Identification and characterization of a novel AA9-type lytic polysaccharide monooxygenase from a bagasse metagenome.

Lytic polysaccharide monooxygenases (LPMOs) are auxiliary enzymes catalyzing oxidative cleavages of cellulose chains in crystalline regions, resulting in their increasing accessibility to the hydrolytic enzyme counterparts and hence higher released sugars from biomass saccharification. In this study, a novel auxiliary protein family 9 LPMO (BgAA9) was identified from a metagenomic library derived from a thermophilic microbial community in bagasse collection site where diverse AA9 and AA10 putative sequences were annotated. The enzyme showed highest similarity to a glycoside hydrolase family 61 from Chaetomium thermophilum. Recombinant BgAA9 expressed in Pichia pastoris cleaved cellohexaose (DP6) into shorter cellooligosaccharides (DP2, DP3, and DP4). Supplementation BgAA9 to a commercial cellulase, Accellerase® 1500 showed strong synergistic effect on saccharification of Avicel® PH101, decrystallized cellulose, filter paper, and alkaline-pretreated sugarcane bagasse, resulting in 63-93% increase in the total reducing sugar yield after incubation at 50 °C for 72 h. Strong synergism was shown between BgAA9 and the cellulase with the highest total fermentable sugar yield obtained from 75:25% of Accellerase®1500:BgAA9 which released 39 mg glucose/FPU (filter paper unit) equivalent to 38.7% higher than Accellerase®1500 alone at the same total protein dosage of 5 mg/g substrate according to the mixture design study. The enzyme represented the first characterized LPMO from environmental metagenome and a potent auxiliary component for biomass saccharification. KEY POINTS: • BgAA9 represents the first characterized LPMO from metagenome. • 12 AA families were annotated in thermophilic bagasse fosmid library by NGS. • BgAA9 showed homology to Cel61 in Chaetomium thermophilum. • BgAA9 oxidized cellohexaose and PASC to DP2, DP4, and DP6. • BgAA9 showed strong synergism to Accellerase on bagasse hydrolysis.

Biochemical characterization of xylanase GH11 isolated from Aspergillus niger BCC14405 (XylB) and its application in xylooligosaccharide production.

OBJECTIVE: To develop an endo-ß-1,4-xylanase with high specificity for production of prebiotic xylooligosaccharides that optimally works at moderate temperature desirable to reduce the energy cost in the production process. RESULTS: The xylB gene, encoding for a glycosyl hydrolase family 11 xylanase from a thermoresistant fungus, Aspergillus niger BCC14405 was expressed in a methylotrophic yeast P. pastoris KM71 in a secreted form. The recombinant XylB showed a high specific activity of 3852 and 169 U mg-1 protein on beechwood xylan and arabinoxylan, respectively with no detectable side activities against different forms of cellulose (Avicel Ò PH101 microcrystalline cellulose, phosphoric acid swollen cellulose and carboxymethylcellulose). The enzyme worked optimally at 45 °C, pH 6.0. It showed a specific cleavage pattern by releasing xylobiose (X2) as the major product from xylooligosaccharides (X3 to X6) substrates. The highest XOS yield of 708 mg g-1 substrate comprising X2, X3 and X6 was obtained from beechwood xylan hydrolysis. CONCLUSION: The enzyme is potent for XOS production and for saccharification of lignocellulosic biomass.

Characterization of cold-active trehalose synthase from Pseudarthrobacter sp. for trehalose bioproduction.

Trehalose is a functional sugar that has numerous applications in food, cosmetic, and pharmaceutical products. Production of trehalose from maltose via a single-step enzymatic catalysis using trehalose synthase (TreS) is a promising method compared with the conventional two-step process due to its simplicity with lower formation of byproducts. In this study, a cold-active trehalose synthase (PaTreS) from Pseudarthrobacter sp. TBRC 2005 was heterologously expressed and characterized. PaTreS showed the maximum activity at 20 °C and maintained 87% and 59% of its activity at 10 °C and 4 °C, respectively. The enzyme had remarkable stability over a board pH range of 7.0-9.0 with the highest activity at pH 7.0. The activity was enhanced by divalent metal ions (Mg2+, Mn2+ and Ca2+). Conversion of high-concentration maltose syrup (100-300 g/L) using PaTreS yielded 71.7-225.5 g/L trehalose, with 4.5-16.4 g/L glucose as a byproduct within 16 h. The work demonstrated the potential of PaTreS as a promising biocatalyst for the development of low-temperature trehalose production, with the advantages of reduced risk of microbial contamination with low generation of byproduct.

