Recombinant production and characterization of an N-acyl-d-amino acid amidohydrolase from Streptomyces sp. 64E6

N-Acyl-d-amino acid amidohydrolases (d-aminoacylases) are often used as tools for the optical resolution of d-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding d-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-)d-Phe (0.05–6.32 μmol min^?1 mg^?1) under conditions without CoCl₂. Addition of 1 mM CoCl₂ enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the d-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 μmol min^?1 mg^?1 for N-Ac-d-Phe and N-Ac-d-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. d-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0–9.0. It was stable at pH 5.5–9.0 up to 30 °C. The enzyme hydrolyzed various N-acetyl-d-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl-d-Phe was 2.1-fold higher than that toward N-Ac-d-Phe, indicating that the structure of N-acylated portion of substrate altered the activity.     

Transport of l-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 μM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate l-[³H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H⁺ gradient-stimulated uptake of l-[³H]proline. Uptake was rapid, independent of a Na⁺ gradient but stimulated by an inwardly directed H⁺ gradient with maximal uptake occurring at pH 6.0. l-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K_t) of 130 ± 10 μM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min^?1 mg of protein^?1. Glycine, l-alanine, and l-tryptophan strongly inhibited l-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H⁺-amino acid cotransport system PAT2.     

First Thermostable Endo-β-1,4-Glucanase from Newly Isolated Xanthomonas sp. EC102

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 μmol min^?1 mg^?1, V _max for CMC was 1.44 μmol mg^?1 min^?1, and K _m values was 25.6 mg mL^?1. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²⁺ and Co²⁺. This is the first report about thermostable endoglucanase from Xanthomonas sp.     

Characterization of a novel GH2 family α-l-arabinofuranosidase from hyperthermophilic bacterium Thermotoga thermarum

The 2,367-bp ORF of TtAFase from Thermotoga thermarum DSM 5069 encodes a calculated 90-kDa α-l-arabinofuranosidase (TtAFase), which does not belonging to any reported glycosyl hydrolase families α-l-arabinofuranosidases in the database and represents a novel one of glycosyl hydrolase family 2. The purified recombinant TtAFase produced in Escherichia coli BL21 (DE3) had optimum activity at pH 5.5 and at 80 °C. It was stable up to 80 °C and from pH 4.5–8.5. Kinetic experiments at 80 °C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a K _m of 0.77 mM, V _max of 2.3 μmol mg^?1 min^?1 and k _cat of 4.5 s^?1. The enzyme had no apparent requirement of metal ions for activity, and its activity was significantly inhibited by Cu²⁺ or Zn²⁺.     

Characterization of a d-psicose-producing enzyme, d-psicose 3-epimerase, from Clostridium sp.

The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co²⁺ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the K _m, turnover number (k _cat), and catalytic efficiency (k _cat/K _m) for d-psicose were 227 mM, 32,185 min^?1, and 141 min^?1 mM^?1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l^?1 was produced from 500 g d-fructose l^?1 after 5 h.     

Catalytic mechanism of serine racemase from Dictyostelium discoideum

The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of d- and l-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to l-serine dehydrase; S81A showed no racemase activity and had significantly reduced d-serine dehydrase activity, but it completely retained its l-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove d-serine dehydration by abstracting the α-hydrogen in d-serine. Our data suggest that the abstraction and addition of α-hydrogen to l- and d-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.     

Synthesis of β-alanine from l-aspartate using l-aspartate-α-decarboxylase from Corynebacterium glutamicum

β-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding l-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADC _C.g catalyzes the α-decarboxylation of l-aspartate to β-alanine. The purified ADC _C.g was optimal at 55 °C and pH 6 with excellent stability at 16–37 °C and pH 4–7. A pH–stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep the pH value within 6–7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g l-aspartate/l and another three feedings of 0.5 % (w/v) l-aspartate at 8 h intervals. The final β-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of β-alanine by using ADC.     

Bioconversion of ginsenoside Rc into Rd by a novel α-l-arabinofuranosidase, Abf22-3 from Leuconostoc sp. 22-3: cloning, expression, and enzyme characterization

A novel α-l-arabinofuranosidase (Abf22-3) that could biotransform ginsenoside Rc into Rd was obtained from the ginsenoside converting Leuconostoc sp. strain 22-3, isolated from the Korean fermented food kimchi. The gene, termed abf22-3, consisting of 1,527 bp and encoding a protein with a predicted molecular mass of 58,486 Da was cloned into the pMAL-c2x (TEV) vector. A BLAST search using the Abf22-3’s amino acid sequence revealed significant homology to that of family 51 glycoside hydrolases. The over-expressed recombinant Abf22-3 in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinofuranoside moiety attached to the C-20 position of ginsenoside Rc under optimal conditions of pH 6.0 and 30 °C. This result indicated that Abf22-3 selectively converts ginsenoside Rc into Rd, but did not catalyze the hydrolysis of glucopyranosyl groups from Rc or other ginsenosides such as Rb₁ and Rb₂. Over-expressed recombinant enzymes were purified by two steps with amylose-affinity and DEAE-cellulose chromatography and then characterized. The kinetic parameters for α-l-arabinofuranosidase showed apparent K_m and V_max values of 0.95 ± 0.02 μM and 1.2 ± 0.1 μmol min^?1 mg of protein^?1 against p-nitrophenyl-α-l-arabinofuranoside, respectively. Using a purified MBP–Abf22-3 (10 μg/ml), 0.1 % of ginsenoside Rc was completely converted to ginsenoside Rd within 20 min.     

