A novel AA14 LPMO from Talaromyces rugulosus with bifunctional cellulolytic/hemicellulolytic activity boosted cellulose hydrolysis.

BACKGROUND: The recently discovered PcAA14A and B from white-rot basidiomycete Pycnoporus coccineus enriched our understanding of the oxidative degradation of xylan in fungi, however, the unusual mode of action of AA14 LPMOs has sparked controversy. The substrate specificity and functionality of AA14 LPMOs still remain enigmatic and need further investigation. RESULTS: In this study, a novel AA14 LPMO was characterized from the ascomycete Talaromyces rugulosus. TrAA14A has a broad substrate specificity with strong oxidative activity on pure amorphous cellulose and xyloglucan. It could simultaneously oxidize cellulose, xylan and xyloglucan in natural hemi/cellulosic substrate such as fibrillated eucalyptus pulp, and released native and oxidized cello-oligosaccharides, xylo-oligosaccharides and xyloglucan oligosaccharides from this substrate, but its cellulolytic/hemicellulolytic activity became weaker as the contents of xylan increase in the alkaline-extracted hemi/cellulosic substrates. The dual cellulolytic/hemicellulolytic activity enables TrAA14A to possess a profound boosting effect on cellulose hydrolysis by cellulolytic enzymes. Structure modelling of TrAA14A revealed that it exhibits a relatively flat active-site surface similar to the active-site surfaces in AA9 LPMOs but quite distinct from PcAA14B, despite TrAA14A is strongly clustered together with AA14 LPMOs. Remarkable difference in electrostatic potentials of L2 and L3 surfaces was also observed among TrAA14A, PcAA14B and NcLPMO9F. We speculated that the unique feature in substrate-binding surface might contribute to the cellulolytic/hemicellulolytic activity of TrAA14A. CONCLUSIONS: The extensive cellulolytic/hemicellulolytic activity on natural hemi/cellulosic substrate indicated that TrAA14A from ascomycete is distinctively different from previously characterized xylan-active AA9 or AA14 LPMOs. It may play as a bifunctional enzyme to decompose some specific network structures formed between cellulose and hemicellulose in the plant cell walls. Our findings shed new insights into the novel substrate specificities and biological functionalities of AA14 LPMOs, and will contribute to developing novel bifunctional LPMOs as the booster in commercial cellulase cocktails to efficiently break down the hemicellulose-cellulose matrix in lignocellulose.

Microbial α-L-arabinofuranosidases: diversity, properties, and biotechnological applications.

Arabinoxylans (AXs) are hemicellulosic polysaccharides consisting of a linear backbone of ß-1,4-linked xylose residues branched by high content of α-L-arabinofuranosyl (Araf) residues along with other side-chain substituents, and are abundantly found in various agricultural crops especially cereals. The efficient bioconversion of AXs into monosaccharides, oligosaccharides and/or other chemicals depends on the synergism of main-chain enzymes and de-branching enzymes. Exo-α-L-arabinofuranosidases (ABFs) catalyze the hydrolysis of terminal non-reducing α-1,2-, α-1,3- or α-1,5- linked α-L-Araf residues from arabinose-substituted polysaccharides or oligosaccharides. ABFs are critically de-branching enzymes in bioconversion of agricultural biomass, and have received special attention due to their application potentials in biotechnological industries. In recent years, the researches on microbial ABFs have developed quickly in the aspects of the gene mining, properties of novel members, catalytic mechanisms, methodologies, and application technologies. In this review, we systematically summarize the latest advances in microbial ABFs, and discuss the future perspectives of the enzyme research.

A novel cellulolytic/xylanolytic SbAA14 from Sordaria brevicollis with a branched chain preference and its synergistic effects with glycoside hydrolases on lignocellulose.