Profiling multi-enzyme activities of Aspergillus niger strains growing on various agro-industrial residues.

Agro-industrial wastes provide potential sources of carbon for production of fungal enzymes applied for various biotechnological applications. In this study, 23 strains of Aspergillus niger were systematically investigated for their capability on production of carbohydrate-processing enzymes used in industries. The strains were grown on glucose or selected agricultural wastes comprising varied chemical compositions as the sole carbon source. As a control, glucose induced basal activities of amylase, pectinase, and xylanase in only a few strains, while the CMCase, ß-glucanase, and invertase activities were detected only when the carbon source was switched to the agro-industrial biomass. According to one-way ANOVA analysis, banana peels containing lignocellulosic components with high pectin and starch contents with its easily digestible nature, were found to be the best carbon source for inducing production of most target enzymes, while the cellulose-rich sugarcane bagasse efficiently promoted maximal levels of ß-glucanase and xylanase activities. The starch fiber-rich cassava pulp also effectively supported the activities of amylase and most other enzymes, but at relatively lower levels compared to those obtained with banana peel. The A. niger TL11 strain was considered the most potent strain for production of all target enzymes with the CMCase, xylanase, pectinase, ß-glucanase, amylase, and invertase activities of 76.15, 601.59, 160.89, 409.20, 426.73, and 1186.94 U/mL, respectively. The results provide insights into the efficiency of various carbon sources with different chemical compositions on inducing the target enzymes as well as the dissimilarity of A. niger strains on the production of different carbohydrate-processing enzymes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03086-y.

A novel low temperature active maltooligosaccharides-forming amylase from Bacillus koreensis HL12 as biocatalyst for maltooligosaccharide production.

Maltooligosaccharide-forming amylases (MFAses) are promising enzymes for a variety of industrial applications. In this study, a maltooligosaccharide-forming amylase (BkAmy) isolated from Bacillus koreensis HL12 was first heterologous expressed and characterized. According to structural-sequence alignment, BkAmy contained seven conserved regions which are the signature of a novel GH13 subfamily. The gene was expressed in Pichia pastoris KM71 as an extracellular protein with a volumetric activity of 3.38 U/mL culture medium after 72 h induction by 3% (w/v) of methanol. The recombinant BkAmy migrated as a single protein band with an expected size approximately of 55 kDa. BkAmy exhibited the highest catalytic activity on soluble starch with a specific activity of 42.2 U/mg at 40 °C, pH 7.0. The enzyme exhibited 65% relative activity at 30 °C, indicating its advantage on application at moderate reaction temperature desirable for energy saving and reduction of side unwanted reactions. The enzyme exhibited a specific cleavage pattern by releasing maltose (G2), maltotriose (G3) and maltotetraose (G4) from cassava starch with the highest yield of 363 mg/g substrate equivalent to 36% conversion using 40 U/g substrate at 60 min. The work demonstrates the potential of this enzyme on maltooligosaccharide production from starch to create high value-added products in starch processing industries. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03188-1.

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris.

Concerns over climate change have led to increased interest in renewable fuels in recent years. Microbial production of advanced fuels from renewable and readily available carbon sources has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the yeast Pichia pastoris, an industrial powerhouse in heterologous enzyme production, to produce the advanced biofuel isobutanol from sugarcane trash hydrolysates. Our strategy involved overexpressing a heterologous xylose isomerase and the endogenous xylulokinase to enable the yeast to consume both C5 and C6 sugars in biomass. To enable the yeast to produce isobutanol, we then overexpressed the endogenous amino acid biosynthetic pathway and the 2-keto acid degradation pathway. The engineered strains produced isobutanol at a titer of up to 48.2 ± 1.7 mg/L directly from a minimal medium containing sugarcane trash hydrolysates as the sole carbon source. To our knowledge, this is the first demonstration of advanced biofuel production using agricultural waste-derived hydrolysates in the yeast P. pastoris. We envision that our work will pave the way for a scalable route to this advanced biofuel and further establish P. pastoris as a versatile production platform for fuels and high-value chemicals.

Functional Characterization of Recombinant Endo-Levanase (LevBk) from Bacillus koreensis HL12 on Short-Chain Levan-Type Fructooligosaccharides Production.

