Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp.

Hydroquinone glycosides were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme^® 3.0L). The reactions were carried out in an aqueous system containing hydroquinone (HQ) and maltodextrin as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutin) were 9 mM hydroquinone, maltodextrin (5%, w/v) in 20 mM citrate phosphate buffer, pH 5.5 and 0.025 mg/ml toruzyme at 40 °C for 24 h. The transfer efficiency of hydroquinone glycosylation was 31.8% and 29.2% respectively, when α-cyclodextrin and maltodextrin were employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-α-d-glucopyranoside (α-arbutin) on the basis of mass spectrometric, nuclear magnetic resonance analysis and component analysis of its enzymatic hydrolysates. The highest molar yield of α-arbutin (21.2%) was obtained when α-cyclodextrin was used as the donor substrate. A two step enzymatic reaction system comprising of CGTase and amyloglucosidase helped to attain a molar yield of 30% for α-arbutin. At room temperature the solubility of α-arbutin in water was determined to be 12.8 g/100 ml which is approximately 1.8 fold higher than that of hydroquinone.     

Synthesis of cellobiose-containing oligosaccharides by intermolecular transglucosylation of cyclodextrin glycosyltransferase from Paenibacillus sp. A11

Intermolecular transglucosylation of cyclodextrin glycosyltransferase (CGTase) was investigated for its use in oligosaccharide synthesis. From the kinetic parameters of the CGTase-catalyzed transglucosylation reaction, using β-cyclodextrin (β-CD) as the glucosyl donor and various saccharides or derivatives as acceptors, the efficient acceptors of the Paenibacillus sp. A11 enzyme were glucose, sorbose, lactose and cellobiose. Amongst these acceptors, cellobiose showed the highest k_cat/K_m value. The transglucosylation yields of the reactions for cellobiose, sorbose and glucose acceptors were 78, 57 and 54%, respectively, making cellobiose the most efficient acceptor of the tested saccharides in coupling with β-CD. The optimal condition for the coupling reaction was determined as: 2% (w/v) β-CD and 0.5% (w/v) cellobiose, incubated with 64 U/mL of CGTase at 30 °C for 2 h. Two main transfer products detected by HPLC, PC1 and PC2, with retention times of 3.81 and 4.42 min, respectively, and a product ratio of 3:1, had a molecular mass of 504 and 666 Da, respectively, as analyzed by mass spectrometry. The structures suggested by NMR were a trisaccharide and a novel tetrasaccharide-containing cellobiose of the structures glc (α1 → 4) glc (β1 → 4) glc and glc (α1 → 4) glc (α1 → 4) glc (β1 → 4) glc, respectively. The products were found to be resistant to hydrolysis by α-amylase.     

Enzymatic transformation of 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) by immobilized α-cyclodextrin glucanotransferase from recombinant Escherichia coli

This work aims to produce 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin with immobilized α-cyclodextrin glucanotransferase (α-CGTase) from recombinant Escherichia coli. Molecular sieve (SBA-15) was used as an adsorbent, and sodium alginate was used as a carrier, and glutaraldehyde (GA) was used as a cross-linker. The effects of several key variables on α-CGTase immobilization were examined, and optimal immobilization conditions were determined as the following: glutaraldehyde (GA, cross-linker) 0.01% (v/v), SBA-15 (adsorbent) 2 g/L, CaCl₂ 3 g/L, sodium alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free α-CGTase, immobilized α-CGTase had a similar optimal pH (5.5) and a higher optimal temperature (45 °C). The continuous production of AA-2G from ascorbic acid and β-cyclodextrin in the presence of immobilized α-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with free α-CGTase. The immobilization procedure developed here was efficient for α-CGTase immobilization, which was proved to be a prospective approach for the enzymatic production of AA-2G.     

Transglucosylation reaction of amylomaltase for the synthesis of anticariogenic oligosaccharides

