Structure of an Alkaline Pectate Lyase and Rational Engineering with Improved Thermo-Alkaline Stability for Efficient Ramie Degumming.

Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed ß-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca2+-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔTm values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T5015 value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T5015 value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions (4 h, 60 °C, pH 10.0), compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases.

Screening of a Novel Polysaccharide Lyase Family 10 Pectate Lyase from Paenibacillus polymyxa KF-1: Cloning, Expression and Characterization.

Pectate lyase (EC 4.2.2.2) catalyzes the cleavage of α-1,4-glycosidic bonds of pectin polymers, and it has potential uses in the textile industry. In this study, a novel pectate lyase belonging to polysaccharide lyase family 10 was screened from the secreted enzyme extract of Paenibacillus polymyxa KF-1 and identified by liquid chromatography-MS/MS. The gene was cloned from P. polymyxa KF-1 genomic DNA and expressed in Escherichia coli. The recombinant enzyme PpPel10a had a predicted Mr of 45.2 kDa and pI of 9.41. Using polygalacturonic acid (PGA) as substrate, the optimal conditions for PpPel10a reaction were determined to be 50 °C and pH 9.0, respectively. The Km, vmax and kcat values of PpPel10a with PGA as substrate were 0.12 g/L, 289 µmol/min/mg, and 202.3 s-1, respectively. Recombinant PpPel10a degraded citrus pectin, producing unsaturated mono- and oligogalacturonic acids. PpPel10a reduced the viscosity of PGA, and weight loss of ramie (Boehmeria nivea) fibers was observed after treatment with the enzyme alone (22.5%) or the enzyme in combination with alkali (26.3%). This enzyme has potential for use in plant fiber processing.

Cloning, evaluation, and high-level expression of a thermo-alkaline pectate lyase from alkaliphilic Bacillus clausii with potential in ramie degumming.

Alkaline pectate lyases (Pels) have potential application in bioscouring of the textile industry. In this study, a thermo-alkaline Pel (BacPelA) gene from an alkaliphilic Bacillus clausii strain was cloned and overexpressed in Escherichia coli. The mature BacPelA exhibited maximum activity at pH 10.5 and 70 °C and showed high cleavage capability on methylated pectins. BacPelA showed the highest specific activity of 936.2 U mg-1 on ≥85% methylated pectin and 675.5 U mg-1 on standard substrate polygalacturonic acid (PGA) upon evaluation of the absorbance at 235 nm (A235). The K m and k cat values for PGA were 0.54 g l-1 and 346.5 s-1, respectively. Moreover, the 3,5-dinitrosalicylic acid (DNS) assay, which detects the released reducing oligogalacturonic acids, was confirmed to be inaccurate and unsuitable for endo-acting pectinase activity assay because of the difference in the reducibility by DNS reagent between the standard galacturonic acid and the catalytic oligomer products. Significant ramie fiber weight loss was observed following treatment with BacPelA (24.8%) and combined enzyme-chemical method (30.9%), which indicated that the degumming efficiency of BacPelA was the highest of all alkaline and thermostable Pels reported to date. The total activity of the recombinant mature BacPelA reached 8378.2 U ml-1 (A235) by high-cell-density cultivation in fed-batch fermentation with productivity of 239.4 U ml-1 h-1 using E. coli as host, which represents the highest Pel yield reported to date. Therefore, BacPelA, with promising properties for bioscouring, shows potential applications for ramie degumming in the textile industry.