Levan-type fructooligosaccharides (L-FOSs) are a prominent class of non-digestible oligosaccharides with potential as nutritional prebiotics. Endo-levanase, which randomly hydrolyzes ß-(2,6)-linkages in fructans, is a promising enzyme for short-chain FOS production. In this work, a recombinant levanase (LevBk) from Bacillus koreensis strain HL12 was characterized. Soluble LevBk protein was produced in Escherichia coli BL21(DE3) system at 40 mg/L of culture medium. Based on sequence and structural analysis, LevBk was classified as a member of endo-levanase in GH32 family containing N-terminal substrate binding pocket and C-terminal ß-sandwich domains. LevBk optimally worked at 45 °C, pH 6.0 with the specific activity of 2.43 U/mg. Based on enzymatic hydrolysis, short-chain L-FOSs with degree of polymerization (DP) of 2-4 were produced from hydrolysis of timothy grass levan under optimal conditions for 9-24 h. With its ability to produce L-FOSs with specific chain lengths, LevBk could be attractively applied for converting of levan containing material to high value-added sweetener in the biorefinery industry.

Enhancement of catalytic activity and alkaline stability of cellobiohydrolase by structure-based protein engineering.

Alkaline cellobiohydrolases have the potential for application in various industries, including pulp processing and laundry where operation under high pH conditions is preferred. In this study, variants of CtCel6A cellobiohydrolase from Chaetomium thermophilum were generated by structural-based protein engineering with the rationale of increasing catalytic activity and alkaline stability. The variants included removal of the carbohydrate-binding module (CBM) and substitution of residues 173 and 200. The CBM-deleted enzyme with Y200F mutation predicted to mediate conformational change at the N-terminal loop demonstrated increased alkaline stability at 60 °C, pH 8.0 for 24 h up to 2.25-fold compared with the wild-type enzyme. Another CBM-deleted enzyme with L173E mutation predicted to induce a new hydrogen bond in the substrate-binding cleft showed enhanced hydrolysis yield of pretreated sugarcane trash up to 4.65-fold greater than that of the wild-type enzyme at the pH 8.0. The variant enzymes could thus be developed for applications on cellulose hydrolysis and plant fiber modification operated under alkaline conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03339-4.

Insights into the phylogeny and metabolic potential of a primary tropical peat swamp forest microbial community by metagenomic analysis.

A primary tropical peat swamp forest is a unique ecosystem characterized by long-term accumulation of plant biomass under high humidity and acidic water-logged conditions, and is regarded as an important terrestrial carbon sink in the biosphere. In this study, the microbial community in the surface peat layer in Pru Toh Daeng, a primary tropical peat swamp forest, was studied for its phylogenetic diversity and metabolic potential using direct shotgun pyrosequencing of environmental DNA, together with analysis of 16S rRNA gene library and key metabolic genes. The community was dominated by aerobic microbes together with a significant number of facultative and anaerobic microbial taxa. Acidobacteria and diverse Proteobacteria (mainly Alphaproteobacteria) constituted the major phylogenetic groups, with minor representation of archaea and eukaryotic microbes. Based on comparative pyrosequencing dataset analysis, the microbial community showed high metabolic versatility of plant polysaccharide decomposition. A variety of glycosyl hydrolases targeting lignocellulosic and starch-based polysaccharides from diverse bacterial phyla were annotated, originating mostly from Proteobacteria, and Acidobacteria together with Firmicutes, Bacteroidetes, Chlamydiae/Verrucomicrobia, and Actinobacteria, suggesting the key role of these microbes in plant biomass degradation. Pyrosequencing dataset annotation and direct mcrA gene analysis indicated the presence of methanogenic archaea clustering in the order Methanomicrobiales, suggesting the potential on partial carbon flux from biomass degradation through methanogenesis. The insights on the peat swamp microbial assemblage thus provide a valuable approach for further study on biogeochemical processes in this unique ecosystem.

Screening, Cloning, Expression and Characterization of New Alkaline Trehalose Synthase from Pseudomonas monteilii and Its Application for Trehalose Production.

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli. Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40°C, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

Identification and characterization of lipolytic enzymes from a peat-swamp forest soil metagenome.

In this work, a metagenomic library was generated from peat-swamp forest soil obtained from Narathiwat Province, Thailand. From a fosmid library of approximately 15,000 clones, six independent clones were found to possess lipolytic activity at acidic pH. Analysis of pyrosequencing data revealed six ORFs, which exhibited 34-71% protein similarity to known lipases/esterases. A fosmid clone, designated LP8, which demonstrated the highest level of lipolytic activity under acidic conditions and demonstrated extracellular activity, was subsequently subcloned and sequenced. The full-length lipase/esterase gene, estPS2, was identified. Its deduced amino acid was closely related to a lipolytic enzyme of an uncultured bacterium, and contained the highly conserved motif of a hormone-sensitive family IV lipase. The EstPS2 enzyme exhibited highest activity toward p-nitrophenyl butyrate (C4) at 37 °C at pH 5, indicating that it was an esterase with activity and secretion characteristics suitable for commercial development.

