Structural Insight into and Mutational Analysis of Family 11 Xylanases: Implications for Mechanisms of Higher pH Catalytic Adaptation

<div><p>To understand the molecular basis of higher pH catalytic adaptation of family 11 xylanases, we compared the structures of alkaline, neutral, and acidic active xylanases and analyzed mutants of xylanase Xyn11A-LC from alkalophilic <i>Bacillus</i> sp. SN5. It was revealed that alkaline active xylanases have increased charged residue content, an increased ratio of negatively to positively charged residues, and decreased Ser, Thr, and Tyr residue content relative to non-alkaline active counterparts. Between strands β6 and β7, alkaline xylanases substitute an α-helix for a coil or turn found in their non-alkaline counterparts. Compared with non-alkaline xylanases, alkaline active enzymes have an inserted stretch of seven amino acids rich in charged residues, which may be beneficial for xylanase function in alkaline conditions. Positively charged residues on the molecular surface and ionic bonds may play important roles in higher pH catalytic adaptation of family 11 xylanases. By structure comparison, sequence alignment and mutational analysis, six amino acids (Glu16, Trp18, Asn44, Leu46, Arg48, and Ser187, numbering based on Xyn11A-LC) adjacent to the acid/base catalyst were found to be responsible for xylanase function in higher pH conditions. Our results will contribute to understanding the molecular mechanisms of higher pH catalytic adaptation in family 11 xylanases and engineering xylanases to suit industrial applications.</p></div

Additional file 1: Figure S1. of Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

Schematic diagram of a high-throughput screening of the positive mutant. Table S1. Primers used for random mutagenesis, site-directed mutagenesis and site saturation mutagenesis. (DOC 325 kb

The type of secondary structure and the number of hydrogen bonds between β6 and β7 of family 11 mesophilic xylanases with known structure and pH-dependent activity.

<p>t = 4.667,P = 0.001.</p><p>The type of secondary structure and the number of hydrogen bonds between β6 and β7 of family 11 mesophilic xylanases with known structure and pH-dependent activity.</p

Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery

Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including enhanced oil recovery (EOR), biodegradation, and bioremediation. Rhamnolipid is composed of rhamnose sugar molecule and β-hydroxyalkanoic acid. The rhamnosyltransferase 1 complex (RhlAB) is the key enzyme responsible for transferring the rhamnose moiety to the β-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid. Through transposome-mediated chromosome integration, the RhlAB gene was inserted into the chromosome of the Pseudomonas aeruginosa PAO1-rhlA- and Escherichia coli BL21 (DE3), neither of which could produce rhamnolipid. After chromosome integration of the RhlAB gene, the constitute strains P. aeruginosa PEER02 and E. coli TnERAB did produce rhamnolipid. The HPLC/MS spectrum showed that the structure of purified rhamnolipid from P. aeruginosa PEER02 was similar to that from other P. aeruginosa strains, but with different percentage for each of the several congeners. The main congener (near 60%) of purified rhamnolipid from E. coli TnERAB was 3-(3-hydroxydecanoyloxy) decanoate (C10-C10) with mono-rhamnose. The surfactant performance of rhamnolipid was evaluated by measurement of interfacial tension (IFT) and oil recovery via sand-pack flooding tests. As expected, pH and salt concentration of the rhamnolipid solution significantly affected the IFT properties. With just 250 mg/L rhamnolipid (from P. aeruginosa PEER02 with soybean oil as substrate) in citrate-Na2HPO4, pH 5, 2% NaCl, 42% of oil otherwise trapped was recovered from a sand pack. This result suggests rhamnolipid might be considered for EOR applications

Structural models of six-point mutation sites around the catalytic center in Xyn11A-LC (PDB: 4IXL).

<p>(A) E16Q. (B) L46V/A/G. (C) R48G. (D) S187G. E16, L46, R48 and S187 are shown in green. The corresponding mutation sites are shown in cyan. V46, A46, and G46 are shown in cyan, magenta, and yellow, respectively. Hydrogen bonds and salt bridges are represented in yellow by dashed lines.</p

Structure-based sequence alignment of family 11 mesophilic xylanases with known structure and pH-dependent activity.

<p>Coils, arrows, and the symbol T represent helices, strands, and turns, respectively.</p

Effect of pH on the activity of wild-type Xyn11A-LC and mutants.

<p>(A) pH-dependent relative activities of the wild type and mutants E16Q, W18Y, N44D, R48G, and S187G. (B) pH-dependent specific activities of the wild type and mutants E16Q, W18Y, N44D, R48G, and S187G. (C) pH-dependent relative activities of the wild type and mutants L46V/A/G. (D) pH-dependent specific activities of the wild type and mutants L46V/A/G.</p

Solvent-exposed residues, hydrogen bonds content and the number of ionic bonds of structure-determined family 11 mesophilic xylanases.

<p>Solvent-exposed residues, hydrogen bonds content and the number of ionic bonds of structure-determined family 11 mesophilic xylanases.</p

Engineering Rhamnolipid Biosurfactants as Agents for Microbial Enhanced Oil Recovery

This investigation considered engineered rhamnolipid biosurfactants as EOR agents that potentially could be manufactured at low cost from renewable resources, and have lower toxicity than synthetic EOR surfactants. This particular biosurfactant comes mainly from the microbe Pseudomonas aeruginosa. Disadvantages of working with this strain include that the chemical structures of the produced rhamnolipids are not easily controlled, plus there is a preference to use instead a completely non-pathogenic microbe. Towards that end, the study took the approach to clone the genetic information from a P. aeruginosa strain into E. coli to manipulate systematically the structure of the created rhamnolipids and evaluate their EOR performance by themselves (no co-surfactant or viscosity chemical added). Six E.coli strains (ETRA, ETRAB, ERAC, ETRABC, ETRhl, ETRhl-RC) that carry different combinations of the genes involoved in rhamnolipid bio-synthesis were successfully engineered and tested for their rhamnolipid production. Sand-pack core flooding tests were run to evaluate and compare the effectiveness of these products as agents for enhanced oil recovery. The brine with optimized pH and salt concentration in which a given biosurfactant product has its lowest IFT was used to saturate the core, perform a waterflood, and prepare the surfactant solution. Injection of 6 PV of only a 250 ppm rhamnolipid biosurfactant solution and 4 PV of a brine chaser could recover as much as half of the waterflood residual hydrocarbon (n-octane). The engineered E. coli strains that include more of the implanted genetic code had the better performance in these oil displacement tests. The IFT, biosurfactant concentration and pH of effluents from core flooding were monitored to address EOR mechanisms and quantify the adsorption of each product in the sand pack