Synthesis of maltooligosylsucroses by the recombinant amylomaltase from Corynebacterium glutamicum as a N terminal (His)₆ chimera is reported. From the analysis of the products, by TLC and HPLC analysis on a Rezex RSO-Oligosaccharide column, the suitable glucosyl donor was found to be raw tapioca starch. The optimal condition was 2.0% (w/v) sucrose, 2.5% (w/v) raw tapioca starch and 9 U/ml of amylomaltase at 30 °C for 48 h, giving an overall 82% yield of maltooligosylsucrose products. After purified by Bio-Gel P-2 size exclusion column chromatography, the main products were determined by MS and NMR analysis to be maltosyl-, maltotriosyl-, maltotretraosyl- and maltopentaosyl-fructosides (G₂F, G₃F, G₄F and G₅F, respectively, where G = glucosyl unit and F = fructose) with an α-1,4 linkage between the added glucosyl unit and the sucrose. The low cariogenic property of these maltooligosylsucrose products was confirmed by analyzing the effect on the synthesis of water insoluble glucan, acid fermentation, plaque formation and cell aggregation of Streptococcus mutans when compared to those exerted by sucrose. Moreover, by adding sucrose to maltooligosylsucrose products at ratios of 1:1, 1:2 and 1:4, the inhibitory effects on glucosyltransferase activity of S. mutans by 7, 33 and 50%, respectively, were observed. These results suggest that the obtained maltooligosylsucrose products have an anticariogenic property and could be used to substitute for sucrose in food or related products.     

Optimization of ultrasound-assisted extraction conditions for total phenols with anti-hyperglycemic activity from Psidium guajava leaves

The high concentration of total phenolic compounds (TPC) in Psidium guajava leaf extracts (GvEx) is correlated to its anti-hyperglycemic activity. In this study, we established the optimum ultrasound extraction conditions for maximizing TPC yield. The response surface methodology (RSM) was employed for empirical model building. The maximum value of TPC (26.12%) was obtained at solvent to solid ratio (v/w) of 12.1, extraction temperature of 59.8 °C, and extraction time of 5.1 min. The anti-hyperglycemic activity of GvEx was compared to the commonly used diabetic drug acarbose. The IC₅₀ of GvEx for α-amylase and α-glucosidase inhibition was 50.5 μg/mL and 34.6 μg/mL, respectively. However, the IC₅₀ of acarbose for α-amylase and α-glucosidase inhibition was 95.3 μg/mL and 1075.2 μg/mL, respectively. In conclusion, GvEx obtained under optimum extraction conditions had higher anti-hyperglycemic activity than acarbose. In addition, the recommended extraction procedures for GvEx save time and are environmentally friendly.     

Production,purification, and characterization of two extremely halotolerant,thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101

The halophilic bacterial strain Chromohalobacter sp. TVSP 101 was shown to produce extracellular, halotolerant, alkali-stable and moderately thermophilic α-amylase activity. The culture conditions for higher amylase production were optimized with respect to NaCl, pH, temperature and substrates. Maximum amylase production was achieved in a medium containing 20% NaCl or 15% KCl at pH 9.0 and 37 °C in the presence of 0.5% rice flour and tryptone. Addition of 50 mM CaCl₂ to the medium increased amylase production by 29%. Two kinds of amylase activity, designated amylase I and amylase II, were purified from culture filtrates to homogeneity with molecular masses of 72 and 62 kDa, respectively. Both enzymes had maximal activity at pH 9.0 and 65 °C in the presence of 0–20% (w/v) NaCl but amylase I was much more stable in the absence of NaCl than amylase II. The enzymes efficiently hydrolyzed carbohydrates to yield maltotetraose, maltotriose, maltose, and glucose as the end products.     

Substrate effects on the mechanism of enantioselective hydrogenation using ruthenium bis(phosphine) complexes as catalyst: A mechanistic investigation of the hydrogenation of α,β-unsaturated acids and esters based on deuterium labeling studies

The mechanism of ruthenium-bis(phosphine) catalyzed enantioselective hydrogenation of olefins was examined using [Ru((R)-BINAP)(H)(MeCN)_n(sol)_3 − n]BF₄ (n = 0–3, sol = solvent used in reaction) as catalyst. Tiglic and angelic acids were used as standard α,β-unsaturated acid substrates; (Z)-methyl α-acetamidocinnamate and dimethyl itaconate were used as standard α,β-unsaturated ester substrates. Isotopic labeling studies (deuterium scrambling) indicate that two distinct mechanisms are in operation for α,β-unsaturated acids versus α,β-unsaturated esters. In each case, 5-membered metallocycle intermediates are formed via olefin-hydride insertion. The mechanisms, however, deviate primarily in the activation of dihydrogen, which is strongly affected by the nature of the substrate. Hydrogenation of α,β-unsaturated acids proceed via heterolytic cleavage of dihydrogen, whereas hydrogenation of α,β-unsaturated esters proceed via homolytic cleavage of dihydrogen. A full discussion of the mechanisms is presented.     

