Optimizing bioconversion processes of rice husk into value-added products: D-psicose, bioethanol, and lactic acid.

Rice husk, rich carbon content, is an agricultural waste produced globally at an amount of 120 million tons annually, and it has high potential as a biorefinery feedstock. Herein, we investigated the feasibility of producing various products as D-psicose, bioethanol and lactic acid from rice husk (RH) through a biorefinery process. Alkali-hydrogen peroxide-acetic acid pretreatment of RH effectively removed lignin and silica, resulting in enzymatic hydrolysis yield of approximately 86.3% under optimal hydrolysis conditions. By using xylose isomerase as well as D-psicose-3-epimerase with borate, glucose present in the RH hydrolysate was converted into D-psicose with a 40.6% conversion yield in the presence of borate. Furthermore, bioethanol (85.4%) and lactic acid (92.5%) were successfully produced from the RH hydrolysate. This study confirmed the high potential of RH as a biorefinery feedstock, and it is expected that various platform chemicals and value-added products can be produced using RH.

Succinic acid production from softwood with genome-edited Corynebacterium glutamicum using the CRISPR-Cpf1 system.

Corynebacterium glutamicum is a useful microbe that can be used for producing succinic acid under anaerobic conditions. In this study, we generated a knock-out mutant of the lactate dehydrogenase 1 gene (ΔldhA-6) and co-expressed the succinic acid transporter (Psod:SucE- ΔldhA) using the CRISPR-Cpf1 genome editing system. The highly efficient HPAC (hydrogen peroxide and acetic acid) pretreatment method was employed for the enzymatic hydrolysis of softwood (Pinus densiflora) and subsequently utilized for production of succinic acid. Upon evaluating a 1%-5% hydrolysate concentration range, optimal succinic acid production with the ΔldhA mutant was achieved at a 4% hydrolysate concentration. This resulted in 14.82 g L-1 succinic acid production over 6 h. No production of acetic acid and lactic acid was detected during the fermentation. The co-expression transformant, [Psod:SucE-ΔldhA] produced 17.70 g L-1 succinic acid in 6 h. In the fed-batch system, 39.67 g L-1 succinic acid was produced over 48 h. During the fermentation, the strain consumed 100% and 73% of glucose and xylose, respectively. The yield of succinic acid from the sugars consumed was approximately 0.77 g succinic acid/g sugars. These results indicate that the production of succinic acid from softwood holds potential applications in alternative biochemical processes.

Comparative Evaluation of Adsorption of Major Enzymes in a Cellulase Cocktail Obtained from Trichoderma reesei onto Different Types of Lignin.

Cellulase adsorption onto lignin decreases the productivity of enzymatic hydrolysis of lignocellulosic biomass. Here, adsorption of enzymes onto different types of lignin was investigated, and the five major enzymes-cellobiohydrolases (CBHs), endoglucanase (Cel7B), ß-glucosidase (Cel3A), xylanase (XYNIV), and mannanase (Man5A)-in a cellulase cocktail obtained from Trichoderma reesei were individually analyzed through SDS-PAGE and zymogram assay. Lignin was isolated from woody (oak and pine lignin) and herbaceous (rice straw and kenaf lignin) plants. The relative adsorption of CBHs compared to the control was in the range of 14.15-18.61%. The carbohydrate binding motif (CBM) of the CBHs contributed to higher adsorption levels in oak and kenaf lignin, compared to those in pine and rice lignin. The adsorption of endoglucanase (Cel7B) by herbaceous plant lignin was two times higher than that of woody lignin, whereas XYNIV showed the opposite pattern. ß-glucosidase (Cel3A) displayed the highest and lowest adsorption ratios on rice straw and kenaf lignin, respectively. Mannanase (Man5A) was found to have the lowest adsorption ratio on pine lignin. Our results showed that the hydrophobic properties of CBM and the enzyme structures are key factors in adsorption onto lignin, whereas the properties of specific lignin types indirectly affect adsorption.