In this study, the novel auxiliary activity (AA) family 14 lytic polysaccharide monooxygenase (LPMO) SbAA14 from Sordaria brevicollis was successfully characterized. It was active against heteroxylan, xyloglucan and cellulose in ß-cellulose and released native oligosaccharides and corresponding C1- and/or C4-oxidized products. SbAA14 showed a branched chain preference, because partial removal of arabinosyl substituents from heteroxylan led to a decrease in activity. SbAA14 had synergistic effects with the debranching enzyme EpABF62C in an enzyme- and ascorbic acid-dependent manner. SbAA14 had synergistic effects with the GH10 endoxylanase EpXYN1, and the degree of synergy was greater with step-by-step addition than with simultaneous addition. SbAA14 could also synergize with Celluclast® 1.5 L on NaOH-pretreated wheat straw and on NaOH-pretreated and hydrogen peroxide-acetic acid (HPAC)-H2SO4-pretreated bamboo substrates. The greatest synergistic effect between SbAA14 and Celluclast® 1.5 L was observed for HPAC-H2SO4-200 mM pretreated bamboo, in which the degree of synergy reached approximately 1.61. The distinctive substrate preference of SbAA14 indicated that it is a novel AA14 LPMO that may act mainly on heteroxylan with numerous arabinosyl substituents between cellulose fibers rather than on recalcitrant xylan tightly associated with cellulose. These findings broaden the understanding of enigmatic AA14 LPMOs and provide new insights into the substrate specificities and biological functionalities of AA14 LPMOs in fungi.

High-level production of cordycepin by the xylose-utilising Cordyceps militaris strain 147 in an optimised medium.

Cordycepin is an important active metabolite of Cordyceps militaris. Xylose, an attractive feedstock for producing chemicals through microbial fermentation, cannot be effectively utilised by many reported C. militaris strains. Herein, a xylose-utilising C. militaris strain 147 produced the highest level of cordycepin (3.03 g/L) in xylose culture. Xylose, alanine, and ammonium citrate were determined as the main affecting factors on the cordycepin production using a Plackett-Burman design. The combination of these factors was optimised using response surface methodology, and the maximal 6.54 g/L of cordycepin was produced by the fungus in the optimal medium. Transcriptome analysis revealed that xylose utilisation upregulated the transcriptional levels of genes participating in purine and energy metabolisms in the fungus, which may facilitate the formation of precursors for cordycepin biosynthesis. This investigation provides new insights into the efficient production of cordycepin and is conducive to the valorisation of biomass rich in xylose.

Insights into the capability of the lignocellulolytic enzymes of Penicillium parvum 4-14 to saccharify corn bran after alkaline hydrogen peroxide pretreatment.

BACKGROUND: Corn bran is a major agro-industrial byproduct from corn starch processing. It contains abundant arabinoxylan that can be converted into value-added chemicals via biotechnology. Corn bran arabinoxylan (CBAX) is one of the most recalcitrant xylans for enzymatic degradation due to its particular heterogeneous nature. The present study aimed to investigate the capability of the filamentous fungus Penicillium parvum 4-14 to enzymatically saccharify CBAX and reveal the fungal carbohydrate-active enzyme (CAZyme) repertoire by genome sequencing and secretome analysis. RESULTS: CBAX1 and CBAX2 with different branching degrees, together with corn bran residue (CBR) were generated from corn bran after alkaline hydrogen peroxide (AHP) pretreatment and graded ethanol precipitation. The protein blends E_CBAX1, E_CBAX2, and E_CBR were produced by the fungus grown on CBAX1, CBAX2, or CBR, respectively. Under the optimal conditions, E_CBAX1 released more than 80% xylose and arabinose from CBAX1 and CBAX2. Almost complete saccharification of the arabinoxylans was achieved by combining E_CBAX1 and a commercial enzyme cocktail Cellic®CTec3. Approximately 89% glucose, 64% xylose, and 64% arabinose were liberated from CBR by E_CBR. The combination of E_CBR with Cellic®CTec3 enhanced the saccharification of CBR, with conversion ratios of 97% for glucose, 81% for xylose, and 76% for arabinose. A total of 376 CAZymes including plentiful lignocellulolytic enzymes were predicted in P. parvum based on the fungal genomic sequence (25.8 Mb). Proteomic analysis indicated that the expression of CAZymes in P. parvum varied between CBAX1 and CBR, and the fungus produced complete cellulases, numerous hemicellulases, as well as high levels of glycosidases under the culture conditions. CONCLUSIONS: This investigation disclosed the CAZyme repertoire of P. parvum at the genomic and proteomic levels, and elaborated on the promising potential of fungal lignocellulolytic enzymes upon saccharification of corn bran biomass after AHP pretreatment.

