Improvement of the thermostability and catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement

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Abstract

To improve the thermostability and catalytic activity of Aspergillus niger xylanase A (AnxA), its N-terminus was substituted with the corresponding region of Thermomonospora fusca xylanase A (TfxA). The constructed hybrid xylanase, named ATx, was overexpressed in Pichia pastoris and secreted into the medium. After 96-h 0.25% methanol induction, the activity of the ATx in the culture supernatant reached its peak, 633 U/mg, which was 3.6 and 5.4 times as high as those of recombinant AnxA (reAnxA) and recombinant TfxA (reTfxA), respectively. Studies on enzymatic properties showed that the temperature and pH optimum of the ATx were 60 °C and 5.0, respectively. The ATx was more thermostable, when it was treated at 70 °C, pH 5.0, for 2 min, the residual activity was 72% which was higher than that of reAnxA and similar to that of reTfxA. The ATx was very stable over a broader pH range (3.0–10.0) and much less affected by acid/base conditions. After incubation at pH 3.0–10.0, 25 °C for 1 h, all the residual activities of the ATx were over 80%. These results revealed that the thermostability and catalytic activity of the AnxA were enhanced. The N-terminus of TfxA contributed to the observed thermostability of itself and the ATx, and to the high activity of the ATx. Replacement of N-terminus between mesophilic eukaryotic and thermostable prokaryotic enzymes may be a useful method for constructing the new and improved versions of biologically active enzymes.

Section snippets

Materials

The P. pastoris expression kit including the pPIC9K vector, P. pastoris strain GS115, and Escherichia coli strain TOP10F′ were purchased from Invitrogen. Medium components were from Difco. Birchwood xylan was from Sigma Chemical. Restriction endonucleases were from TaKaRa. T4 DNA ligase and PCR kit were from Promega. Primers were synthesized by Sangon. The recombinant pBS-T anx plasmids containing the AnxA gene and pBS-T tfx plasmids containing TfxA gene were stored at −20 °C in our laboratory.

Construction of pPIC9k-atx plasmid and expression of ATx

Amino acid sequence comparison of catalytic domains of TfxA and AnxA is shown in Fig. 1. Fragments A, B, and atx were separated by electrophoresis and recovered (Fig. 2). It was confirmed that the nucleotide sequences of the amplified atx were 573 bp and identical with that of the theoretically designed by DNA sequencing. The nucleotide sequences of atx and its deduced amino acid sequences are shown in Fig. 3. Fig. 4 shows the schematic map of pPIC9K-atx plasmid.

The pPIC9K-atx plasmid was used

Discussion

As a eukaryote, P. pastoris is an attractive host for expression and production of heterologous protein for research and even for industrial purposes. The increasing popularity of this expression system can be attributed to several factors, most importantly: the techniques needed for the molecular genetic manipulation of P. pastoris are relatively simple; it can grow conveniently to high density levels in a simple and inexpensive medium; it is able to form a number of posttranslational

Acknowledgment

This work was supported by the National Natural Science Foundation of China (No. 30471254).

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