Synthesis and characterization of carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrid for amylase immobilization

Carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrids have been synthesized in a modified Stöber process. The hybrid synthesis was optimized to obtain an efficient immobilization matrix for diastase alpha amylase, a multimeric enzyme of high technological significance. The synthesized hybrids were characterized using FTIR, XRD, SEM, TGA and BET studies. The enzyme immobilization was done by adsorption and using the immobilized enzyme, the hydrolysis of soluble starch has been optimized in comparison to free enzyme. The optimum usable pH for the immobilized enzyme ranged from pH 4 to 5, while pH 5 was optimum pH for the free enzyme activity. The kinetic parameters for the immobilized, (K _M = 3.4610 mg ml^?1; V _max = 6.3540 mg ml^?1 min^?1) and free enzyme (K _M = 4.1664 mg ml^?1; V _max = 4.291 mg ml^?1 min^?1) hydrolysis indicated that the immobilization at the nanohybrid has significantly improved the catalytic property of the enzyme. In the immobilized state, the enzyme remained usable for many repeated cycles like our previous material, gum acacia-gelatin-AgNp-silica. Storage experiments indicated that the immobilization has increased the stability of the enzyme and also that AgNps play a role in stabilizing the immobilized enzyme.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe₃O₄) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of K_m and V_max. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The V_max values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg^?1 min^?1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.     

Bamboo (<Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>) as a Natural Support for Neutral Protease Immobilization

Lignin polymers in bamboo (Phyllostachys pubescens) were decomposed into polyphenols at high temperatures and oxidized for the introduction of quinone groups from peroxidase extracted from bamboo shoots and catalysis of UV. According to the results of FT-IR spectra analysis, neutral proteases (NPs) can be immobilized on the oxidized lignin by covalent bonding formed by amine group and quinone group. The optimum condition for the immobilization of NPs on the bamboo bar was obtained at pH 7.0, 40 °C, and duration of 4 h; the amount of immobilized enzyme was up to 5 mg g^?1 bamboo bar. The optimal pH for both free NP (FNP) and INP was approximately 7.0, and the maximum activity of INP was determined at 60 °C, whereas FNP presented maximum activity at 50 °C. The K_m values of INP and FNP were determined as 0.773 and 0.843 mg ml^?1, respectively; INP showed a lower K_m value and V_max, than FNP, which demonstrated that INP presented higher affinity to substrate. Compared to FNP, INP showed broader thermal and storage stability under the same trial condition. With respect to cost, INP presented considerable recycling efficiency for up to six consecutive cycles.     

Development and Characterization of a Solid-Phase Biocatalyst Based on Cyclodextrin Glucantransferase Reversibly Immobilized onto Thiolsulfinate-Agarose

Reduction of disulfide bonds and introduction of ??de novo?? thiol groups in cyclodextrin glucantransferase from Thermoanaerobacter sp. were assessed in order to perform reversible covalent immobilization onto thiol-reactive supports (thiolsulfinate-agarose). Only the thiolation process dramatically improved the immobilization yield, from 0?% for the native and reduced enzyme, up to nearly 90?% for the thiolated enzyme. The mild conditions of the immobilization process (pH 6.8?C7.0 and 22?°C) allowed the achievement of 100?% coupling efficiencies when low loads were applied. Ionic strength was a critical parameter for the immobilization process; for high activity recoveries, 50?mM phosphate buffer supplemented with 0.15?M NaCl was required. The kinetic parameters, pH and thermal stabilities for the immobilized biocatalyst were similar to those for the native enzyme. For ??-cyclization activity, optimal pH range and temperature were 4.0?C5.4 and 85?°C. The possibility of reusing the support was demonstrated by the reversibility of enzyme?Csupport binding.     

Improved Covalent Immobilization of Horseradish Peroxidase on Macroporous Glycidyl Methacrylate-Based Copolymers

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g^?1 dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K _m) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.     