Comparison of the Biochemical Properties and Roles in the Xyloglucan-Rich Biomass Degradation of a GH74 Xyloglucanase and Its CBM-Deleted Variant from Thielavia terrestris.

Xyloglucan is closely associated with cellulose and still retained with some modification in pretreated lignocellulose; however, its influence on lignocellulose biodegradation is less understood. TtGH74 from Thielavia terrestris displayed much higher catalytic activity than previously characterized fungal GH74 xyloglucanases. The carbohydrate-binding module 1 (CBM1) deleted variant (TtGH74ΔCBM) had the same optimum temperature and pH but an elevated thermostability. TtGH74 displayed a high binding affinity on xyloglucan and cellulose, while TtGH74ΔCBM completely lost the adsorption capability on cellulose. Their hydrolysis action alone or in combination with other glycoside hydrolases on the free xyloglucan, xyloglucan-coated phosphoric acid-swollen cellulose or pretreated corn bran and apple pomace was compared. CBM1 might not be essential for the hydrolysis of free xyloglucan but still effective for the associated xyloglucan to an extent. TtGH74 alone or synergistically acting with the CBH1/EG1 mixture was more effective in the hydrolysis of xyloglucan in corn bran, while TtGH74ΔCBM showed relatively higher catalytic activity on apple pomace, indicating that the role and significance of CBM1 are substrate-specific. The degrees of synergy for TtGH74 or TtGH74ΔCBM with the CBH1/EG1 mixture reached 1.22-2.02. The addition of GH10 xylanase in TtGH74 or the TtGH74ΔCBM/CBH1/EG1 mixture further improved the overall hydrolysis efficiency, and the degrees of synergy were up to 1.50-2.16.

Functional characterization of a new 3-dehydroshikimate dehydratase from Eupenicillium parvum and its potential for protocatechuic acid production.

Protocatechuic acid (PCA) is an important phenolic compound with diverse industrial values. Conversion of 3-dehydroshikimate (DHS) to PCA by dehydroshikimate dehydratase (DSD) provides an efficient approach for the production of the molecule. Herein, a new DSD from fungus Eupenicillium parvum was functionally investigated after recombinant expression in Escherichia coli. The DSD displayed 30%-35% sequence identities with the known fungal DSDs. The recombinant protein showed catalysis activity against DHS, with the optimal temperature of 40 °C and pH of 7.5. The specific activity and Km of the protein were 910 mU per mg protein and 0.83 m m, respectively. Metal ion (Mg2+ or Mn2+) played a critical role in the enzymatic activity. Meanwhile, the thermal stability of the protein was improved by Mg2+ or Mn2+. Furthermore, the expression of the protein in E. coli resulted in de novo synthesis of 491 mg/L PCA in a modified M9 medium with glycerol as a carbon source.

Functional characterization of a GH62 family α-L-arabinofuranosidase from Eupenicillium parvum suitable for monosaccharification of corncob arabinoxylan in combination with key enzymes.

Corncob rich in arabinoxylan is an important raw material widely used in bio-refinery. Complete saccharification of arabinoxylan depends on the synergism of different enzymes including α-L-arabinofuranosidase (ABF). This study aimed to investigate the functional characteristics of a new ABF EpABF62A belonging to glycoside hydrolase (GH) 62 family from the fungus Eupenicillium parvum, and to explore its potential in the saccharification of corncob arabinoxylan. The recombinant EpABF62A showed high activity against wheat arabinoxylan and rye arabinoxylan, with the optimal temperature of 55 °C and pH of 4.5. The protein contains an N-terminal cellulose-binding domain family 1 (CBM_1) domain, and displayed a 59.5% absorption rate to phosphoric acid swollen cellulose. Regioselectivity analysis indicated that the enzyme selectively removed α-1,2 or α-1,3 linked arabinofuranosyl residues on mono-substituted xylose residues on arabinoxylan. Corncob arabinoxylans (CAX1 or CAX2) with different (low or high) branching degrees were extracted from the raw material by alkaline hydrogen peroxide pretreatment and graded ethanol precipitation. Single EpABF62A removed 69.5% or 67.1% arabinose from CAX1 or CAX2, respectively. EpABF62A combined with a GH10 xylanase, a GH43 ß-D-xylosidase and a GH67 α-glucuronidase released 75.0% or 64.5% xylose from CAX1 or CAX2, respectively. The addition of the four hemicellulases enhanced the saccharification the solid fraction of the pretreated corncob by the commercial cellulase Cellic® CTec2, and the conversion ratios of glucose, xylose and arabinose were up to 94.0%, 91.8% and 82.6%, respectively.