Hydrolysis pattern analysis of xylem tissues of woody plants pretreated with hydrogen peroxide and acetic acid: rapid saccharification of softwood for economical bioconversion.

BACKGROUND: Woody plants with high glucose content are alternative bioresources for the production of biofuels and biochemicals. Various pretreatment methods may be used to reduce the effects of retardation factors such as lignin interference and cellulose structural recalcitrance on the degradation of the lignocellulose material of woody plants. RESULTS: A hydrogen peroxide-acetic acid (HPAC) pretreatment was used to reduce the lignin content of several types of woody plants, and the effect of the cellulose structural recalcitrance on the enzymatic hydrolysis was analyzed. The cellulose structural recalcitrance and the degradation patterns of the wood fibers in the xylem tissues of Quercus acutissima (hardwood) resulted in greater retardation in the enzymatic saccharification than those in the tracheids of Pinus densiflora (softwood). In addition to the HPAC pretreatment, the application of supplementary enzymes (7.5 FPU cellulase for 24 h) further increased the hydrolysis rate of P. densiflora from 61.42 to 91.94% whereas the same effect was not observed for Q. acutissima. It was also observed that endoxylanase synergism significantly affected the hydrolysis of P. densiflora. However, this synergistic effect was lower for other supplementary enzymes. The maximum concentration of the reducing sugars produced from 10% softwood was 89.17 g L-1 after 36 h of hydrolysis with 15 FPU cellulase and other supplementary enzymes. Approximately 80 mg mL-1 of reducing sugars was produced with the addition of 7.5 FPU cellulase and other supplementary enzymes after 36 h, achieving rapid saccharification. CONCLUSION: HPAC pretreatment removed the interference of lignin, reduced structural recalcitrance of cellulose in the P. densiflora, and enabled rapid saccharification of the woody plants including a high concentration of insoluble substrates with only low amounts of cellulase. HPAC pretreatment may be a viable alternative for the cost-efficient production of biofuels or biochemicals from softwood plant tissues.

Development of an advanced integrative process to create valuable biosugars including manno-oligosaccharides and mannose from spent coffee grounds.

Spent coffee grounds (SCG) or coffee residue wastes (CRW) provide excellent raw material for mannose and bioethanol production. In this study, SCG were used to produce valuable biosugars, including oligosaccharides (OSs), manno-oligosaccharides (MOSs), mannose, and bioethanol. SCG were subjected to delignification and defatting, producing SCG-derived polysaccharides. Two-stage enzymatic hydrolysis (short- and long-term) was performed to produce short-chain manno-oligosaccharides (MOSs) and monosaccharides (MSs), respectively. From 100 g dry weight (DW) amounts of SCG, approximately 77 g delignified SCG and 61 g SCG-derived polysaccharides, amounts of 15.9 g of first biosugars (mostly MOSs), 25.6 g of second biosugars (mostly MSs), and 3.1 g of bioethanol, were recovered. This technique may aid in the production of high-value mannose and OSs from SCG and other lignocellulosic biomasses that contain specific polysaccharides.

Evaluation of biomass quality in short-rotation bamboo (Phyllostachys pubescens) for bioenergy products.