Protocorm culture of Dendrobium candidum in balloon type bubble bioreactors

Protocorm cultures of Dendrobium candidum were established in balloon type bubble bioreactors using Murashige and Skoog (MS) medium with 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5% (w/v) sucrose, 5:25 mM NH₄:NO₃ and 1% (v/v) banana homogenate for the production of biomass and bioactive compounds. In 3 l bioreactor containing 2 l medium, a maximum protocorm biomass (21.0 g l⁻¹ dry biomass) and also optimum quantities of total polysaccharides (389.3 mg g⁻¹ DW), coumarins (18.0 mg g⁻¹ DW), polyphenolics (11.9 mg g⁻¹ DW), and flavonoids (4.5 mg g⁻¹ DW) were achieved after 7 weeks of culture. Based on these studies, 5 and 10 l bioreactor cultures were established to harvest 80 g and 160 g dry biomass. In 10 l bioreactors, the protocorms grown were accumulated with optimal levels of polysaccharides (424.1 mg g⁻¹ DW), coumarins (15.8 mg g⁻¹ DW), polyphenols (9.03 mg g⁻¹ DW) and flavonoids (4.7 mg g⁻¹ DW). The bioreactor technology developed here will be useful for the production of important bioactive compounds from D. candidum.     

Inulinase production by the marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase

The marine yeast strain G7a isolated from sediment of China South Sea was found to secrete a large amount of inulinase into the medium. This marine yeast strain was identified to be a strain of Cryptococcus aureus according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast showed the highest activity at pH 5.0 and 50 °C. The optimal medium for inulinase production was artificial seawater containing inulin 4.0% (w/v), K₂HPO₄ 0.3% (w/v), yeast extract 0.5% (w/v), KCl 0.5% (w/v), CaCl₂ 0.12% (w/v), NaCl 4.0% (w/v) and MgCl₂·6H₂O 0.6% (w/v), while the optimal cultivation conditions for inulinase production were pH 5.0, a temperature of 28 °C and a shaking speed of 170 rpm. Under the optimal conditions, over 85.0 U/ml of inulinase activity was produced within 42 h of fermentation at shake flask level. This is very high level of inulinase activity produced by yeasts. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.     

Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched α-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin–Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS–PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K_m,app = 0.16 ± 0.02 mg/mL and k_cat,app = 79 ± 10 s^?1 by fitting the uncompetitive substrate inhibition Michaelis–Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k_cat,app/K_m,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with K_d of 27.2, 0.70, and 34.7 μM, respectively.     

Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar

Extraction of bromelain from pineapple peel (Nang Lae cultv.) using aqueous two phase system (ATPS) was optimized. Some biochemical properties including collagen hydrolysis were also investigated. Bromelain predominantly partitioned to the polyethylene glycol (PEG)-rich phase. The highest enzyme activity recovery (113.54%) and purification fold (2.23) were presented in the top phase of 15% PEG2000–14% MgSO₄. Protein pattern and activity staining showed the molecular weight (MW) of bromelain to be about 29 kDa. The extracted bromelain showed the highest relative activity at pH 7.0 and 55 °C. Its activity was decreased continuously by increasing NaCl concentration (up to 1.5% (w/v)). The bromelain extract was applied to hydrolyze the skin collagen of beef and giant catfish (0–0.3 units). The β, α₁, α₂ of giant catfish skin collagen extensively degraded into small peptides when treated with 0.02 units of the bromelain extract. Bovine collagen was hydrolyzed using higher bromelain up to 0.18 units. This study showed the ATPS can be employed to partially purify bromelain from Nang Lae pineapple peel and the enzyme effectively hydrolyzed the collagens.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Class II α-mannosidase from Aspergillus fischeri: Energetics of catalysis and inhibition

Energetics of the catalysis of Class II α-mannosidase (E.C.3.2.1.24) from Aspergillus fischeri was studied. The enzyme showed K_cat/K_m for Man (α1-3) Man, Man (α1-2) Man and Man (α1-6) Man as 7488, 5376 and 3690 M^?1 min^?1, respectively. The activation energy, E_a was 15.14, 47.43 and 71.21 kJ/mol for α1-3, α1-2 and α1-6 linked mannobioses, respectively, reflecting the energy barrier in the hydrolysis of latter two substrates. The enzyme showed K_cat/K_m as 3.56 × 10⁵ and 4.61 × 10⁵ M^?1 min^?1 and E_a as 38.7 and 8.92 kJ/mol, towards pNPαMan and 4-MeUmbαMan, respectively. Binding of Swainsonine to the enzyme is stronger than that of 1-deoxymannojirimycin.     