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties

A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50?°C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30?°C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg²⁺ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55?°C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K _m and V _max values of β-galactosidase using ONPG as substrate at 50?°C were 2.805 mM and 37.45?×?10^?3?mM/min/mg, respectively.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Design and Properties of an Immobilization Enzyme System for Inulin Conversion

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0–5.5 and 45–50 °C for the immobilized enzyme. The kinetic parameters of V _max and K _m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.     

Kinetic studies of lipase from Candida rugosa

The search for an in expensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Canada rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of oliveoil. The binding of the lipase onto the support was performed by physicalad sorption using hexane as the dispersion medium. A comparativestudy between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h (K _d=0.98 h⁻¹), whereas for the immobilized lipase it was 1.10 h (K _d=0.63 h⁻¹). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation. The K _m was 0.15 mM and the V _max was 51 μmol/(min·mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.     

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH?7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40?°C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.     

Production of Xylooligosaccharides by Immobilized His-tagged Recombinant Xylanase from Penicillium occitanis on Nickel-Chelate Eupergit C

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0–4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.     

Highly Efficient Method Towards In Situ Immobilization of Invertase Using Cryogelation

A novel method was developed for the immobilization of Saccharomyces cerevisiae invertase within supermacroporous polyacrylamide cryogel and was used to produce invert sugar. First, the cross-linking of invertase with soluble polyglutaraldehyde (PGA) was carried out prior to immobilization in order to increase the bulkiness of invertase and thus preventing the leakage of the cross-linked enzyme after immobilization by entrapment. And then, in situ immobilization of PGA cross-linked invertase within cryogel synthesis was achieved by free radical polymerization in semi-frozen state. The method resulted in 100 % immobilization and 74 % activity yields. The immobilized invertase retained all the initial activity for 30 days and 30 batch reactions. Immobilization had no effect on optimum temperature and it was 60 °C for both free and immobilized enzyme. However, optimum pH was affected upon immobilization. Optimum pH values for free and immobilized enzyme were 4.5 and 5.0, respectively. The immobilized enzyme was more stable than the free enzyme at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes and microorganisms.     

Layer-by-Layer Self-Assembly Immobilization of Catalases on Wool Fabrics

A new immobilization strategy of catalases on natural fibers was reported in this paper. Catalase (CAT) from Bacillus subtilis was assembled into multiple layers together with poly(diallyldimethylammonium chloride) (PDDA) on wool fabrics via layer-by-layer (LBL) electrostatic self-assembly deposition. The mechanism and structural evaluation of LBL electrostatic self-assembly were studied in terms of scanning electron microscopy (SEM), surface zeta potential, and apparent color depth (K/S). The SEM pictures showed obvious deposits absorbed on the wool surfaces after LBL self-assembly. The surface zeta potential and dyeing depth of CAT/PDDA-assembled wool fabrics presented a regular layer-by-layer alternating trend along with the change of deposited materials, revealing the multilayer structure of the wool fiber immobilized catalases. The V _max values were found to be 2,500?±?238 U/mg protein for the free catalase and 1,000?±?102 U/mg protein for the immobilized catalase. The K _m value of free catalase (11.25?±?2.3 mM) was found to be lower than that of the immobilized catalase (222.2?±?36.5 mM). The immobilized catalase remained high enzymatic activity and showed a measureable amount of reusability, which proved that LBL electrostatic self-assembly deposition is a promising approach to immobilize catalases.     

Production, Purification, Immobilization, and Characterization of a Thermostable β-Galactosidase from Aspergillus alliaceus

A fungal strain isolated from rotten banana and identified as Aspergillus alliaceus was found capable of producing thermostable extracellular ??-galactosidase enzyme. Optimum cultural conditions for ??-galactosidase production by A. alliaceus were as follows: pH?4.5; temperature, 30?°C; inoculum age, 25?h; and fermentation time, 144?h. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 45?°C, 20?min, and 7.2, respectively, for crude and partially purified enzyme. For immobilized enzyme?Csubstrate reaction, these three variable, temperature, time, and pH were optimized at 50?°C, 40?min, and 7.2, respectively. Glucose was found to inhibit the enzyme activity. The K _m values of partially purified and immobilized enzymes were 170 and 210?mM, respectively. Immobilized enzyme retained 43?% of the ??-galactosidase activity of partially purified enzyme. There was no significant loss of activity on storage of immobilized beads at 4?°C for 28?days. Immobilized enzyme retained 90?% of the initial activity after being used four times.     

