matter degradation during composting. Hence, in the present study species such as Actinomadura vinacea, Thermomonospora curvata, Actinoallomurus spadix, Actinomadura rubrobrunea. T. curvata were identified from the compost mixture, which can utilize many organic compounds such as cellulose starch, xylose or pectin. The other biggest group in compost sample was Actinobacteria with Thermoleophilum album as the most abundant species followed by Collinsella aerofaciens. The compost was stabilized with higher volatile solids reduction, lower OUR (4.49mg/g VS/day) and CO2 (2.28mg/g VS/day) values at the end of 20days. The final compost was observed with 2.31% of TKN and 4.3% of phosphorus. Finally the results indicate that degradation of agricultural waste using drum composter was dominated by Bacilli, γ, β-proteobacteria, and actinobacteria.

Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis

Bacillus subtilis as an important host has been widely used in synthetic biology, metabolic engineering, and production of industrial enzymes. To fully take advantage of this organism, 114 endogenous putative promoters were measured with a green fluorescent protein reporter and four classes of phase-dependent promoters (class I: exponential phase; class II: middle-log and early stationary phases; class III: lag-log and stationary phases; class IV: stationary phase) with different strengths were identified. The transcriptional strengths ranged from 0.03 to 2.03-fold of that of the commonly used strong promoter P43. On this basis, the temperature (for instance P bltD , P ydaD , and P gerBC ) and pH (such as P abrB , P ydjO , and P opuE ) inducible phase-dependent promoters were further identified and characterized. In comparison, P abrB (class I), P spoVG (class II), and P lytR (class III) achieved the highest expression levels of esterase, keratinase, and alkaline polygalacturonate lyase, respectively. The constructed phase-dependent promoter library should have great application potentials for enzyme production, metabolic engineering, and synthetic biology.

Immobilization of alkaline polygalacturonate lyase from Bacillus subtilis on the surface of bacterial polyhydroxyalkanoate nano-granules.

Alkaline polygalacturonate lyase (PGL), one of the pectinolytic enzymes, has been widely used for the bioscouring of cotton fibers, biodegumming, and biopulp production. In our study, PGL from Bacillus subtilis was successfully immobilized on the surface of polyhydroxyalkanoate (PHA) nanogranules by fusing PGL to the N-terminal of PHA synthase from Ralstonia eutropha via a designed linker. The PGL-decorated PHA beads could be simply achieved by recombinant fermentation and consequent centrifugation. The fused PGL occupied 0.985% of the total weight of purified PHA granules, which was identified by mass spectrometer-based quantitative proteomics. The activity of immobilized PGL (184.67U/mg PGL protein) was a little lower than that of the free PGL (215.93U/mg PGL protein). The immobilization process did not affect the optimal pH and the optimal temperature of the PGL, but it did enhance the thermostability as well as the pH stability at certain conditions, which will extend the practicability of the immobilized PGL-PHA beads in the alkaline and generally harsh bioscouring process. Furthermore, the immobilized PGL still retained more than 60% of its initial activity after 8cycles of reuse. Our study provided a novel and promising approach for cost-efficient in vivo PGL immobilization, contributing to wider commercialization of this environmental-friendly biocatalyst.

Enhancing production of alkaline polygalacturonate lyase from Bacillus subtilis by fed-batch fermentation.

Alkaline polygalacturonate lyase (PGL, EC 4.2.2.2) is an enzyme used in many industries. We developed a fed-batch fermentation process that combines the enzymatic pretreatment of the carbon source with controlling the pH of the fermentative broth to enhance the PGL production from Bacillus subtilis 7-3-3 to decrease the production cost. Maintaining the fermentation broth at pH 6.5 prior to feeding with ammonia and at pH 6.0 after feeding significantly improved PGL activity (743.5 U mL−1) compared with the control (202.5 U mL−1). The average PGL productivity reached 19.6 U mL−1 h−1 after 38 h of fermentation. The crude PGL was suitable for environmentally friendly ramie enzymatic degumming.

Characteristics of Polygalacturonate Lyase C from Bacillus subtilis 7-3-3 and Its Synergistic Action with PelA in Enzymatic Degumming

An alkaline polygalacturonate lyase (PGL) from Bacillus subtilis 7-3-3, PelC, with diverse depolymerization abilities for different pectin substrates was found. The PGL activity of PelC decreased with increasing degree of methyl esterification of the substrate. PelA and PelC displayed notable synergistic effects in the enzymatic degumming of ramie fibers. Gum loss rates increased by 62% when PelC was used to replace up to three-eighths of the PelA dose (PelC, 60 U g−1 ramie fibers). To the best of our knowledge, this study is the first to report the synergistic action of members of polysaccharide lyase families 1 and 3, represented by PelA and PelC, respectively. The present paper provides new insights into the improvement and production of enzymes used in enzymatic degumming.

