Cloning, purification, and characterization of an organic solvent-tolerant chitinase, MtCh509, from Microbulbifer thermotolerans DAU221

Abstract

Background: The ability to use organic solvents in enzyme reactions offers a number of industrially useful advantages. However, most enzymes are almost completely inactive in the presence of organic solvents. Thus, organic solvent-tolerant enzymes have potential applications in industrial processes.

Results: A chitinase gene from Microbulbifer thermotolerans DAU221 (mtch509) was cloned and expressed in Escherichia coli BL21 (DE3). The molecular weight of the expressed MtCh509 protein was approximately 60 kDa, and it was purified by His-tag affinity chromatography. Enzymatic assays showed that the optimum temperature for MtCh509 chitinase activity was 55 °C, and the enzyme was stable for 2 h at up to 50 °C. The optimum pH for MtCh509 activity was in the sub-acidic range, at pH 4.6 and 5.0. The activity of MtCh509 was maintained in presence of 1 M salt, gradually decreasing at higher concentrations, with residual activity (20%) detected after incubation in 5 M salt. Some organic solvents (benzene, DMSO, hexane, isoamyl alcohol, isopropyl alcohol, and toluene; 10-20%, v/v) increased the reactivity of MtCh509 relative to the aqueous system. When using NAG₃, as a substrate, MtCh509 produced NAG₂ as the major product, as well as NAG₄, demonstrating that MtCh509 has transglycosylation activity. The K _m and V _max values for MtCh509 using colloidal chitin as a substrate were 9.275 mg/mL and 20.4 U/mg, respectively. Thus, MtCh509 could be used in extreme industrial conditions.

Conclusion: The results of the hydrolysate analysis and the observed increase in enzyme activity in the presence of organic solvents show that MtCh509 has industrially attractive advantages. This is the first report on an organic solvent-tolerant and transglycosylating chitinase from Microbulbifer species.

Keywords: DAU221; Microbulbifer thermotolerans; Organic solvent-tolerant chitinase; Transglycosylation.

Fig. 1

Alignment of the MtCh509 amino acid sequence with other bacterial chitinases. Similar sequences are marked by boxes and identical sequences are highlighted in red. The chitin-binding domain and the SXGG and DXXDXDXE motifs are marked with black, red, and blue lines under the sequences, respectively. The conserved catalytic proton donor is marked with a black inverted triangle. The conserved α + β insertion domain is marked with a gray line. Secondary structural elements (i.e., alpha helix [α], beta sheet [β], random coil [ƞ], and beta turn [T]) are marked on the MtCh509 sequence. MtCh509: chitinase from M. thermotolerance DAU221 (WP_06714446); S. aga: glycosyl hydrolase from Simiduia agarivorans (WP_015046629); C. jap: glycosyl hydrolase from Cellvibrio japonicus (WP_012488573); C. mix: glycosyl hydrolase from Cellvibrio mixtus (WP_039915554); G. aga: glycosyl hydrolase from Gilvimarinus agarilyticus (WP_041522559); and S. deg: glycosyl hydrolase from Saccharophagus degradans (WP_011468184)

Fig. 2

SDS-PAGE analysis of MtCh509 from M. thermotolerans DAU221. Lane 1, Protein molecular mass marker; lane 2, soluble protein extracted from E. coli cells over-expressing His₆-MtCh509; lane 3, MtCh509 purified by His-tag affinity chromatography; lane 4, zymogram of MtCh509

Fig. 3

Effect of temperature, pH, and NaCl on MtCh509. a Optimal temperature (solid circles) and thermostability (open circles) of MtCh509 under preincubation for 30 min. b Further thermostability of MtCh509 preincubated at 40 °C (open circles), 50 °C (open squares), and 55 °C (open diamonds) for 1–4 h. c Optimal pH (solid) and pH stability (open) of MtCh509. Reactions were performed in the following 50 mM buffers: citrate buffer for pH 3.0–6.0 (circles), sodium phosphate buffer for pH 6.0–8.0 (squares), Tris–HCl buffer for pH 8.0–9.0 (diamonds), and glycine–NaOH buffer for pH 9.0–10.0 (triangles). The means of the relative values (n = 3) and standard deviations are shown

Fig. 4

Effect of NaCl on MtCh509. MtCh509 was preincubated on ice for 30 min with various concentrations of NaCl (0.5–5 M). The negative control was a reaction performed in 50 mM citrate buffer (pH 5.0) at 55 °C without NaCl. The means of the relative values (n = 3) and standard deviations are shown

Fig. 5

TLC analysis of the hydrolysis products of colloidal chitin and chitooligosaccharide (NAG₃–NAG₆) generated by purified MtCh509. The reaction mixture (25 μL), which contained 0.3 μg of purified MtCh509, 50 mM citrate buffer (pH 5.0), and substrates, was incubated at 55 °C for various times (0, 1, 15, 30, 60, 180, 360, 540, or 720 min). A Colloidal chitin was used as a substrate; B NAG₃ was used as a substrate; C NAG₄ was used as a substrate; D NAG₅ was used as a substrate; E NAG₆ was used as a substrate; F NAG₃ was used as a substrate and reacted using 0.6 μg of purified MtCh509. Lane STD is a standard mixture of NAG₁–NAG₆. Lane C is a substrate control without purified MtCh509

Fig. 6

HPLC analysis of the hydrolysate of colloidal chitin generated by purified MtCh509. a Standard peaks of NAG₁–NAG₆. b Hydrolysate of colloidal chitin generated by purified MtCh509 (black arrow, buffer; red arrow, NAG₁; white arrow, NAG₃). The reaction mixture (500 μL), which contained 6 μg of purified MtCh509, 50 mM citrate buffer (pH 5.0), and 1% colloidal chitin, was incubated at 55 °C for 12 h