Ontogeny of hypoxic modulation of cardiac performance and its allometry in the African clawed frog Xenopus laevis

The ontogeny of cardiac hypoxic responses, and how such responses may be modified by rearing environment, are poorly understood in amphibians. In this study, cardiac performance was investigated in Xenopus laevis from 2 to 25 days post-fertilization (dpf). Larvae were reared under either normoxia or moderate hypoxia (PO₂ = 110 mmHg), and each population was assessed in both normoxia and acute hypoxia. Heart rate (f _h ) of normoxic-reared larvae exhibited an early increase from 77 ± 1 beats min^?1 at 2 dpf to 153 ± 1 beats min^?1 at 4 dpf, followed by gradual decreases to 123 ± 3 beats min^?1 at 25 dpf. Stroke volume (SV), 6 ± 1 nl, and cardiac output (CO), 0.8 ± 0.1 μl min^?1, at 5 dpf both increased by more than 40-fold to 25 dpf with rapid larval growth (~30-fold increase in body mass). When exposed to acute hypoxia, normoxic-reared larvae increased f _h and CO between 5 and 25 dpf. Increased SV in acute hypoxia, produced by increased end-diastolic volume (EDV), only occurred before 10 dpf. Hypoxic-reared larvae showed decreased acute hypoxic responses of EDV, SV and CO at 7 and 10 dpf. Over the period of 2–25 dpf, cardiac scaling with mass showed scaling coefficients of ?0.04 (f _h ), 1.23 (SV) and 1.19 (CO), contrary to the cardiac scaling relationships described in birds and mammals. In addition, f _h scaling in hypoxic-reared larvae was altered to a shallower slope of ?0.01. Collectively, these results indicate that acute cardiac hypoxic responses develop before 5 dpf. Chronic hypoxia at a moderate level can not only modulate this cardiac reflex, but also changes cardiac scaling relationship with mass.     

Characterization of ribose-5-phosphate isomerase converting d-psicose to d-allose from Thermotoga lettingae TMO

The gene coding for ribose-5-phosphate isomerase (Rpi) from Thermotoga lettingae TMO was cloned and expressed in E. coli. The recombinant enzyme was purified by Ni-affinity chromatography. It converted d-psicose to d-allose maximally at 75 °C and pH 8.0 with a 32 % conversion yield. The k _m, turnover number (k _cat), and catalytic efficiency (k _cat k _m ^?1 ) for substrate d-psicose were 64 mM, 6.98 min^?1 and 0.11 mM^?1 min^?1 respectively.     

Characterization and constitutive expression of an acidic mesophilic endo-1,4-β-d-xylanohydrolase with high thermotolerance and catalytic efficiency in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl11 from Aspergillus niger, encoding a 188-residue xylanase of glycosyl hydrolase family 11, was constitutively expressed in Pichia pastoris. The recombinant Xyl11 exhibited optimal activity at pH 5.0 and 50 °C, and displayed more than 68 % of the maximum activity over the temperature range 35–65 °C and 33 % over the pH range 2.2–7.0. It maintained more than 40 % of the original activity after incubation at 90 °C (pH 5.0) for 10 min and more than 75 % of the original activity after incubation at pH 2.2–11.0 (room temperature) for 2 h. The specific activity, K _m and V _max of purified Xyl11 were 22,253 U mg^?1, 6.57 mg ml^?1 and 51,546.4 μmol min^?1 mg^?1. It could degrade xylan to a series of xylooligosaccharides and no xylose was detected. The recombinant enzyme with high stability and catalytic efficiency could work over wide ranges of pH and temperature and thus has the potential for various industrial applications.     

Characterization and constitutive expression of a novel endo-1,4-β-d-xylanohydrolase from Aspergillus niger in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl10 from Aspergillus niger, encoding a 308-residue mature xylanase belonging to glycosyl hydrolase family 10, was constitutively expressed in Pichia pastoris. The recombinant Xyl10 exhibited optimal activity at pH 5.0 and 60 °C with more than 50 % of the maximum activity from 40 to 70 °C. It retained more than 90 % of the original activity after incubation at 60 °C (pH 5.0) for 30 min and more than 74 % after incubation at pH 3.0–13.0 for 2 h (25 °C). The specific activity, K _m and V _max values for purified Xyl10 were, respectively, 3.2 × 10³ U mg^?1, 3.6 mg ml^?1 and 5.4 × 10³ μmol min^?1 mg^?1 towards beechwood xylan. The enzyme degraded xylan to a series of xylooligosaccharides and xylose. The recombinant enzyme with these properties has the potential for various industrial applications.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium

The l-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of d-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting l-alanine to the germination inhibitor d-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of l- to d-alanine. Unlike ATCC 14579 spores, l-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.     

