Molecular cloning, sequence analysis, expression and characterization of the endochitinase gene from Trichoderma sp. in Escherichia coli BL21

Abstract

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5-8. Significant activity stimulation by 1 mM Mg 2+ and 5 mM Fe 2+ and inhibition by 5 mM Co 2+ and 5 mM Hg 2+ were observed. The kinetic constants K m , V max and K cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 lmol min -1 and 6.23 min -1 , respectively.

Thermophilic 4-α-Glucanotransferase from Thermoproteus Uzoniensis Retards the Long-Term Retrogradation but Maintains the Short-Term Gelation Strength of Tapioca Starch

“…The disproportionation activity of TuαGT was determined following the method reported by Yu et al 17 by incubating 1% maltooligosaccharides (maltose (G2), maltotriose (G3) and maltotetraose (G4)) in 900 μL assay buffer (50 mM, pH 6.0 sodium citrate buffer) with 100 μL TuαGT (0.2 g/mL) at 75°C 6 h. The rate of glucose released was determinated by high performance liquid chromatography (HPLC) using a Shimadzu LC-20AT pump, a Rheodyne injector and a Shimadzu RID-10A detector, equipped with an Ultimate XB-NH2 column at 30°C using as mobile phase acetonitrile: ultrapure water (7:3, v/v) at a flow rate of 1 mL/min. 14 One unit of disproportionation activity was defined as the amount of enzyme which released 1 μmol of glucose per minute under the above conditions. Effects of Temperature, pH and Metal Ions on the Activity of TuαGT.…”

Codon optimisation improves the expression of Trichoderma viride sp. endochitinase in Pichia pastoris

“…Chitin comprises a large family of glycans that are composed of unbranched homopolymers of β-1,4-linked N-acetyl-glucosamine (GlcNAc), constituting a constantly recycled mass of 10 11 tons in the biosphere per year1234. Chitin is a major component of the cell walls of some microorganisms, including fungi, and invertebrate exoskeletons, including those of insects and crustaceans5678.…”

“…Chitin is a major component of the cell walls of some microorganisms, including fungi, and invertebrate exoskeletons, including those of insects and crustaceans5678. Chitin can also be degraded to functional chito-oligomers that play a role in enhancing immunity, promoting intestinal health, eliminating toxins from the body and inhibiting the growth of tumour cells2910. The degradation of chitin currently involves acidic hydrolysis; however, some of the disadvantages, including the production of acid wastes due to the use of highly concentrated hydrochloric acid, high production costs and serious environmental pollution, cannot be easily overcome2.…”

“…Chitin can also be degraded to functional chito-oligomers that play a role in enhancing immunity, promoting intestinal health, eliminating toxins from the body and inhibiting the growth of tumour cells2910. The degradation of chitin currently involves acidic hydrolysis; however, some of the disadvantages, including the production of acid wastes due to the use of highly concentrated hydrochloric acid, high production costs and serious environmental pollution, cannot be easily overcome2. Therefore, the enzymatic degradation of chitin is gradually becoming the preferred method.…”

“…Chitin can be degraded by chitinases, which are enzymes that are generally divided into two categories: endochitinases and exochitinases211. Endochitinases cleave chitin polymers at random internal sites, whereas exochitinases progressively cleave chitin beginning at the non-reducing end of the chitin chain, releasing N-acetyl-D-glucosamine monomers and diacetyl-chitobiose in the process12.…”