Two C1-oxidizing AA9 lytic polysaccharide monooxygenases from Sordaria brevicollis differ in thermostability, activity, and synergy with cellulase.

Cellulolytic fungi usually have multiple genes for C1-oxidizing auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) in their genomes, but their potential functional differences are less understood. In this study, two C1-oxidizing AA9 LPMOs, SbLPMO9A and SbLPMO9B, were identified from Sordaria brevicollis, and their differences, particularly in terms of thermostability, reducing agent specificity, and synergy with cellulase, were explored. The two enzymes exhibited weak binding to cellulose and intolerance to hydrogen peroxide. Their oxidative activity was influenced by cellulose crystallinity and surface morphology, and both enzymes tended to oxidize celluloses of lower crystallinity and high surface area. Comparably, SbLPMO9A had much better thermostability than SbLPMO9B, which may be attributed to the presence of a carbohydrate binding module 1 (CBM1)-like sequence at its C-terminus. In addition, the two enzymes exhibited different specificities and responsivities toward electron donors. SbLPMO9A and SbLPMO9B were able to boost the catalytic efficiency of endoglucanase I (EGI) on physically and chemically pretreated substrates but with different degrees of synergy. Substrate- and enzyme-specific synergism was observed by comparing the synergistic action of SbLPMO9A or SbLPMO9B with commercial Celluclast 1.5L on three kinds of cellulosic substrates. On regenerated amorphous cellulose and PFI (Papirindustriens Forskningsinstitut)-fibrillated bleached eucalyptus pulp, SbLPMO9B showed a higher synergistic effect than SbLPMO9A, while on delignified wheat straw, the synergistic effect of SbLPMO9A was higher than that of SbLPMO9B. On account of its excellent thermostability and boosting effect on the enzymatic hydrolysis of delignified wheat straw, SbLPMO9A may have high application potential in biorefineries for lignocellulosic biomass. KEY POINTS: • C1-oxidizing SbLPMO9A displayed higher thermostability than SbLPMO9B, probably due to the presence of a CBM1-like module. • The oxidative activity of the two SbLPMO9s on celluloses increased with decreasing cellulose crystallinity or increasing beating degree. • The two SbLPMO9s boosted the catalytic efficiency of cellulase, but the synergistic effect was substrate- and enzyme-specific.

Comparison of C4-oxidizing and C1/C4-oxidizing AA9 LPMOs in substrate adsorption, H2O2-driven activity and synergy with cellulase on celluloses of different crystallinity.

Two C1/C4-oxidizing AA9 lytic polysaccharide monooxygenases (AA9 LPMOs), AoLPMO9A and AoLPMO9B, and one C4-oxidizing AoLPMO9C from Aspergillus oryzae, were characterized and compared with the well-studied C4-oxidizing NcLPMO9C. NcLPMO9C and AoLPMO9C harboring carbohydrate-binding module 1 (CBM1) exhibited much stronger adsorption capacity than AoLPMO9A and B without CBM1. The binding affinity is crucial for the efficacy of H2O2 as cosubstrate and oxidative activity of AA9 LPMOs on crystalline cellulose. C4-oxidizing AA9 LPMOs had a striking boosting effect on cellobiohydrolase I (CBHI), while C1/C4-oxidizing AA9 LPMOs boosted CBHII and endoglucanase I (EGI) activity. Our results indicated that two types of AA9 LPMOs with different modularities and regioselectivities varied in cellulose adsorption, H2O2-driven activity and synergy with cellulase on celluloses of different crystallinity which could complement each other in lignocellulose degradation. C4-oxidizing AA9 LPMOs with CBM1 were particularly essential in cellulase cocktail due to high H2O2-driven activity and a striking boosting effect on CBHI.

