Production of L-alanyl-L-glutamine by immobilized Escherichia coli expressing amino acid ester acyltransferase.

Production of Ala-Gln by the E. coli expressing α-amino acid ester acyltransferase was a promising technical route due to its high enzyme activity, but the continuous production ability still needs to improve. Therefore, the immobilized E. coli expressing α-amino acid ester acyltransferase was applied for the continuous production of Ala-Gln. Four materials were selected as embedding medium for the whole cell entrapment of recombinant bacteria. Calcium alginate beads were found to be the most proper entrapment carrier for production of Ala-Gln. The temperature, pH, and repeatability of the immobilized cell were compared with free cells. Results showed that immobilization cell could maintain a wider range of temperature/pH and better stability than free cell (20-35 °C/pH 8.0-9.0, and 25 °C/pH 8.5, respectively). On this basis, continuous production strategy was put forward by filling the immobilized cell in the tubular reactor with multiple control conditions. The Ala-Gln by immobilization cell achieved the productivity of 2.79 mg/(min*mL-CV) without intermittent time. Consequently, these findings suggest that the immobilization technique has potential applications in the production of Ala-Gln by biotechnological method. KEY POINTS: • Immobilization helps to achieve high efficiency production of Ala-Gln. • Immobilized cells have better stability than free cells. • Sodium alginate is the most suitable immobilized material.

The production of ethanol from lignocellulosic biomass by Kluyveromyces marxianus CICC 1727-5 and Spathaspora passalidarum ATCC MYA-4345.

Efficient bioconversion of lignocellulosic biomass is one of the key challenges for the production of bioethanol and chemicals. Therefore, the present work focuses on finding a robust microorganism able to convert all sugars in lignocellulosic hydrolysates efficiently. The fermentation performance showed that Kluyveromyces marxianus CICC 1727-5 could produce ethanol from glucose with productivity 4.2 g/L/h and higher ethanol yields (0.44 g/g) under 40 °C, outdistance the productivity 0.258 g/L/h of S. passalidarum ATCC MYA-4345. The xylose utilization of S. passalidarum ATCC MYA-4345 was faster than K. marxianus CICC 1727-5 with the ethanol yield 0.31 g/g at 30 °C. However, K. marxianus CICC 1727-5 could produce xylitol from xylose with the yield 0.58 g/g at 40 °C. Meanwhile, the two yeasts both had the ability to use arabinose naturally, but K. marxianus CICC 1727-5 could consume arabinose completely and quickly. Furthermore, the two yeasts both could ferment glucose and xylose simultaneously, but K. marxianus CICC 1727-5 showed much better performance in the cofermentation. The peak ethanol concentration of K. marxianus CICC 1727-5 and S. passalidarum ATCC MYA-4345 was 42.6 and 31.9 g/L, respectively. In the saccharification and cofermentation (SSCF) process using non-detoxificated corncob, K. marxianus CICC 1727-5 showed better performance. K. marxianus CICC 1727-5 was more tolerant in the presence of formic acid, acetic acid, and mix inhibitors and even was capable to grow in the medium with the acetic acid concentration up to 15 g/L. K. marxianus CICC 1727-5 is a promising candidate strain for further metabolic engineering to develop robust industrial strains for the lignocellulosic ethanol.

Production of L-alanyl-L-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase.

BACKGROUND: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln. RESULTS: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles. CONCLUSIONS: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.

[Cloning and expression of alginate lyase genes from Vibrio alginolyticus and characterization of the alginate lyase].

With the discovery of the significant medicinal value of alginate oligosaccharides and bioethanol produced by microalgae, alginate lyase has been the focus of research in all fields. Five alginate lyase genes in cluster from Vibrio alginolyticus were cloned and expressed in Escherichia coli. SDS-PAGE and enzyme activity showed that four of the five genes have the activity to degrade alginate. Optimization of the induction conditions, protein purification and enzyme properties of rAlgV3 with the highest enzyme activity were studied. The results showed that the enzyme activity of recombinant enzyme rAlgV3 increased from 2.34×104 U/L to 1.68×105 U/L, which was 7.3 times higher than before. The optimal reaction temperature was 40 °C, and the enzyme was relatively stable between 4 °C and 20 °C. The enzyme had a higher activity between pH 6.5 and 9.0, with the optimum pH 8.0. It showed a wide range of pH that the alginate lyase can exist stably between pH 4.5 and 9.5. Appropriate concentrations of NaCl and Fe²âº, Fe³âº ions promoted enzyme activity. SDS and Cu²âº ions inhibited the enzyme activity. The enzyme degraded Poly-M fragments and Poly-G fragments, with a wide range of substrate properties. The degraded product of sodium alginate of rAlgV3 analyzed by ESI-MS mainly was oligosaccharides with a polymerization degree of 2 to 3, which means that rAlgV3 was an endo-type alginate lyase. This enzyme has the potential in the development of third-generation bioethanol and the production of alginate oligosaccharides.