Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002

Abstract

Background Chitinases are enzymes which degrade β-1,4-glycosidid linkages in chitin. The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. However, chitinases are currently produced with low efficiency and poor tolerance, limiting the industrial utility. Therefore, identifying chitinases with higher enzymatic activity and tolerance is of great importance. Methods Primers were designed using the genomic database of Paenibacillus chitinolyticus NBRC 15660. An exochitinase (CHI) was cloned into the recombinant plasmid pET-22b (+) to form pET-22b (+)-CHI, which was transformed into Escherichia coli TOP10 to construct a genomic library. Transformation was confirmed by colony-polymerase chain reaction and electrophoresis. The target sequence was verified by sequencing. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Recombinant protein was purified by Ni-NTA affinity chromatography and enzymatic analysis was carried out. Results The exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). Purification yielded a 13.36-fold enrichment and recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg−1. The optimum pH and temperature for degradation of colloidal chitin were 5.0 and 45 °C, respectively. The enzyme showed high stability, retaining >70% activity at pH 4.0–10.0 and 25–45 °C (maximum of 90 min). The activity of CHI strongly increased with the addition of Ca2+, Mn2+, Tween 80 and urea. Conversely, Cu2+, Fe3+, acetic acid, isoamyl alcohol, sodium dodecyl sulfate and β-mercaptoethanol significantly inhibited enzyme activity. The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc)2 as products. Conclusions This is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste.

Production of Thermophilic Chitinase by Paenibacillus sp. TKU052 by Bioprocessing of Chitinous Fishery Wastes and Its Application in N-acetyl-D-glucosamine Production

“…TKU052 chitinase is specific to the linkages of GlcNAc-GlcNAc. Likewise, a similar phenomenon was observed earlier in chitinases from P. chitinolyticus UMBR 0002 [1], P. barengoltzii CAU904 [49], and P. elgii HOA73 [33]. In addition, the physical form of chitin significantly affected the activity of Paenibacillus sp.…”

Gene Cloning, Characterization, and Molecular Simulations of a Novel Recombinant Chitinase from Chitinibacter Tainanensis CT01 Appropriate for Chitin Enzymatic Hydrolysis

“…The optimum temperature for chitinase 1198 activity was determined by incubating enzyme preparations with p-NP-(GlcNAc) 2 substrate at various temperatures between 30 and 90 °C at pH 7.0. Briefly, 21-μL protein samples (0.153 M) were added into 300 μL of 0.18 mM p-NP-(GlcNAc) 2 in 50 mM Tris-HCl buffer (pH = 7.0) and incubated at different temperatures for 1 h. The reaction was ended by adding 10 μL of 1 M NaOH, and the amount of p-nitrophenol released from p-NP-(GlcNAc) 2 was measured by recording absorbance at 405 nm [ 20 ]. To determine the thermostability of chitinase 1198 activity, 21 μL of protein samples (0.153M) were preincubated at different temperatures (20 °C to 90 °C) without p-NP-(GlcNAc)2 substrate for 1 h. After preincubation, protein samples were placed in ice for 30 min.…”

“…To determine the thermostability of chitinase 1198 activity, 21 μL of protein samples (0.153M) were preincubated at different temperatures (20 °C to 90 °C) without p-NP-(GlcNAc)2 substrate for 1 h. After preincubation, protein samples were placed in ice for 30 min. Then, these samples were followed by the addition of 300 μL of 0.18 mM p-NP-(GlcNAc)2 to 50 mM Tris-HCl buffer (pH = 7.0) and then incubated for another 1 h [ 20 ]. Moreover, the activity of the non-heated enzyme was defined as 100%.…”

Bioprocessing of Squid Pens Waste into Chitosanase by Paenibacillus sp. TKU047 and Its Application in Low-Molecular Weight Chitosan Oligosaccharides Production

“…Endo-chitosanase degrades chitosan at a random position and releases COS, thus it is the major enzyme responsible for COS production [ 2 ]. Until now, numerous bacteria have been explored for their chitosanase producing ability, such as Bacillus [ 10 , 15 , 18 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ], Paenibacillus [ 8 , 13 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ], Acinetobacter [ 42 , 43 ], Streptomyces [ 14 , 44 ], Serratia [ 45 ], and Pseudomonas [ 46 ]. For the production of chitosanase via bacterial fermentation, chitin is the common carbon and nitrogen (C/N) supplement.…”

“…Paenibacillus was separated from the Bacillus genus in 1993 with around 200 species [ 58 ]. Many strains of this genus have revealed various biological activities, with potential applications in medical, agricultural, and industrial sectors [ 8 , 13 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 54 , 57 , 58 ]. Recently, marine chitinous wastes including shrimp heads, crab shells, and squid pens were extensively used for the production of chitinolytic enzymes, proteases, antidiabetic drugs, and exopolysaccharides by Paenibacillus species [ 4 ].…”