A highly xyloglucan active lytic polysaccharide monooxygenase EpLPMO9A from Eupenicillium parvum 4-14 shows boosting effect on hydrolysis of complex lignocellulosic substrates.

The recently identified lytic polysaccharide monooxygenases (LPMOs) are important auxiliary proteins which contribute to lignocellulose biodegradation by oxidatively cleaving the glycosidic bonds in cellulose and other polysaccharides. The vast differences in terms of substrate specificity and regioselectivity within LPMOs provide us new possibilities to find promising candidates for the use in enzyme cocktails in biorefinery applications. In this study, a highly xyloglucan active family AA9 lytic polysaccharide monooxygenase EpLPMO9A was identified from Eupenicillium parvum 4-14. EpLPMO9A exhibited a mixed C1/C4 oxidative cleavage activity on cellulose and xyloglucan with a broad range of pH stability and good thermal stability at 40 °C. It showed a higher boosting effect on the enzymatic saccharification of complex lignocellulosic substrates associated with xyloglucan than on the lignocellulosic substrates without xyloglucan particularly in low commercial cellulase dosage cases. The oxidative cleavage of xyloglucan by EpLPMO9A may facilitate to open up the sterical hindrance of cellulose by xyloglucan and thereby increase accessibility for cellulase to lignocellulosic substrates. The discovery of more and more hemicellulose-active LPMOs and their contribution to breaking down the barriers by oxidatively acting on hemicellulose may expand our knowledge for their functions of LPMOs in lignocellulose biodegradation.

Characterization and functional analysis of two novel thermotolerant α-L-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum.

Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-L-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-L-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. 1H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone. KEY POINTS: • Two novel α-l-arabinofuranosidases (ABFs) displayed high thermal stability. • The thermal stability of GH62 family EpABF62C was dependent on calcium. • Buffer pH affects the thermal stability of the two ABFs. • Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

Characterization of an L-Arabinose Isomerase from Bacillus velezensis and Its Application for L-Ribulose and L-Ribose Biosynthesis.

L-Ribulose and L-ribose are two high-value unnatural sugars that can be biosynthesized by sugar isomerases. In this paper, an L-arabinose isomerase (BvAI) from Bacillus velezensis CICC 24777 was cloned and overexpressed in Escherichia coli BL21 (DE3) strain. The maximum activity of recombinant BvAI was observed at 45 °C and pH 8.0, in the presence of 1.0 mM Mn2+. Approximately 207.2 g/L L-ribulose was obtained from 300 g/L L-arabinose in 1.5 h by E. coli harboring BvAI. In addition, approximately 74.25 g/L L-ribose was produced from 300 g/L L-arabinose in 7 h by E. coli co-expressing BvAI and L-RI from Actinotalea fermentans ATCC 43279 (AfRI). This study provides a feasible approach for producing L-ribose from L-arabinose using a co-expression system harboring L-Al and L-RI.

Alkaline organosolv pretreatment of different sorghum stem parts for enhancing the total reducing sugar yields and p-coumaric acid release.

BACKGROUND: The sorghum stem can be divided into the pith and rind parts with obvious differences in cell type and chemical composition, thus arising the different recalcitrance to enzyme hydrolysis and demand for different pretreatment conditions. The introduction of organic solvents in the pretreatment can reduce over-degradation of cellulose and hemicellulose, but significance of organic solvent addition in pretreatment of different parts of sorghum stem is still unclear. Valorization of each component is critical for economy of sorghum biorefinery. Therefore, in this study, NaOH-ethanol pretreatment condition for different parts of the sorghum stem was optimized to maximize p-coumaric acid release and total reducing sugar recovery. RESULT: Ethanol addition improved p-coumaric acid release and delignification efficiency, but significantly reduced hemicellulose deconstruction in NaOH-ethanol pretreatment. Optimization using the response surface methodology revealed that the pith, rind and whole stem require different NaOH-ethanol pretreatment conditions for maximal p-coumaric acid release and xylan preservation. By respective optimal NaOH-ethanol pretreatment, the p-coumaric acid release yields reached 94.07%, 97.24% and 95.05% from pith, rind and whole stem, which increased by 8.16%, 8.38% and 8.39% compared to those of NaOH-pretreated samples. The xylan recoveries of pith, rind and whole stem reached 76.80%, 88.46% and 85.01%, respectively, which increased by 47.75%, 15.11% and 35.97% compared to NaOH pretreatment. Adding xylanase significantly enhanced the enzymatic saccharification of pretreated residues. The total reducing sugar yields after respective optimal NaOH-ethanol pretreatment and enzymatic hydrolysis reached 84.06%, 82.29% and 84.09% for pith, rind and whole stem, respectively, which increased by 29.56%, 23.67% and 25.56% compared to those of NaOH-pretreated samples. Considering the separation cost of the different stem parts, whole sorghum stem can be directly used as feedstock in industrial biorefinery. CONCLUSION: These results indicated that NaOH-ethanol is effective for the efficient fractionation and pretreatment of sorghum biomass. This work will help to understand the differences of different parts of sorghum stem under NaOH-ethanol pretreatment, thereby improving the full-component utilization of sorghum stem.