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

Alanine racemase catalyzes the interconversion of d- and l-alanine and plays an important role in supplying d-alanine, a component of peptidoglycan biosynthesis, to most bacteria. Alanine racemase exists mostly in prokaryotes and is generally absent in higher eukaryotes; this makes it an attractive target for the design of new antibacterial drugs. Here, we present the cloning and characterization of a new gene-encoding alanine racemase from Pseudomonas putida YZ-26. An open reading frame (ORF) of 1,230 bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44,217.3 Da, was cloned into modified vector pET32M to form the recombinant plasmid pET–alr. After introduction into E.coli BL21, the strain pET-alr/E.coli BL21 expressed His₆-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni²⁺–NTA and a gel filtration column, with 82.5% activity recovery. The amino acid sequence deduced from the alanine racemase gene revealed identity similarities of 97.0, 93, 23, and 22.0% with from P. putida F1, P. putida200, P. aeruginosa, and Salmonella typhimurium, respectively. The recombinant alanine racemase is a monomeric protein with a molecular mass of 43 kDa. The enzyme exhibited activity with l-alanine and l-isoleucine, and showed higher specificity for the former compared with the latter. The enzyme was stable from pH 7.0–11.0; its optimum pH was at 9.0. The optimum temperature for the enzyme was 37°C, and its activity was rapidly lost at temperatures above 40°C. Divalent metals, including Sr²⁺, Mn²⁺, Co²⁺, and Ni²⁺ obviously enhanced enzymatic activity, while the Cu²⁺ ion showed inhibitory effects.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

Heterologous expression of β-xylosidase gene from Paecilomyces thermophila in Pichia pastoris

β-xylosidase from thermophilic fungi Paecilomyces thermophila was functionally expressed in Pichia pastoris with a his tag in the C-terminal under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 0.22 mg l^?1. Its molecular mass was estimated to be 52.3 kDa based on the SDS-PAGE analysis, which is 1.3 times higher than the predicted 39.31 kDa from its amino acid compositions, although no potential N- or O- glycosylation sites were predicted from its amino acid sequence. This is presumed to be caused by some unpredictable posttranslational modifications based on mass spectrum analysis of the recombinant protein. The enzyme was most active at 60 °C and pH 7. It showed not only a β-xylosidase activity with a K_m of 8 mM and a V_max of 54 μmol min^?1 mg^?1 for hydrolysis of p-nitrophenyl β-d-xylopyranoside but also an arabinofuranosidase activity (6.2 U mg^?1) on p-nitrophenyl arabinofuranoside.     

Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif ‘GXSXG’. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K _m and V _max of enzyme was determined to be 21.29 U mg^?1 protein, 714.28 μM and 62.5 μmol ml^?1 min^?1, respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser⁵⁰, Asp¹⁸⁰ and His²⁰³ residues by site-directed mutagenesis.     

Biochemical and thermodynamic characteristics of thermo-alkali-stable xylanase from a novel polyextremophilic Bacillus halodurans TSEV1

The purified extracellular xylanase of polyextremophilic Bacillus halodurans TSEV1 has been visualized as a single band on SDS-PAGE and eluted as single peak by gel filtration, with a molecular mass of 40 kDa. The peptide finger print and cloned xylanase gene sequence analyses indicate that this enzyme belongs to GH family 10. The active site carboxyl residues are mainly involved in catalysis, while tryptophan residues are involved in substrate binding. The enzyme is optimally active at 80 °C and pH 9.0, and stable in the pH range of 7.0–12.0 with T _1/2 of 35 min at 80 °C (pH 9.0). Activation energy for birch wood xylan hydrolysis is 30.51 kJ mol^?1. The K _m, V _max and k _cat (birchwood xylan) are 2.05 mg ml^?1, 333.33 μmol mg^?1 min^?1 and 3.33 × 10⁴ min^?1, respectively. The pKa₁ and pKa₂ of ionizable groups of the active site that influence V _max are 8.51 and 11.0. The analysis of thermodynamic parameters for xylan hydrolysis suggests this as a spontaneous process. The enzyme is resistant to chemical denaturants like urea and guanidinium-HCl. The site-directed mutagenesis of catalytic glutamic acid residues (E196 and E301) resulted in a complete loss of activity. The birch wood xylan hydrolyzate contained xylobiose and xylotriose as the main products without any trace of xylose, and the enzyme hydrolyzes xylotetraose and xylopentaose rapidly to xylobiose. Thermo-alkali-stability, resistance to various chemical denaturants and mode of action make it a useful biocatalyst for generating xylo-oligosaccharides from agro-residues and bleaching of pulp in paper industries.     

In vitro and in vivo analyses of the role of the carboxysomal β-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 μmol mg^?1 protein min^?1) was only 30 % of that observed for the wild-type carboxysomes (0.71 μmol mg^?1 protein min^?1), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO₂ in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO₂ for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO₂ by the cytoplasmically localized Rubisco by counteracting the action of the CO₂ hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO₂ fixation by the cyanobacterium at CO₂ levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.