Comparison of visual performance with iTrace analyzer following femtosecond laser-assisted cataract surgery with bilateral implantation of two different trifocal intraocular lenses.

AIM: To compare the postoperative binocular visual performance with an iTrace analyzer following femtosecond laser-assisted cataract surgery (FLACS) combined with bilateral implantation of two different types of diffractive trifocal intraocular lenses (IOL). METHODS: During this retrospective observational study, patients who received bilateral FLACS combined with implantation of two different types of diffractive trifocal IOLs were evaluated. According to the IOLs' different types and design, the patients were divided into AT LISA tri839MP group (tri839 group) and AcrySof PanOptix TFNT00 group (TFNT group). Study parameters included preoperative and postoperative uncorrected distance visual acuity (UDVA) at 5 m, uncorrected near visual acuity (UNVA) at 30 cm and 40 cm, uncorrected intermediate visual acuity (UIVA) at 60 cm and 80 cm, postoperative refractive status, objective visual qualities and total high order aberrations (HOAs) postoperatively. The postoperative complications were also recorded. RESULTS: Totally 56 eyes of 28 patients (tri839 group, n=26; TFNT group, n=30) were included. Preoperative baseline characteristics between groups were not statistically significantly different. UDVA was not significantly different between groups except for 1wk follow-up due to the postoperative corneal edema. TFNT group showed statistically significant better UNIA at 60 cm than tri839 group at the 1wk (0.05±0.19 vs 0.15±0.10 logMAR, P=0.013), 1mo (0.05±0.12 vs 0.15±0.09 logMAR, P=0.001) and 3mo (0.04±0.12 vs 0.15±0.11 logMAR, P=0.001) follow-up, while tri839 group showed statistically significant better UNIA at 80 cm than TFNT group at the 1d (0.14±0.15 vs 0.20±0.14 logMAR, P=0.041) and 1mo (0.09±0.07 vs 0.14±0.10 logMAR, P=0.042) follow-up. Postoperative refractive status showed stable at every visit. Modulated transfer function (MTF) values and strehl ratio (SR) values were improved and HOAs were lower significantly after surgery. CONCLUSION: FLACS with bilateral implantations of both tri839 and TFNT00 can achieve satisfactory natural whole-course vision, high postoperative refractive stability and good visual quality but without significantly difference. iTrace aberration instrument can accurately evaluate the visual quality under different status.

Engineering of reaction specificity, enantioselectivity, and catalytic activity of nitrilase for highly efficient synthesis of pregabalin precursor.

Simultaneous evolution of multiple enzyme properties remains challenging in protein engineering. A chimeric nitrilase (BaNITM0 ) with high activity towards isobutylsuccinonitrile (IBSN) was previously constructed for biosynthesis of pregabalin precursor (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA). However, BaNITM0 also catalyzed the hydration of IBSN to produce by-product (S)-3-cyano-5-methylhexanoic amide. To obtain industrial nitrilase with vintage performance, we carried out engineering of BaNITM0 for simultaneous evolution of reaction specificity, enantioselectivity, and catalytic activity. The best variant V82L/M127I/C237S (BaNITM2 ) displayed higher enantioselectivity (E = 515), increased enzyme activity (5.4-fold) and reduced amide formation (from 15.8% to 1.9%) compared with BaNITM0 . Structure analysis and molecular dynamics simulations indicated that mutation M127I and C237S restricted the movement of E66 in the catalytic triad, resulting in decreased amide formation. Mutation V82L was incorporated to induce the reconstruction of the substrate binding region in the enzyme catalytic pocket, engendering the improvement of stereoselectivity. Enantio- and regio-selective hydrolysis of 150 g/L IBSN using 1.5 g/L Escherichia coli cells harboring BaNITM2 as biocatalyst afforded (S)-CMHA with >99.0% ee and 45.9% conversion, which highlighted the robustness of BaNITM2 for efficient manufacturing of pregabalin.

Constitutive expression of nitrilase from Rhodococcus zopfii for efficient biosynthesis of 2-chloronicotinic acid.

2-chloronicotinic acid (2-CA) is a key precursor for the synthesis of a series of pesticides and pharmaceuticals. Nitrilase-catalyzed bioprocess is a promising method for 2-CA production from 2-chloronicotinonitrile (2-CN). In this study, a mutant of nitrilase from Rhodococcus zopfii (RzNIT/W167G) was constitutively overexpressed with Escherichia coli as host, which exhibited a onefold increase in enzymatic activity compared with inducible expression. Biosynthesis of 2-CA using whole cells harboring nitrilase as biocatalysts were investigated and 318.5 mM 2-CA was produced, which was the highest level for 2-CA production catalyzed by nitrilase to date. 2-CA was recovered from the reaction mixture through a simple acidification step with a recovery yield of 90%. This study developed an efficient bioprocess for 2-CA with great potential for industrial application. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03119-0.

Rational Regulation of Reaction Specificity of Nitrilase for Efficient Biosynthesis of 2-Chloronicotinic Acid through a Single Site Mutation.

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for the efficient synthesis of 2-chloronicotinic acid (2-CA). The development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potential. Herein, a nitrilase from Rhodococcus zopfii (RzNIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN. Two residues (N165 and W167) significantly affecting the reaction specificity were precisely identified. By tuning these two residues, a single mutation of W167G with abolished hydration activity and 20-fold improved hydrolysis activity was obtained. Molecular dynamics simulation and molecular docking revealed that the mutation generated a larger binding pocket, causing the substrate 2-CN to bind more deeply in the pocket and form a delocalized π bond between the residues W190 and Y196, which reduced the negative influence of steric hindrance and electron effect caused by chlorine substituent. With mutant W167G as biocatalyst, 100 mM 2-CN was exclusively converted into 2-CA within 16 h. The study provides useful guidance in nitrilase engineering for simultaneous improvement of reaction specificity and catalytic activity, which are highly desirable in value-added carboxylic acids production from nitriles hydrolysis. IMPORTANCE 2-CA is an important building block for agrochemicals and pharmaceuticals with a rapid increase in demand in recent years. It is currently manufactured from 3-cyanopyridine by chemical methods. However, during the final step of 2-CN hydrolysis under high temperature and strong alkaline conditions, the byproduct 2-CM was generated except for the target product, leading to low yield and tedious separation steps. Nitrilase-mediated hydrolysis is regarded as a promising alternative for 2-CA production, which proceeded under mild conditions. Nevertheless, nitrilase capable of efficient hydrolysis of 2-CN has not been reported because the enzymes showed either extremely low activity or surprisingly high hydration activity toward 2-CN. Herein, the reaction specificity of RzNIT was precisely tuned through a single site mutation. The mutant exhibited remarkably enhanced hydrolysis activity without the formation of byproducts, providing a robust biocatalyst for 2-CA biosynthesis with industrial potential.

Analysis of the effects of different nitrogen sources and calcium on the production of amphotericin by Streptomyces nodosus based on comparative transcriptome.

Streptomyces nodosus is known as the main manufacturer of amphotericin B (AmB), which is an effective antifungal drug. It is verified that the optimization of fermentation conditions and key growth factors have a great impact on the yield of AmB. The AmB production of S. nodosus in cotton-seed meal (CM) medium was 1.6 times than that of beef-paste medium. The transcriptome analysis was used to analyze the effects of different nitrogen media and calcium on S. nodosus. Related genes of the EMP and TCA pathways, such as phosphofructokinase, pyruvate dehydrogenase, and citrate synthase, were upregulated in CM medium. The expression level of the PKS modules of the amphotericin synthesis gene cluster in beef-paste medium was higher. Other functional genes, such as amphGH and amphRIV, have the advantage of expressing in CM medium. Ca2+ promoted the upregulation of genes in metabolic pathways such as EMP pathway (pyruvate dehydrogenase), TCA pathway (citrate synthase), and amphotericin synthesis genes (PKS modules). The expression of WhiB family genes SNOD_RS 13310 and SNOD_RS 17625 was positively correlated with Ca2+ concentration. In addition, in the presence of calcium, the expression level of Sec transport system genes of S. nodosus was lower.

Enhancing the production of amphotericin B by Strepyomyces nodosus in a 50-ton bioreactor based on comparative genomic analysis.

Amphotericin, as an important macrolide antibiotic, is synthesized by Streptomyces nodosus. A high-yield S. nodosus ZJB2016050 was obtained by mutagenesis in our lab with the advantages of high yield, short fermentation cycle and few by-products, which was more suitable for industrial production. The fermentation differences in 50-tons bioreactor between S. nodosus ATCC14899 and S. nodosus ZJB2016050 were compared. The amphotericin B (AmB) yield of S. nodosus ZJB2016050 was 9.73 mg/g at 96 h, which was 30% higher than that of S. nodosus ATCC14899. The by-product amphotericin A (AmA) production of S. nodosus ZJB2016050 was 78% lower than that of S. nodosus ATCC14899. By performing whole-genome sequencing of S. nodosus ZJB2016050 and comparative genome analysis with the wild-type S. nodosus ATCC14899, it was found that the two strains have high synteny, but each has a special gene fragment. The genes functions of fragment were identified in the amino acid transport and metabolism, carbohydrate metabolism and lipid transport and metabolism. The gene functions of SNP (single nucleotide polymorphism) genes were identified in amino acid transport and metabolism, carbohydrate metabolism, coenzyme metabolism and secondary metabolites biosynthesis. The difference in signal-regulation and transcription may be the main reason for the differences between these two strains. Three GntR family egulatory factors of S. nodosus ATCC14899 may reduce the synthesis of amphotericin. Based on the analysis of comparative genomes, the effects of corn oil in S. nodosus ATCC14899 and S. nodosus ZJB2016050 were also compared. The results showed that corn oil can promote the fermentation of S. nodosus ZJB2016050. The S. nodosus ZJB2016050 may degrade fatty acids faster, and the degraded acyl-coenzyme can be used to synthesize amphotericin. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02844-2.

Trifocal diffractive intraocular lens implantation in patients after previous corneal refractive laser surgery for myopia.

BACKGROUND: With the difficulties in IOL power calculation and the potential side effects occurring postoperatively, multifocal IOL implantation after previous corneal refractive surgery are rarely reported especially for the trifocal IOL. Herein we report the clinical observation of trifocal IOL implantation in patients with previous myopia excimer laser correction. In this study, a multi-formula average method was performed for the IOLs power calculation to improve the accuracy. Visual and refractive outcomes were analyzed, and the subjective quality of patients' life was evaluated by questionnaires survey. METHODS: This retrospective case series included patients with previous myopia excimer laser correction who underwent femtosecond laser assisted phacoemulsification and trifocal IOL (AT LISA tri 839 MP) implantation. Follow-up was done at 1-day, 1-month and 3-month to assess the visual outcomes. Outcome measures were uncorrected distance, intermediate and near visual acuity (UDVA, UIVA, UNVA), manifest refraction, defocus curve, and subjective quality of vision. RESULTS: Twenty-one Eyes from sixteen patients (14 eyes with previous laser in situ keratomileusis and 7 eyes with previous photorefractive keratectomy) were included. Mean postoperative spherical equivalent (SE) at 3-month was - 0.56 D ± 0.49 SD, wherein, 10 eyes (47.6%) were within ±0.50 D of the desired emmetropia and 19 eyes (90.5%) were within ±1.0 D. Mean monocular UDVA, UIVA and UNVA (logMAR) at last visit were 0.02 ± 0.07, 0.10 ± 0.10, and 0.15 ± 0.11 respectively. Three patients (19%) reported halos and glare in postoperative 3 months, two of them needed to use spectacles to improve the intermediate visual acuity. Fifteen patients (94%) reported a satisfaction score of ≥3.5 out of 4.0, without any difficulty in daily activity. Thirteen patients (81%) did not need spectacles at all distances, while the other 3 patients (19%) used spectacles for near-distance related visual activity. Mean composite score of the VF-14 questionnaire was 95.00 ± 7.29 out of 100. CONCLUSIONS: Trifocal IOL implantation after myopia excimer laser correction could restore good distance, intermediate visual acuity and acceptable near visual acuity, and provide accurate refractive outcomes as well as high spectacles independence rate.

Changes in ocular surface status and dry eye symptoms following femtosecond laser-assisted cataract surgery.

AIM: To observe the changes in ocular surface and the dry eye symptoms following femtosecond laser-assisted cataract surgery (FLACS). METHODS: Patients with no eye signs or symptoms in Guangzhou Aier Eye Hospital between October 2017 and September 2018, who underwent FLACS and intraocular lens (IOL) implantation for age-related cataract were enrolled. Tear film stability assessed with OCULUS Keratograph 5M, Schirmer's I test (SIT), and corneal fluorescein staining (CFS) were evaluated before and after surgery at 1d, 1wk, 1, and 3mo in order. Ocular Surface Disease Index scores (OSDI) and Subjective Symptom Questionnaires (SSQs) were recorded at the same time point. RESULTS: Thirty-eight eyes of 38 patients were enrolled. The noninvasive tear film break-up time (first break-up time and average break-up time) decreased in a peak at the 1wk visit, and then increased to basic levels at 1mo. The tear meniscus height (TMH) increased transiently at 1d, and declined in the following 3mo visits. The SIT had a transient increase at 1d (P=0.357) and a decrease at 1wk and 1mo (both P<0.05) but returned to the preoperative levels at 3mo after surgery (P=0.062). CFS scores were significantly improved compared with those before surgery, and had a statistical difference (P<0.05). OSDI scores and SSQs after surgery were obviously higher, and had a statistical difference (P<0.001) but didn't return to the basic level by 3mo. CONCLUSION: Dry eye signs and symptoms can occur immediately following FLACS and have a peak severity on day 7 postoperatively. Most signs of dry eye can return to preoperative basic levels within 3mo postoperatively. However, all cases can not recover from CFS and dry eye symptoms at 3mo postoperatively.

Highly regio- and enantioselective synthesis of chiral intermediate for pregabalin using one-pot bienzymatic cascade of nitrilase and amidase.

Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. In this study, a robust nitrilase from Arabis alpina (AaNIT) was screened and engineered. The N258D mutant was obtained with high catalytic activity and excellent enantioselectivity (E > 300) towards IBSN at a high substrate concentration of 100 g L-1. Byproduct (S)-3-cyano-5-methyl hexanoic amide ((S)-CMHM) was detected and identified for the first time during the catalytic process. By employing a feasible one-pot bienzymatic cascade of mutant N258D and amidase from Pantoea sp. (Pa-Ami) expressed separately in recombinant Escherichia coli cells, the byproduct (S)-CMHM was eliminated and (S)-CMHA was obtained with a conversion of 45.0% and eep of 99.3%. These results provided the novel plant-derived nitrilase as a promising biocatalyst for (S)-CMHA biosynthesis and demonstrated the feasibility of one-pot bienzymatic cascade reaction for large-scale production of the pregabalin precursor.

Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor.

To develop a highly efficient method for aprepitant chiral intermediate (S)-4-fluorophenylglycine, a continuous reaction system was established in packed bed bioreactor using amidase covalently immobilized on epoxy resin as biocatalyst. The epoxy resin was firstly modified by metal-chelate method and functional groups (Cu2+-IDA) generated were able to rapidly adsorb amidases, which were further covalently bound onto the modified resin with 90.1% immobilization yield and 80.2% activity recovery. The immobilized amidase exhibited excellent thermal stability with the longest half-life of 1456.8 h at 40 °C ever reported. (S)-4-fluorophenylglycine was continuously produced using the reaction system with 49.9% conversion, 99.9% ee, and an outstanding space-time yield of 5.29 kg L-1 d-1. Moreover, the efficient reaction system exhibited a high operational stability and retained 86.3% catalytic activity after 25-day continuous operation. This efficient continuous bioprocess presents great industrial potential for large-scale production of (S)-4-fluorophenylglycine.

Structure-Based Engineering of Amidase from Pantoea sp. for Efficient 2-Chloronicotinic Acid Biosynthesis.

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from a Pantoea sp. (Pa-Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with kcat/Km values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 gproduct liter-1 day-1 These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships.IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa-Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa-Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa-Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid.

Biocatalytic production of (S)-2-aminobutanamide by a novel d-aminopeptidase from Brucella sp. with high activity and enantioselectivity.

As the important chiral building block of levetiracetam, the synthesis of (S)-2-aminobutanamide has attracted a great deal of attention. The d-aminopeptidase catalyzed kinetic resolution of 2-aminobutanamide was demonstrated as an effective strategy for (S)-2-aminobutanamide production. In this study, a novel d-aminopeptidase from Brucella sp. (Bs-Dap) was screened and systematically characterized. The enzyme exhibited maximum activity at 45°C, pH 8.0 and it showed relatively low Km value toward 2-aminobutanamide, indicating its high affinity to the substrate. Kinetic resolution of 300g/L 2-aminobutanamide by recombinant Escherichia coli whole cells (4g/L wet cell weight) resulted in 50% conversion and >99% e.e. within 80min. The catalytic properties of Bs-Dap demonstrated its great potential for industrial production of (S)-2-aminobutanamide.

Biocatalytic hydrolysis of chlorinated nicotinamides by a superior AS family amidase and its application in enzymatic production of 2-chloronicotinic acid.

2-Chloronicotinic acid (2-CA) is an important building block for a series of agrochemicals and pharmaceuticals. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is one of the most attractive approaches for 2-CA production. However, development of the bioprocess was plagued by low activity of amidase for 2-chloronicotinamide. In this work, an amidase signature (AS) family amidase from Pantoea sp. (Pa-Ami), with superior activity for nicotinamide and its chlorinated derivatives, was exploited and characterized. Kinetic analysis and molecular docking clearly indicated that chlorine substitution in the pyridine ring of nicotinamide, especially the substitution at 2-position led to a dramatic decrease of Pa-Ami activity. The productivity of the bioprocess was significantly improved using fed-batch mode at low reaction temperature and 2-CA was produced as high as 370 mM with a substrate conversion of 94.2%. These results imply that Pa-Ami is potentially promising biocatalyst for industrial production of 2-CA.

[Multifocal electroretinography for therapeutic effect evaluation of intravitreal injection Lucentis for wet age-related macular degeneration].

OBJECTIVE: To evaluate the changes in retinal functions using multifocal electroretinography (mfERG) following intravitreal injection of Lucentis for treatment of wet age-related macular degeneration. METHODS: This prospective study was conducted in 14 patients (9 men and 5 women, 14 eyes) with wet age-related macular degeneration receiving treatment with intravitreal injections of ranibizumab (Lucentis) in our hospital between October, 2014 and January, 2016. All the patients received the treatment following a 1+PRN protocol and after the initial injection, the patients were followed up monthly for 6 months to decide if additional injections were needed. The corrected visual acuity and mfERG findings of the patients were assessed before and at l, 3 and 6 months after the initial injection. RESULTS: At the last follow-up, the patients received injections for a mean of 2.86∓1.58 times. The best corrected visual acuity (BCVA) at 1 month after the initial treatment was not significantly different from that before treatment (P=0.07), but showed significant improvements at 3 and 6 months (P<0.05). In mfERG, the implicit time of the 6 rings showed no significant decrease after the treatment, but the amplitude density of P1 and N1 in rings 1 and 2 improved significantly at 1, 3, and 6 months after the initial injection (P<0.05). CONCLUSION: Multifocal electroretinography can serve as a useful modality for evaluating visual function changes in patients receiving intravitreal injection of Lucentis for wet age-related macular degeneration.

Identification and characterization of a novel amidase signature family amidase from Parvibaculum lavamentivorans ZJB14001.

Amidase signature (AS) family amidases are known to exhibit broad substrate specificity. According to the available genome sequence data, a novel AS family amidase, Pl-Ami, was identified and cloned from the genome of Parvibaculum lavamentivorans ZJB14001. The recombinant amidase was overexpressed in Escherichia coli BL21, purified and functionally characterized. The optimal pH and temperature for Pl-Ami were 9.5 and 45 °C, respectively. Pl-Ami preferred long chain aliphatic amides as substrates, while no activity was detected towards aromatic, heterocyclic and other amides. The highest enzyme activity of 128 U/mg was obtained when hexanoamide was used as substrate. Kinetic analysis indicated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover number (kcat) was higher with long chain aliphatic amides as substrates. The obtained results provided a distinct understanding of substrate specificity of AS family amidases.

Identification and characterization of a thermostable and cobalt-dependent amidase from Burkholderia phytofirmans ZJB-15079 for efficient synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.

Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co2+, with a specific activity of 753.5 U/mg, K m value of 24.73 mM, and k cat /K m value of 22.47 mM-1 s-1. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (ee p) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.

Exploitation and characterization of three versatile amidase super family members from Delftia tsuruhatensis ZJB-05174.

Amidases can be assigned into two families according to their amino acid sequences. Three amidases (Dt-Amis) were mined and identified from genome of Delftia tsuruhatensis. Homology analysis demonstrated that Dt-Ami 2 and Dt-Ami 6 belonged to amidase signature (AS) family, while Dt-Ami 7 belonged to nitrilase superfamily. AS amidases were shown to hydrolyze a wide spectrum of amides. Kinetic analysis demonstrated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover numbers (kcat) were high with linear aliphatic amides as substrates. Dt-Ami 2 showed maximum activity near a quite alkaline pH (11.0) and exhibited opposite enantioselectivity to Dt-Ami 6. Furthermore, a novel bioprocess for hydrolysis of 1-cyanocyclohexaneacetamide was developed using Dt-Ami 6 as biocatalyst, resulting in >99% conversion within 1.5h at a substrate loading of 100g/L by 0.5g/L of Escherichia coli cells. On the other hand, nitrilase superfamily amidase only hydrolyzed aliphatic amides. The Km values of Dt-Ami 7 were considerably increased with the extension of chain length of aliphatic amides. The characterized enzymes from different families showed distinct biochemical characteristics and catalytic properties, leading to a better understanding of the two super amidase family members.

Thermophilic esterase from Thermomyces lanuginosus: molecular cloning, functional expression and biochemical characterization.

A novel esterase encoding gene, tle, was cloned from the thermophilic fungus Thermomyces lanuginosus DSM 10635. The tle had an open reading frame of 945bp encoding TLE of 314 amino acids with a theoretical molecular mass of 34.5kDa. The putative catalytic triad of TLE was consisted of Ser151, His279, and Asp249. TLE was heterologously expressed in Escherichia coli in biologically active form and purified to homogeneity. Several biochemical properties of TLE were studied: Among the tested p-nitrophenol esters, TLE showed the highest hydrolytic activity with p-nitrophenyl butyrate (C4) and exhibited the maximum activity at 60°C and pH 8.5. The enzyme was stable at temperatures below 60°C and retained 53% of the maximum activity after treatment at 70°C for 60min. Esterase activity was notably enhanced by addition of Ca(2+) and Ba(2+), respectively. Furthermore, TLE showed high enantioselectivity (E=95) in the kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE), which produce a valuable chiral intermediate-(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid for Pregabalin. These unique properties of the esterase indicate that TLE is a potential candidate for industrial application.