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

Abstract

Background: γ-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.

Results: We 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 V(max) and k(cat) value of Avicel immobilized Gla-linker-Cbd were 381 U mg⁻¹ and 4.7 × 10⁵ s⁻¹ respectively. And the values of the free Gla-linker-Cbd were 151 U mg⁻¹ and 1.8 × 10⁵ s⁻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 48 h. It remained stable in catalyzing 20-batch reactions. After optimization, the immobilized enzyme concentration in transformation was set as 200 mg/mL. We found out that there was inhibition that occurred to the immobilized enzyme when substrate concentration exceeded 60 mM. Finally a 10 mL-volume transformation was conducted, in which 0.6 M substrate was hydrolyzed and the resolution was completed within 9 h with a 99.5% ee value.

Conclusions: Cellulose 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.

Figure 1

Construction of cbd-linker-gla and gla-linker-cbd fusion genes (A) and expression of the two constructs (B) and adsorption isotherm measurements for crude fusion proteins (C). A. his6: his-tag encoding gene; thr and s tag: thrombin cleavage site and S peptide tag on pET30; B. 1.E. coli rosetta (DE3) harboring pET30; 2.E. coli rosetta (DE3) harboring pETcbdgla; 3.E. coli rosetta (DE3) harboring pETglacbd; 4.Purified Cbd-Linker-Gla; 5.Purified Gla-Linker-Cbd C. The total activity of Gla-Linker-Cbd supernatant at 0 h (5500 U) was set as 100% relative activity.

Figure 2

Comparison of the optimal temperatures (A) and thermostabilities (B) between free Gla-Linker-Cbd protein and its immobilized counterpart, and thermostability of immobilized Gla-Linker-Cbd at 60°C and 70°C (C) and batch stability (D). A. Optimal temperature; B. Thermostability. The activity of immobilized Gla-Linker-Cbd at 70°C (A) or firstly incubated at 30°C for 30 min (B, 145 ± 12 U mg^-1) was set as 100% relative activity. In B, firstly enzymes were incubated at designated temperature for 30 min then applied for catalysis. The activity of immobilized Gla-Linker-Cbd in cubated at 60°C (C) or the first batch (D, 116 ± 6 U mg^-1) was set as 100% relative activity.

Figure 3

Optimal concentration of immobilized enzyme (A) and substrate (B), and time course of the optimized transformation (C) and chiral HPLC analyses of the (rac)-γ-lactam control (D) and sample after the 9 h transformation (E). The activity of 200 mg/ml immobilized Gla-Linker-Cbd at 60°C (A, 120.6 ± 5.2 μmol/min) and immobilized Gla-Linker-Cbd to 60 mM substrate (B, 338.6 ± 15.2 μmol/min) was set as 100% relative activity. The line in dot in B represents no substrate inhibition to enzyme. C: Substrate concentration 0.6 M, immobilized enzyme concentration 200 mg/ml, transformation temperature 60°C.