Enzymatic synthesis of cyclohexyl-α and β-d-glucosides catalyzed by α- and β-glucosidase in a biphase system

Two secondary alcohol glucosides, cyclohexyl-α-d-glucoside and cyclohexyl-β-d-glucoside, were synthesized via the condensation reaction of cyclohexanol with d-glucose in a biphase system catalyzed by α-glucosidase and β-glucosidase, respectively. The effects of pH, water content, glucose concentration and metal ions on the yield of glucosides were studied. The optimum catalytic conditions established for α-glucosidase was 25% (v/v) water content, 2.5 mol/L glucose concentration and pH 2.0, and for β-glucosidase was 30% (v/v) water content, 2.0 mol/L glucose and pH 5.0. The maximum yield of glucoside was 13.3 mg/mL for cyclohexyl-α-d-glucoside and 8.9 mg/mL for cyclohexyl-β-d-glucoside. Synthesis progress was monitored by TLC and quantitatively analyzed by pre-derived capillary gas chromatography (GC). The retention time was 12.34 min for the α isomer and 12.96 min for the β isomer, respectively. With an anomeric purity of more than 99.5%, the two glucosides display excellent site-specific catalysis by α- and β-glucosidase. Herein, we present a general method to produce anomerically pure glucosides via a one-step bio-reaction in a biphase system. This method could potentially be applied in glucosylation of primary and secondary alcohols or other reactions requiring glucosylation.     

Efficient production of α-arbutin by whole-cell biocatalysis using immobilized hydroquinone as a glucosyl acceptor

The aim of the present study is to develop an efficient and cost-effective method for α-arbutin production by using whole-cell of Xanthomonas maltophilia BT-112 as a biocatalyst. Hydroquinone (HQ), substrate for the bioconversion as glucosyl acceptor, was immobilized on H107 macroporous resin to reduce its toxic effect on the cells, and the optimal reaction conditions for α-arbutin synthesis were investigated. When 350 g/L H107 resin (254.5 mM HQ) and 20 g/L (4.2 U/g) of cells were shaken in 10 mL Na₂HPO₄–KH₂PO₄ buffer (50 mM, pH 6.5) containing 509 mM sucrose at 35 °C with 150 rpm for 48 h, the final yield of α-arbutin reached 65.9 g/L with a conversion yield of 95.2% based on the amount of HQ supplied. The α-arbutin production was 202% higher than that of the control (free HQ) and the cells maintained its full activity for almost six consecutive batch reactions, indicating a potential for reducing production costs. Additionally, the product was one-step isolated and identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the combination of whole cells and immobilized hydroquinone (IMHQ) is a promising approach for economical and industrial-scale production of α-arbutin.     

Synthesis and α-glucosidase inhibitory activity evaluation of N-substituted aminomethyl-β-d-glucopyranosides

A series of N-substituted 1-aminomethyl-β-d-glucopyranoside derivatives was prepared. These novel synthetic compounds were assessed in vitro for inhibitory activity against yeast α-glucosidase and both rat intestinal α-glucosidases maltase and sucrase. Most of the compounds displayed α-glucosidase inhibitory activity, with IC₅₀ values covering the wide range from 2.3 μM to 2.0 mM. Compounds 19a (IC₅₀ = 2.3 μM) and 19b (IC₅₀ = 5.6 μM) were identified as the most potent inhibitors for yeast α-glucosidase, while compounds 16 (IC₅₀ = 7.7 and 15.6 μM) and 19e (IC₅₀ = 5.1 and 10.4 μM) were the strongest inhibitors of rat intestinal maltase and sucrase. Analysis of the kinetics of enzyme inhibition indicated that 19e inhibited maltase and sucrase in a competitive manner. The results suggest that the aminomethyl-β-d-glucopyranoside moiety can mimic the substrates of α-glucosidase in the enzyme catalytic site, leading to competitive enzyme inhibition. Moreover, the nature of the N-substituent has considerable influence on inhibitory potency.     