Highly Efficient Extraction of Ferulic Acid from Cereal Brans by a New Type A Feruloyl Esterase from Eupenicillium parvum in Combination with Dilute Phosphoric Acid Pretreatment.

Feruloyl esterase (FAE) is a critical enzyme in bio-extraction of ferulic acid (FA) from plant cell wall. A new FAE (EpFAE1) encoding gene was isolated from Eupenicillium parvum and heterologously expressed in Pichia pastoris cells. Based on phylogenetic tree analysis, the protein EpFAE1 belongs to type A of the seventh FAE subfamily. Using methyl ferulate as substrate, the optimum temperature and pH for the catalytic activity of EpFAE1 were 50 °C and 5.5, respectively. The enzyme exhibited high stability at 50 °C, in a wide pH range (3.0-11.0), or in the presence of 2 M of NaCl. Together with the endo-xylanase EpXYN1, EpFAE1 released 72.32% and 4.00% of the alkali-extractable FA from de-starched wheat bran (DSWB) or de-starched corn bran (DSCB), respectively. Meanwhile, the substrates were pretreated with 1.75% (for DSWB) or 1.0% (for DSCB) of phosphoric acid (PA) at 90 °C for 12 h, followed by enzymatic hydrolysis of the soluble and insoluble fractions. The release efficiencies of FA were up to 84.64% for DSWB and 66.73% for DSCB. Combined dilute PA pretreatment with enzymatic hydrolysis is a low-cost and highly efficient method for the extraction of FA from cereal brans.

Characterization of a D-lyxose isomerase from Bacillus velezensis and its application for the production of D-mannose and L-ribose.

D-Mannose and L-ribose are two important monosaccharides, which have attracted public attention recently because of their great application potentials in food, cosmetic and pharmaceutical industries. Sugar isomerases catalyze the sugar isomerization and therefore can be used as the biocatalysts for production of the high-value sugars from inexpensive sugars. L-arabinose isomerase catalyzes the conversion of L-arabinose to L-ribulose, while D-lyxose isomerase catalyzes L-ribulose and D-fructose to L-ribose and D-mannose, respectively. In this paper, a putative D-LI from Bacillus velezensis (BvLI) was identified, characterized and used to produce D-mannose and L-ribose from D-fructose and L-arabinose, respectively. The recombinant BvLI exhibited a maximum activity at 55 °C and pH 6.5, in the presence of 0.1 mM Co2+. Approximately 110.75 g/L D-mannose was obtained from 500 g/L D-fructose in 6 h by the recombinant BvLI, and approximately 105 g/L L-ribose was obtained from 500 g/L L-arabinose in 8 h by the successive biocatalysis of L-arabinose isomerase from Bacillus licheniformis (BlAI) and BvLI.

Expression and Characterization of Carotenoid Cleavage Oxygenases From Herbaspirillum seropedicae and Rhodobacteraceae bacterium Capable of Biotransforming Isoeugenol and 4-Vinylguaiacol to Vanillin.