High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements

The present work aims to construct a robust recombinant Bacillus subtilis to achieve secretory production of alkaline polygalacturonate lyase (PGL). First, 6 signal peptides (amyX, bpr, vpr, yvgO, wapA and nprE) were screened with a semi-rational approach and comparatively investigated their effects on the production of PGL. The signal peptide bpr directed efficient PGL secretory expression and increased PGL titer to 313.7 U mL(-1). By optimizing and applying strong promoter P43 and Shine-Dalgarno sequence, higher titer of 446.3 U mL(-1) PGL was achieved. Finally, the capacity of the recombinant B. subtilis WB43CB was evaluated with a fed-batch strategy in 3 L fermentor. The PGL titer reached 632.6 U mL(-1) with a productivity of 17.6 U mL(-1) h(-1), which was the highest secretory production of PGL by the B. subtilis system. The recombinant B. subtilis strain WB43CB constructed in the present work has great potential in production of alkaline PGL.

Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis

A polygalacturonate lyase (PGL), PelA, was purified from the culture broth of Bacillus subtilis 7-3-3, with a molecular weight, optimal temperature, and pH of approximately 45kDa, 55°C, and 9.4, respectively. The PGL gene (pelA) was homologously overexpressed in B. subtilis 7-3-3 to increase the gene copies and enhance the PGL production. The resulting PGL activity was 2138UmL−1 at 44h, and the productivity reached 48.58U(mLh)−1 through the homologous overexpression of strain B-pN-pelA in a 7.5L fermentor, the highest PGL production compared to those reported in literature to the best of our knowledge. Crude enzyme has high PGL and PGase activity, which can remove 50.58% of pectin in unpretreatment ramie fibers at 50°C for 4h. Meanwhile, the enzyme system with a low level hemicellulase and almost no cellulase will further help in enhancing the efficiency of degumming besides maintaining tenacity of plant fiber. The B. subtilis B-pN-pelA shows high genetic stability and has great potential in the textile industry.

Increased production of alkaline polygalacturonate lyase in the recombinant Pichia pastoris by controlling cell concentration during continuous culture

Recombinant alkaline polygalacturonate lyase (PGL) production by recombinant Pichia pastoris GS115 was selected as a model to study as a continuous culture strategy for enhancing heterologous protein production based on controlling methanol feeding (CCCM culture) or on dual carbon source feeding (CCCD culture). Using the CCCM process with a dry cell weight of 75g/L regulated by controlling methanol concentration in the induction media, the final PGL activity was 441.9U/mL. The PGL productivity (Qv) and the average specific enzyme production rate (Qx) were 4.65UmL−1h−1 and 84.5Ug−1h−1, an increase of 42.1% and 191.2%, respectively, over what was achieved with traditional fed-batch culture with high cell density. The control strategies also reduced proteolytic degradation by 84.1% in the fermentation broth and increased cell viability by 12.2%.

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04–02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12mlh−1 at post-induction phase. The total PGL yield reached 371.86UmL−1, which is the highest PGL production by recombinant E. coli expression system.

Improved production of polygalacturonate lyase by combining a pH and online methanol control strategy in a two-stage induction phase with a shift in the transition phase

Polygalacturonate lyase is a kind of enzyme that is abundantly used in the textile industry for cotton scouring. Previously, we reconstructed the polygalacturonate lyase gene in Pichia pastoris for the expression of this enzyme. To enhance the production of polygalacturonate lyase (PGL), a combined strategy was formulated by combining online methanol control and two-stage pH control strategies. For the two-stage pH control strategy during the growth phase, the pH was controlled at 5.5, and in the induction phase different pH levels were investigated for the optimum enzyme production. During the online methanol control strategy, the different levels of methanol (v/v) were investigated for the best enzyme production at pH 5.5. These two strategies were combined together for enhanced PGL productivity, and the induction phase was divided into two stages in which methanol concentrations were maintained at different levels online. The transition phase was introduced during the induction phase instead of introducing it after the growth phase. The two-stage combination strategy was formulated on the bases of methanol consumption of cells, optimal pH, cell viability and the production of polygalacturonate lyase by P. pastoris. By using this strategy, the production was doubled compared with common conditions, and the highest polygalacturonate lyase activity reached 1,631 U/ml. This strategy proved to be very useful for the enhancement of polygalacturonate lyase production by achieving higher cell viability, alcohol oxidase activity and phosphate-related compounds of the cells during the induction phase.

Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation

Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL −1, which was enhanced 1.85-fold compared to the control (863 U mL −1) cultured with sorbitol added at a constant rate of 3.6 g h −1 L −1 after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.