Purification and characterization of cyclodextrinase from Paenibacillus sp. A11

Paenibacillus sp. A11 produced an intracellular cyclodextrinase (CDase), its presence was confirmed by activity detection on an agar plate with specific screening medium containing β-cyclodextrin (β-CD) and phenolphthalein. The CDase was purified up to 22-fold with a 28% yield. The enzyme was a single polypeptide with a molecular weight of 80 kDa. Optimum activity was at pH 7.0 and 40 °C. The enzyme had an isoelectric point of 5.4 and N-terminal sequence was M F L E A V Y H R P R K N W S. When relative hydrolytic activities of the CDase on different substrates were compared, it was found that high specificity was exerted by β-CD while maltoheptaose, its linear counterpart, was only 40% as active. The enzyme recognized α-1,4-glucose units and the hydrolysis depended on the size of oligosaccharides. Highly branched carbohydrates such as glycogen or dextran or other heteropolymers as glucomannan could not be hydrolyzed. This enzyme was different from other CDases in its ability to hydrolyze maltose and trehalose, though with very low hydrolytic activity. The major product from all substrates was maltose. The k_cat/K_m value for β-CD was 8.28 × 10⁵ M⁻¹min⁻¹. The enzyme activity was completely inactivated by 1 mM N-bromosuccinimide and diethylpyrocarbonate suggesting the crucial importance of Trp and His for its catalytic activity. Essential Trp was confirmed to be at enzyme active site by substrate protection experiment. Partial inactivation by 5 mM phenylglyoxal suggests the involvement of Arg, which has never been reported in other CDases.     

Bioreactor leaching of uranium from a low grade Indian silicate ore

Bio-leaching studies were carried out in a 2 L bioreactor- BIOSTAT-B^® equipped with a PLC based controller at 20–40% (w/v) pulp density using enriched culture of A.ferrooxidans for Turamdih uranium ore (Jharkhand, India). With the enriched culture of A.ferrooxidans adapted on Fe(II) at pH 2.0, 35 °C and 20% (w/v) pulp density, a 98.3% uranium recovery was recorded in 14 days. The leaching of uranium in the bioreactor improved the dissolution rate by reducing the time from 40 days in shake flask as per our earlier studies to 14 days. While investigating the importance of biogenic Fe(III) in the bio-leaching process a maximum recovery of 84.7% U₃O₈ was observed at pH 2.0 and 20% (w/v) pulp density in 10 h as compared to the uranium leaching of 38.3% in the control experiments. On raising the pulp density to 30%, uranium bio-recovery increased to 87.6% in 10 h at pH 2.0 with <76 μm size material. This showed a distinct advantage because of better mixing of slurry in the bioreactor with auto-controlled conditions that improved the kinetics.     

Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides: Control of product distribution by flow rate adjustment

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) was covalently immobilised on Eupergit C and used in a packed-bed reactor to investigate the continuous production of long-carbohydrate-chain alkyl glycosides from α-cyclodextrin (α-CD) and n-dodecyl-(1,4)-β-maltopyranoside (C₁₂G₂β). The effects of buffer ion strength and pH, and enzyme loading on the immobilisation yield and the enzyme activity were evaluated. Approximately 98% of the protein and 33% of the total activity were immobilised. At pH 5.15, the enzymatic half-life was 132 min at 60 °C and 18 min at 70 °C. The immobilised enzyme maintained 60% of its initial activity after 28 days storage at 4 °C. The degree of conversion was controlled by simple regulation of the flow rate through the reactor, making it possible to optimise the product distribution. It was possible to achieve a yield of the primary coupling product n-dodecyl-(1,4)-β-maltooctaoside (C₁₂G₈β) of about 50%, with a ratio between the primary and the secondary coupling product of about 10. Thermoanaerobacter sp. CGTase (Toruzyme 3.0 L) immobilised on Eupergit C had good operational stability at 60 and 70 °C thus showing the advantages of using more thermostable enzymes in biocatalysis. However, this enzyme was unsuitable for the production of C₁₂G₈β due to extensive disproportionation reactions, giving a broad product range.     

Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria

An extracellular protease from a newly isolated seawater haloalkaliphilic bacterium, haloalkaliphilic bacteria Ve₂-20-9₁ [HM047794], was purified and characterized. The enzyme is a monomer with a 37.2 kDa estimated molecular weight. It catalyzed reactions in the pH range 8–11 and performed optimally at pH 10. While maximal activity occurred at 50 °C, the temperature profile shifted from 50 to 80 °C in 1–3 M NaCl. The enzyme's thermal stability was probed using circular dichroism (CD) spectroscopy with NaCl at 50 and 70 °C. The changes in the enzyme's secondary structure were also analyzed using Fourier transform infrared spectroscopy (FTIR). The N-terminal amino acid sequence GKDGPPGLCGFFGCI exhibited low homology with other bacterial proteases, which highlights the enzyme's novelty. The enzyme was labile in anionic surfactant (1% w/v SDS) but showed stability in non-ionic surfactants (Tween 20, Tween 80 and Triton X-100 all 1% v/v), commercial detergents, and oxidizing and reducing agents. The enzyme's excellent stability in commercial detergents highlights its potential as a detergent additive.