BACKGROUND: In order to improve the availability of biomass, the concept of growing high yield biomass with short rotations and intensive culture has been introduced. Bamboo has become a feedstock of potential interest for future energy production due to its high productivity and short rotation time. The growth age of biomass is an important factor affecting the efficiency of bioconversion and pretreatment for bioenergy production. In this regard, more information is required on the morphology and chemical composition of bamboo for short-rotation biomass production. In this study, we used a compositional assay to compare a bamboo of two different growth ages. RESULTS: Bamboo of two different ages showed characteristics patterns of morphology, chemical composition, and bioconversion. In young-age (2-month-old) bamboo, the pattern of tissue organization was similar to that of old-age (3-year-old) bamboo, indicating that the former had reached its full height. There were significant differences between young-age and old-age bamboo in terms of chemical composition. The glucose contents in old-age bamboo did not differ significantly among its internodes. For young-age bamboo, the lignin contents were 14.6-18.3%, whereas those of old-age bamboo were considerably higher, ranging from 25.4 to 27.1% with increasing syringyl-to-guaiacyl ratio. The yield of total sugars following enzymatic hydrolysis of young-age bamboo was approximately eight times. However, following hydrogen peroxide-acetic acid pretreatment, the results of separate hydrolysis and fermentation and simultaneous saccharification and fermentation did not differ significantly between young- and old-age bamboo. However, ethanol production was higher in 2-month old than in 3-year old from initial raw biomass. CONCLUSION: Our data show that the production of total sugar from raw material was high in young bamboo with low lignin content. With respect to short-rotation biomass, bamboo culm harvested after termination of height growth is more appropriate for use as a biomass resource to achieve a high yield for bioconversion process.

Production of bio-sugar and bioethanol from coffee residue (CR) by acid-chlorite pretreatment.

Nowadays, coffee residue (CR) after roasting is recognized as one of the most useful resources in the world for producing the biofuel and bio-materials. In this study, we evaluated the potential of bio-sugar and bioethanol production from acid-chlorite treated CR. Notably, CR treated three times with acid-chlorite after organic solvent extraction (OSE-3), showed the high monosaccharide content, and the efficient sugar conversion yield compared to the other pretreatment conditions. The OSE-3 (6% substrate loading, w/v) can produce bio-sugar (0.568g/g OSE-3). Also, simultaneous saccharification and fermentation (SSF) produced ethanol (0.266g/g OSE-3), and showed an ethanol conversion yield of 73.8% after a 72-h reaction period. These results suggest that acid-chlorite pretreatment can improve the bio-sugar and bioethanol production of CR by removing the phenolic and brown compounds.

Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Vacuole-Targeted Thermotoga maritima BglB Related to Elevated Levels of Liberated Hormones.

The hyperthermostable ß-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified ß-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the ß-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production.

Lignocellulose conversion for biofuel: a new pretreatment greatly improves downstream biocatalytic hydrolysis of various lignocellulosic materials.

BACKGROUND: Lignocellulosic biomass is an attractive renewable resource for future liquid transport fuel. Efficient and cost-effective production of bioethanol from lignocellulosic biomass depends on the development of a suitable pretreatment system. The aim of this study is to investigate a new pretreatment method that is highly efficient and effective for downstream biocatalytic hydrolysis of various lignocellulosic biomass materials, which can accelerate bioethanol commercialization. RESULTS: The optimal conditions for the hydrogen peroxide-acetic acid (HPAC) pretreatment were 80 °C, 2 h, and an equal volume mixture of H2O2 and CH3COOH. Compared to organo-solvent pretreatment under the same conditions, the HPAC pretreatment was more effective at increasing enzymatic digestibility. After HPAC treatment, the composition of the recovered solid was 74.0 % cellulose, 20.0 % hemicelluloses, and 0.9 % lignin. Notably, 97.2 % of the lignin was removed with HPAC pretreatment. Fermentation of the hydrolyzates by S. cerevisiae resulted in 412 mL ethanol kg(-1) of biomass after 24 h, which was equivalent to 85.0 % of the maximum theoretical yield (based on the amount of glucose in the raw material). CONCLUSION: The newly developed HPAC pretreatment was highly effective for removing lignin from lignocellulosic cell walls, resulting in enhanced enzymatic accessibility of the substrate and more efficient cellulose hydrolysis. This pretreatment produced less amounts of fermentative inhibitory compounds. In addition, HPAC pretreatment enables year-round operations, maximizing utilization of lignocellulosic biomass from various plant sources.

Pronounced Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Sucrose Synthase May Reveal a Novel Sugar Signaling Pathway.