Designing cellulolytic enzyme systems for biorefinery: From nature to application.

Cellulolytic enzymes play a key role on conversion of lignocellulosic plant biomass to biofuels and biochemicals in sugar platform biorefineries. In this review, we survey composite carbohydrate-active enzymes (CAZymes) among groups of cellulolytic fungi and bacteria that exist under aerobic and anaerobic conditions. Recent advances in designing effective cellulase mixtures are described, starting from the most complex microbial consortium-based enzyme preparations, to single-origin enzymes derived from intensively studied cellulase producers such as Trichoderma reesei, Talaromyces cellulolyticus, and Penicellium funiculosum, and the simplest minimal enzyme systems comprising selected sets of mono-component enzymes tailor-made for specific lignocellulosic substrates. We provide a comprehensive update on studies in developing high-performance cellulases for biorefineries.

Development of tailor-made synergistic cellulolytic enzyme system for saccharification of steam exploded sugarcane bagasse.

Designing a tailor-made synergistic system is a promising strategy for developing an effective enzyme for saccharification of lignocellulosic materials. In this study, a cellulolytic enzyme mixture comprising selected core recombinant enzymes for hydrolysis of sugarcane bagasse pretreated by alkaline-catalyzed steam explosion was optimized using a mixture design approach. The optimized enzyme system comprised a cellobiohydrolase (Cel7A) from Talaromyces cellulolyticus, an endo-glucanase (Cel7B) from Thielavia terrestris, a ß-glucosidase (BGL) and an endo-ß1,4-xylanase (XYN) from Aspergillus aculeatus at the ratio of 0.34:0.27:0.14:0.25. The maximum reducing sugar yield of 797 mg/g biomass, comprising 543 and 96.8 mg/g glucose and xylose, respectively were achieved, equivalent to 92.44% and 47.50% recoveries, respectively from the pretreated substrate at the enzyme dosage of 20 mg/g biomass. The sugar yield from the quaternary enzyme mixture was 17.37% higher than that obtained with Accellerase 1500.

Binding characteristics and synergistic effects of bacterial expansins on cellulosic and hemicellulosic substrates.

Expansins are non-catalytic proteins which loosen plant cell wall structure. In this study, binding kinetics and synergistic action of five bacterial expansins on cellulosic and hemicellulosic polysaccharides were studied. The expansins differed in binding capacity (Bmax) and affinity (Kd) for different substrates. A common pattern of binding efficiency (Bmax/Kd) was found among the expansins tested, in which efficiency was greatest for the phosphoric acid-swollen cellulose (PASC), then the hemicellulose arabinoxylan followed by the microcrystalline cellulose (Avicel PH101). The expansins enhanced the action of Trichoderma reesei cellulase/hemicellulase mixture for degrading all three substrates to varying degrees. Among the substrates and expansins tested, BpEX from Bacillus pumilus and CmEX from Clavibacter michiganensis showed the greatest enhancement effect on arabinoxylan with 11.4 and 12.2-fold greater reducing sugar yield than the reaction with enzyme alone. The work gives insights into the wider application of expansins on enhancing polysaccharide hydrolysis, particularly on hemicellulosic substrates.

Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation.

Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173 gglc/FPU, higher than the binary ACR:XYL mixture (0.122 gglc/FPU) and ACR alone (0.081 gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry.

Identification of novel bacterial expansins and their synergistic actions on cellulose degradation.

Novel expansins, non-catalytic proteins which induce weakening of the rigid cellulose structure, have been identified in this study. A pipeline of bioinformatics was implemented for sequence and structure-based prediction of putative bacterial expansin-like group × family from NR databases. All putative expansins had no detectable activity against cellulosic and hemicellulosic substrates but showed varying degrees of synergy (2.0-7.6 folds) with the commercial Trichoderma reesei cellulase (Celluclast™ 1.5L) on degradation of filter paper in order of BpEX ≈ CmEX > MaEX > PcEX > SaEX. A mixture design with full cubic model predicted optimal formulation comprising Celluclast™: CmEX from Clavibacter michiganensis = 72.4%: 27.6%, with no synergy of ß-glucosidase on degradation of alkaline pretreated rice straw. Under these conditions, the reducing sugar yield was 163.6% compared with the reaction containing cellulase alone. This work demonstrated the potential benefit of novel bacterial expansins on enhancing cellulose degradation efficiency in lignocellulosic biomass degradation.

Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design.

Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation.