Jianjun Wang

Characterization of a recombinant (+)-γ-lactamase from Microbacterium hydrocarbonoxydans which provides evidence that two enantiocomplementary γ-lactamases are in the strain

A two-step method, i.e., the transfer acyl analysis and then the chiral HPLC analysis, was employed in the screening of the cosmid library of Microbacterium hydrocarbonoxydans genome. Two enantiocomplementary γ-lactamase clones were found. A 40-kb cosmid showed (−)-γ-lactamase activity, and the activity was from Mhg which was reported previously according to the results of PCR identifying experiment. The 37-kb (+)-γ-lactamase cosmid was further constructed into a pUC18 plasmid library and screened by the same two-step method. A plasmid clone harboring a 1.6-kb fragment showed (+)-γ-lactamase activity. A 555-bp ORF in the 1.6-kb fragment showed high (+)-γ-lactamase activity when it was expressed under the control of T7 promoter. The coding protein showed significant homology with bacterial isochorismatase. The (+)-γ-lactamase was characterized and compared with the (−)-γ-lactamase Mhg. This is another report that two enantiocomplementary γ-lactamases are present in the same strain.

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44xa0h, glycerol (20xa0gxa0L−1) and corn steep liquor (CSL) (30xa0gxa0L−1) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2xa0gxa0L−1xa0h−1) and continuous feeding of CSL (1xa0gxa0L−1xa0h−1) at a fermentation time of 28xa0h. After optimization, the maximum dry cell weight achieved was 24.5±0.8xa0gxa0L−1; maximum EH production was 351.2±13.1xa0Uxa0L−1 with a specific activity of 14.3±0.5xa0Uxa0g−1. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1xa0M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.

Immobilization on macroporous resin makes E. coli RutB a robust catalyst for production of (−) Vince lactam

A novel (+) γ-lactamase gene (rutB) was cloned from Escherichia coli JM109 and expressed in E. coli BL21 (DE3), and the recombinant protein was characterized. The optimal conditions for the enzyme were pH 7.0 and temperature 30xa0°C, which indicated that it was a mesophilic protein. The free purified enzyme was deactivated when incubated at 50xa0°C for 30xa0min. However, the kcat value of RutB at its optimal temperature was about 2.5 times that of the archaeal enzyme from Sulfolobus sofataricus at its optimal temperature (85xa0°C). After immobilization on macroporous resin using glutaraldehyde cross-linkage, the thermostability of the crude enzyme was greatly enhanced and the deactivating temperature was raised to 70xa0°C. After immobilization, the minimal substrate inhibition concentration for RutB also improved from 0.75 to 1.5xa0M. The optimal concentrations of immobilized enzyme and substrate were determined to be 250xa0mg/ml and 1.5xa0M, when the initial reaction velocity was the response variable in batch transformations. This immobilization of RutB on macroporous resins provides another feasible approach for the preparation of optically active Vince lactam. As a member of the isochorismatase superfamily, RutB was demonstrated to be another typical γ-lactamase that showed catalytic promiscuity.

CBD binding domain fused γ-lactamase from Sulfolobus solfataricus is an efficient catalyst for (-) γ-lactam production

Backgroundγ-lactamase is used for the resolution of γ-lactam which is utilized in the synthesizing of abacavir and peramivir. In some cases, enzymatic method is the most utilized method because of its high efficiency and productivity. The cellulose binding domain (CBD) of cellulose is often used as the bio-specific affinity matrix for enzyme immobilization. Cellulose is cheap and it has excellent chemical and physical properties. Meanwhile, binding between cellulose and CBD is tight and the desorption rarely happened.ResultsWe prepared two fusion constructs of the γ-lactamase gene gla, which was from Sulfolobus solfataricus P2. These two constructs had Cbd (cellulose binding domain from Clostridium thermocellum) fused at amino or carboxyl terminus of the γ-lactamase. These two constructs were heterogeneously expressed in E. coli rosetta (DE3) as two fusion proteins. Both of them were immobilized well on Avicel (microcrystalline cellulose matrix). The apparent kinetic parameters revealed that carboxyl terminus fused protein (Gla-linker-Cbd) was a better catalyst. The Vmax and kcat value of Avicel immobilized Gla-linker-Cbd were 381 U mg-1 and 4.7u2009×u2009105xa0s-1 respectively. And the values of the free Gla-linker-Cbd were 151 U mg-1 and 1.8u2009×u2009105xa0s-1 respectively. These data indicated that the catalytic efficiency of the enzyme was upgraded after immobilization. The immobilized Gla-linker-Cbd had a 10-degree temperature optimum dropping from 80°C to 70°C but it was stable when incubated at 60°C for 48xa0h. It remained stable in catalyzing 20-batch reactions. After optimization, the immobilized enzyme concentration in transformation was set as 200xa0mg/mL. We found out that there was inhibition that occurred to the immobilized enzyme when substrate concentration exceeded 60xa0mM. Finally a 10xa0mL-volume transformation was conducted, in which 0.6xa0M substrate was hydrolyzed and the resolution was completed within 9xa0h with a 99.5% ee value.ConclusionsCellulose is the most abundant and renewable material on the Earth. The absorption between Cbd domain and cellulose is a bio-green process. The cellulose immobilized fusion Gla exhibited good catalytic characters, therefore we think the cellulose immobilized Gla is a promising catalyst for the industrial preparation of (-) - γ-lactam.

Characterization of a recombinant (−)γ-lactamase from Microbacterium hydrocarbonoxydans

A (−)γ-lactamase, Mhg, from Microbacterium hydrocarbonoxydans was over-expressed in E. coli and was characterized after purification. The maximum activity was at pH 8.0 and 60°C and the half life of Mhg was ~30xa0min at 75°C. The enzyme was activated by DTT. The catalytic triad of the (−)γ-lactamase is comprised of residues Ser98, Asp230, and His259 and an oxyanion hole was formed by Tyr32 and Met99 according to the alignment results. Under native conditions, the (−)γ-lactamase consists of two 31xa0kDa homodimers.

Autodisplay of an archaeal γ-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (-) vince lactam.

At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter Xcc_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of Xcc_Est as an anchoring motif for surface display of γ-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of γ-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated Xcc_Est could efficiently transport γ-lactamase to the cell surface. Compared with the free enzyme, the displayed γ-lactamase exhibited optimum temperature of 30xa0°C other than 90xa0°C, with a substantial decrease of 60xa0°C. Under the preparation system, the engineered E. coli with autodisplayed γ-lactamase converted 100xa0g racemic vince lactam to produce 49.2xa0g (−) vince lactam at 30xa0°C within 4xa0h. By using chiral HPLC, the ee value of the produced (−) vince lactam was determined to be 99.5xa0%. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed γ-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (−) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature.

Transformation of 2-aminoacetophenone to (S)-2-amino-1-phenylethanol by Arthrobacter sulfureus

A new isolate of Arthrobacter sulfureus, when incubated at 50xa0g resting cells (dry cell wt)xa0l-1 with 50xa0g glucosexa0l-1 and 1xa0g 2-aminoacetophenonexa0l-1 in 50xa0mm potassium buffer (pHxa07, 4xa0ml) at 30u2009°C, produced (S)-2-amino-1-phenylethanol (e.e. >99%) with 75% yield in 6xa0h.

Identification and regulation of the catalytic promiscuity of (−)-γ-lactamase from Microbacterium hydrocarbonoxydans

Mhg, a previously reported (−)-γ-lactamase from Microbacterium hydrocarbonoxydans, was identified to have perhydrolase activity by combining structure similarity search with activity assays. Kinetic studies illustrated that perhydrolysis was the native activity owing to lower Km and higher kcat/Km values. Experimental evidence showed that both hydrolysis and perhydrolysis reactions took place at the same active center. Engineering of the putative substrate-binding pocket revealed that Leu233 site played a vital role in the aspects of selective catalysis, soluble protein expression level and optimum temperature shift, etc. The mutants L233A, L233P, and L233T retained (−)-γ-lactamase activity but lost perhydrolase activity, while L233M only kept perhydrolase activity. Substitutions of Leu233 could dramatically influence the state of expressed protein. Computational analysis explicitly explained the relationships between mutations and γ-lactamase activity changes. Our investigations demonstrated that it was an efficient method to identify the enzyme catalytic promiscuity by combining 3D structure alignment with activity validations, and engineering of substrate-binding pocket could serve as a promising way to regulate activities of promiscuous enzymes.

Purification and characterization of a novel NADPH-dependent 2-aminoacetophenone reductase from Arthrobacter sulfureus

A novel 2-aminoacetophenone reductase was purified to homogeneity from Arthrobacter sulfureus BW1010. The enzyme is a monomer with a molecular weight of approximately 60xa0kDa. Using NADPH as coenzyme, it catalyzes the reduction of ketones, especially amine phenyl ketones, and stereospecifically reduces 2-aminoacetophenone to (S)-2-amino-1-phenylethanol (e.exa0>xa099.8%) with the optimal pH at 7.5.