Soluble sugars not only serve as nutrients, but also act as signals for plant growth and development, but how sugar signals are perceived and translated into physiological responses in plants remains unclear. We manipulated sugar levels in transgenic plants by overexpressing sucrose synthase (SuSy), which is a key enzyme believed to have reversible sucrose synthesis and sucrose degradation functions. The ectopically expressed SuSy protein exhibited sucrose-degrading activity, which may change the flux of sucrose demand from photosynthetic to non-photosynthetic cells, and trigger an unknown sucrose signaling pathway that lead to increased sucrose content in the transgenic plants. An experiment on the transition from heterotrophic to autotrophic growth demonstrated the existence of a novel sucrose signaling pathway, which stimulated photosynthesis, and enhanced photosynthetic synthesis of sucrose, which was the direct cause or the sucrose increase. In addition, a light/dark time treatment experiment, using different day length ranges for photosynthesis/respiration showed the carbohydrate pattern within a 24-h day and consolidated the role of sucrose signaling pathway as a way to maintain sucrose demand, and indicated the relationships between increased sucrose and upregulation of genes controlling development of the shoot apical meristem (SAM). As a result, transgenic plants featured a higher biomass and a shorter time required to switch to reproduction compared to those of control plants, indicating altered phylotaxis and more rapid advancement of developmental stages in the transgenic plants.

Rapid saccharification for production of cellulosic biofuels.

The economical production of biofuels is hindered by the recalcitrance of lignocellulose to processing, causing high consumption of processing enzymes and impeding hydrolysis of pretreated lignocellulosic biomass. We determined the major rate-limiting factor in the hydrolysis of popping pre-treated rice straw (PPRS) by examining cellulase adsorption to lignin and cellulose, amorphogenesis of PPRS, and re-hydrolysis. Based on the results, equivalence between enzyme loading and the open structural area of cellulose was required to significantly increase productive adsorption of cellulase and to accelerate enzymatic saccharification of PPRS. Amorphogenesis of PPRS by phosphoric acid treatment to expand open structural area of the cellulose fibers resulted in twofold higher cellulase adsorption and increased the yield of the first re-hydrolysis step from 13% to 46%. The total yield from PPRS was increased to 84% after 3h. These results provide evidence that cellulose structure is one of major effects on the enzymatic hydrolysis.

Efficient function and characterization of GH10 xylanase (Xyl10g) from Gloeophyllum trabeum in lignocellulose degradation.

The xylanase gene from Gloeophyllum trabeum was cloned and expressed in Pichia pastoris GS115. Xyl10g has a molecular weight of approximately 50kDa, and exhibits maximum specific activity at 70°C and a broad range of pH 4.0-7.0. Purified recombinant Xyl10g efficiently degraded popping-pretreated corn stover and newspaper waste at 50°C and pH 4.0 after 24h, and showed synergistic effects with Cel5B (endoglucanase) and BglB (ß-glucosidase) to increase reduced sugar levels by about 1.71- to 1.88-fold and 2.26- to 2.48-fold, respectively. Although Xyl10g has low specific activity for beechwood xylan, as compared to XynA, Xyl10g more efficiently degraded corn stover than did XynA. According to immunogold labeling analysis, Xyl10g can attack highly substituted, unsubstituted, and low-substituted xylans, whereas XynA cannot efficiently attack highly substituted xylans, which is important for lignocellulose degradation. These results suggest that GH10 Xyl10g can be used for lignocellulose degradation.

Improved recombinant cellulase expression in chloroplast of tobacco through promoter engineering and 5' amplification promoting sequence.