HsCCO and RbCCO from Herbaspirillum seropedicae and Rhodobacteraceae bacterium were selected and characterized from five putative bacterial carotenoid cleavage oxygenase gene sequences, due to merits in expression solubility and catalytic properties. Both enzymes can convert 4-vinylguaiacol and isoeugenol to vanillin. HsCCO showed maximum activity at 40°C and pH 7.0 and was stable at pH 6.5-10 and temperature around 25°C, retaining over 90 and 80% of initial activity, respectively. RbCCO showed maximum activity at 35°C and pH 9.0 and was stable at pH 6-11 and temperatures of 25-30°C, retaining over 80% of initial activity. The kinetic constants K m of HsCCO for isoeugenol and 4-vinylguaiacol were 1.55 and 1.65 mM and V max were 74.09 and 27.91 nmol min-1 mg-1, respectively. The kinetic constants K m of RbCCO for isoeugenol and 4-vinylguaiacol were 2.24 and 0.85 mM and V max were 76.48 and 19.96 nmol min-1 mg-1, respectively. The transformed Escherichia coli cells harboring HsCCO converted isoeugenol and 4-vinylguaiacol at molar conversion yields of 80 and 55% and the maximum vanillin concentrations were up to 1.22 and 0.84 g L-1, respectively. Comparably, the molar conversion yields of the transformed E. coli cells harboring RbCCO against isoeugenol 4-vinylguaiacol were 75 and 58%, and the maximum vanillin yields were up to 1.14 and 0.88 g L-1, respectively.

Bioproduction of High-Concentration 4-Vinylguaiacol Using Whole-Cell Catalysis Harboring an Organic Solvent-Tolerant Phenolic Acid Decarboxylase From Bacillus atrophaeus.

The compound 4-vinyl guaiacol (4-VG) is highly valued and widely applied in the pharmaceutical, cosmetic, and food industries. The bioproduction of 4-VG from ferulic acid (FA) by non-oxidative decarboxylation using phenolic acid decarboxylases is promising but has been hampered by low conversion yields and final product concentrations due to the toxicities of 4-VG and FA. In the current study, a new phenolic acid decarboxylase (BaPAD) was characterized from Bacillus atrophaeus. The BaPAD possessed excellent catalytic activity and stability in various organic solvents. Whole Escherichia coli cells harboring intracellular BaPAD exhibited greater tolerances to FA and 4-VG than those of free BaPAD. A highly efficient aqueous-organic biphasic system was established using 1-octanol as the optimal organic phase for whole-cell catalysis. In this system, a very high concentration (1580 mM, 237.3 g/L) of 4-VG was achieved in a 2 L working volume bioreactor, and the molar conversion yield and productivity reached 98.9% and 18.3 g/L/h in 13 h, respectively.

Expression and characterization of two glucuronoyl esterases from Thielavia terrestris and their application in enzymatic hydrolysis of corn bran.

The thermophilic fungus Thielavia terrestris when cultured on cellulose produces a cocktail of thermal hydrolases with potential application in saccharification of lignocellulosic biomass and other biotechnological areas. Glucuronoyl esterases are considered to play a unique role as accessory enzymes in lignocellulosic material biodegradation by cleaving the covalent ester linkage between 4-O-methyl-D-glucuronic acid (MeGlcA) and lignin in lignin-carbohydrate complexes (LCCs). Two glucuronoyl esterases from T. terrestris named TtGE1 and TtGE2 were expressed in Pichia pastoris. Both esterases displayed features of thermophilic enzymes, with the optimal temperature at 45 °C and 55 °C. TtGE1 and TtGE2 exhibited activity towards methyl (4-nitrophenyl ß-D-glucopyranosid) uronate (Me-GlcA-pNP) but no catalytic activity to benzyl-D-glucuronate (BnzGlcA), indicating the difference in substrate specificity from previously studied fungal GEs. A substantial increase in the release of monomeric sugars and glucuronic acid from autohydrolysis of corn bran was observed by the supplementing TtGEs into commercial xylanase; the results clearly demonstrated that the TtGEs played a significant role in this degradation process. This research on TtGEs enriches our knowledge of this novel class of fungal GEs. These newly characterized TtGEs could be used as promising accessory enzymes to improve the hydrolysis efficiency of commercial enzymes in saccharification of lignocellulosic materials due to their thermophilic characteristics.

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties.

BACKGROUND: The non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited. RESULTS: Eight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1. CONCLUSIONS: Our results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.