Screening of Dowex® anion-exchange resins for invertase immobilization

Commercial yeast invertase (Bioinvert^®) was immobilized by adsorption on anion-exchange resins, collectively named Dowex^® (1×8:50–400, 1×4:50–400, and 1×2:100–400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1×4–200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/invertase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The K _m and V _max for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL.     

Preparation and characterization of lipase immobilized on reversibly soluble-insoluble N-(2-carboxylbenzoyl) chitosan

N-(2-carboxylbenzoyl) chitosan (CBC), a reversibly soluble-insoluble polymer with pH change, was prepared by modifying chitosan backbone with phthalic anhydride and employed as carrier for lipase immobilization. The obtained CBC exhibited reversible solubility in aqueous solution; it was soluble at pH above 3.8 and precipitated at pH below 3.4. The porcine pancreatic lipase was covalently immobilized on CBC with glutaraldehyde as the crosslinking agent. Under the optimal immobilization condition, the retention activity of the immobilized lipase was found to be 69.8 %. The maximum activity of lipase immobilized on CBC was observed at 40 °C, pH 8.0, while the free lipase presented maximum activity at 37 °C, pH 7.5. The lipase immobilized on CBC exhibited improved thermal and storage stabilities and retained 58.7 % of its initial activity after 9 times of repeated use.     

Biomimetic Mineralization of the Alginate/Gelatin/Calcium Oxalate Matrix for Immobilization of Pectinase: Influence of Matrix on the Pectinolytic Activity

Pectinases catalyze the degradation of pectic substances and are used in several processes, mainly in food and textile industries. In this study, a biomimetic matrix of alginate/gelatin/calcium oxalate (AGOCa) was synthesized for the in situ immobilization via encapsulation of crude pectinase from Aspergillus niger ATCC 9642, obtaining an immobilization efficiency of about 61.7 %. To determine the performance of AGOCa matrix, this was compared to control matrices of alginate/calcium oxalate (AOxal) and alginate/water (ACa). By the evaluation of pH and temperature effects on the enzyme activity, it was observed an increase on pectinolytic activity for both three tested matrices with an increase on pH and temperature. The kinetic parameters for pectinase immobilized in the three matrices were determined using citric pectin as substrate. Values of K _m of 0.003, 0.0013, and 0.0022 g mL^?1 and V _max of 3.85, 4.32, and 3.17 μmol min^?1 g^?1 for AGOCa, AOxal, and ACa matrices were obtained, respectively. After 33 days of storage, the pectinase immobilized in the three different matrices kept its initial activity, but that immobilized in AGOCa presented high stability to the storage with a relative activity of about 160 %. The enzyme immobilized in AGOCa, AOxal, and ACa could be used in 10, 8, and 7 cycles, respectively, keeping 40 % of its initial activity.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Immobilization and Characterization of <Emphasis Type="Italic">E. gracilis</Emphasis> Extract with Enriched Laminaribiose Phosphorylase Activity for Bienzymatic Production of Laminaribiose

Immobilization methods and carriers were screened for immobilization of Euglena gracilis extract with laminaribiose phosphorylase activity. The extract was successfully immobilized on three different carriers via covalent linkage. Suitable immobilization carriers were Sepabeads EC-EP/S and ECR 8209M with epoxy groups and ECR 8309M with amino groups as functional units. Immobilization on Sepabeads EC-EP/S resulted in highest retained activity (65%). The immobilizates were characterized for pH, temperature, and buffer molarity preferences. The immobilized enzyme lost 48% of its activity when used seven times. Together with sucrose phosphorylase, laminaribiose phosphorylase was successfully applied for bienzymatic production of laminaribiose from sucrose and glucose with a final laminaribiose concentration of 14.3 ± 2.1 g/L (20% yield).