Economical production of bioethanol from lignocellulosic biomass still faces many technical limitations. Cost-effective production of fermentable sugars is still not practical for large-scale production of bioethanol due to high costs of lignocellulolytic enzymes. Therefore, plant molecular farming, where plants are used as bioreactors, was developed for the mass production of cell wall degrading enzymes that will help reduce costs. Subcellular targeting is also potentially more suitable for the accumulation of recombinant cellulases. Herein, we generated transgenic tobacco plants (Nicotiana tabacum cv. SR1) that accumulated Thermotoga maritima BglB cellulase, which was driven by the alfalfa RbcsK-1A promoter and contained a small subunit of the rubisco complex transit peptide. The generated transformants possessed high specific BglB activity and did not show any abnormal phenotypes. Furthermore, we genetically engineered the RbcsK-1A promoter (MRbcsK-1A) and fused the amplification promoting sequence (aps) to MRbcsK-1A promoter to obtain high expression of BglB in transgenic plants. AMRsB plant lines with aps-MRbcsK-1A promoter showed the highest specific activity of BglB, and the accumulated BglB protein represented up to 9.3 % of total soluble protein. When BglB was expressed in Arabidopsis and tobacco plants, the maximal production capacity of recombinant BglB was 0.59 and 1.42 mg/g wet weight, respectively. These results suggests that suitable recombinant expression of cellulases in subcellular compartments such as chloroplasts will contribute to the cost-effective production of enzymes, and will serve as the solid foundation for the future commercialization of bioethanol production via plant molecular farming.

Characterization and pH-dependent substrate specificity of alkalophilic xylanase from Bacillus alcalophilus.

The gene of endo-beta-1-4 xylanase, xynT, was cloned from Bacillus alcalophilus AX2000 and expressed in Escherichia coli. This XynT, which belongs to glycoside hydrolase (GH) family 10, was found to have a molecular weight of approximately 37 kDa and exhibit optimal activity at pH 7-9 and 50 °C. It exhibits a high activity towards birchwood xylan and has the ability to bind avicel. Under optimal conditions, XynT hydrolyzes all xylooligomers into xylobiose as an end product with a preference for cleavage sites at the second or third glycosidic bond from the reducing end. XynT has a different substrate affinity on xylooligomers at pH 5.0, which contributes to its low activity toward xylotriose and its derived intermediate products. This low activity may be due to an unstable interaction with the amino acids that constitute subsites of the active site. Interestingly, the addition of Co(2+) and Mn(2+) led to a significant increase in activity by up to 40 and 50 %, respectively. XynT possesses a high binding affinity and hydrolytic activity toward the insoluble xylan, for which it exhibits high activity at pH 7-9, giving rise to its efficient biobleaching effect on Pinus densiflora kraft pulp.

Synergistic effects of 2A-mediated polyproteins on the production of lignocellulose degradation enzymes in tobacco plants.

Cost-effective bioethanol production requires a supply of various low-cost enzymes that can hydrolyse lignocellulosic materials consisting of multiple polymers. Because plant-based enzyme expression systems offer low-cost and large-scale production, this study simultaneously expressed ß-glucosidase (BglB), xylanase (XylII), exoglucanase (E3), and endoglucanase (Cel5A) in tobacco plants, which were individually fused with chloroplast-targeting transit peptides and linked via the 2A self-cleaving oligopeptideex from foot-and-mouth disease virus (FMDV) as follows: [RsBglB-2A-RaCel5A], [RsXylII-2A-RaCel5A], and [RsE3-2A-RaCel5A]. The enzymes were targeted to chloroplasts in tobacco cells and their activities were confirmed. Similarly to the results of a transient assay using Arabidopsis thaliana protoplasts, when XylII was placed upstream of the 2A sequence, the [RsXylII-2A-RaCel5A] transgenic tobacco plant had a more positive influence on expression of the protein placed downstream. The [RsBglB-2A-RaCel5A] and [RsE3-2A-RaCel5A] transgenic lines displayed higher activities towards carboxylmethylcellulose (CMC) compared to those in the [RsXylII-2A-RaCel5A] transgenic line. This higher activity was attributable to the synergistic effects of the different cellulases used. The [RsBglB-2A-RaCel5A] lines exhibited greater efficiency (35-74% increase) of CMC hydrolysis when the exoglucanase CBHII was added. Among the various exoglucanases, E3 showed higher activity with the crude extract of the [RsBglB-2A-RaCel5A] transgenic line. Transgenic expression of 2A-mediated multiple enzymes induced synergistic effects and led to more efficient hydrolysis of lignocellulosic materials for bioethanol production.

Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum.

The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.

High cadmium-binding ability of a novel Colocasia esculenta metallothionein increases cadmium tolerance in Escherichia coli and tobacco.

Experimental evidence in vivo as to the functional roles and binding properties to cadmium (Cd) of type-2 plants metallothionein (MT) has been limited thus far. We investigated the biological role of metallothionein from Colocasia esculenta (CeMT2b) in Escherichia coli and tobacco, and developed a new model for the relationship between Cd tolerance and Cd-binding ability. Heterologous expression of CeMT2b in Escherichia coli greatly enhanced Cd tolerance and accumulated Cd content as compared to control cells. The molecular weight of CeMT2b increased with Cd, and CeMT2b bound up to 5.96±1 molar ratio (Cd/protein). Under Cd stress, transgenic tobacco plants displayed much better seedling growth and high Cd accumulation than the wild type. The presence of an extra CXC motif in CeMT2b contributed to the enhanced Cd-tolerance. The present study provides the first insight into the ability of type-2 plant MT to bind physiological Cd.

Characterization of a new α-L: -arabinofuranosidase from Penicillium sp. LYG 0704, and their application in lignocelluloses degradation.

A gene (arf) encoding an α-L: -arabinofuranosidase (ARF) that hydrolyzes arabinose substituted on xylan was isolated from Penicillium sp. The gene was predicted to encode 339 amino acid residues showing 71-75% homology to GH family 54. E. coli expressed ARF showed optimal activity at 50°C and pH 5-6 on wheat arabinoxylan. The hydrolysis activities on oat spelt xylan by ARF and xylanase were 1.67-fold higher than that of xylanase alone. The synergistic effects of ARF and commercial enzymes (xylanase and cellulase) on popping-pretreated rice straw were 1.15-1.51-fold higher amounts of sugars released in the [ARF + xylanase + cellulase] mixture than in the mixtures [ARF + xylanase], [ARF + cellulase], and [xylanase + cellulase]. Moreover, the liberation of arabinose by ARF was enhanced 2.1-2.9-fold in a reaction with xylanase and cellulase as compared with [xylanase + cellulase] and ARF alone.

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization.

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on D-lyxose, suggesting that the enzyme is D-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²+. The apparent K(m) values for D-lyxose and D-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k(cat)/K(m)) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for D-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of D-lyxose and D-glucose from d-xylose and D-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM D-lyxose and D-mannose, 3.7 mM and 3.8 mM D-lyxose and D-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.

Arabidopsis thaliana Rubisco small subunit transit peptide increases the accumulation of Thermotoga maritima endoglucanase Cel5A in chloroplasts of transgenic tobacco plants.

Over the past decade various approaches have been used to increase the expression level of recombinant proteins in plants. One successful approach has been to target proteins to specific subcellular sites/compartments of plant cells, such as the chloroplast. In the study reported here, hyperthermostable endoglucanase Cel5A was targeted into the chloroplasts of tobacco plants via the N-terminal transit peptide of nuclear-encoded plastid proteins. The expression levels of Cel5A transgenic lines were then determined using three distinct transit peptides, namely, the light-harvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA). RS:Cel5A transgenic lines produced highly stable active enzymes, and the protein accumulation of these transgenic lines was up to 5.2% of the total soluble protein in the crude leaf extract, remaining stable throughout the life cycle of the tobacco plant. Transmission election microscopy analysis showed that efficient targeting of Cel5A protein was under the control of the transit peptide.