Lujia Zhang

A method to rationally increase protein stability based on the charge-charge interaction, with application to lipase LipK107.

We report a suite of enzyme redesign protocol based on the surface charge–charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with ∼10°C higher half‐inactivation temperature than wild‐type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14‐fold increase in the half‐inactivation time at 50°C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.

New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases

Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered K m values from 0.32 (wild type PG8fn) to 0.75–4.74 mg/ml, all mutants displayed remarkably lowered k cat (~3–20,000 fold) and k cat/K m (~8–187,500 fold) values and significantly increased △(△G) values (5.92–33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.

Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

ABSTRACT Improving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) from Achaetomium sp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues—D244 and D299—were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (T max), temperature at which 50% of the maximal activity of an enzyme is retained (T 50), and melting temperature (Tm ), of about 10, 17, and 10.2°C upward, respectively, with the half-life (t 1/2) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ± 130 U/mg versus 28,000 ± 293 U/mg); on the other hand, it showed more residual activity (8,400 ± 83 U/mg versus 1,400 ± 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability.

The Important Role of Halogen Bond in Substrate Selectivity of Enzymatic Catalysis.

The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis.

A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H)

Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen‐bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our novel strategy, Gox2181 was reconstructed from exhibiting complete NADPH preference to exhibiting dual cofactor specificity for NADH and NADPH. Structure‐guided Gox2181 mutants were designed in silico and molecular dynamics simulations were performed to evaluate the strength of hydrogen‐bond interactions between the enzyme and the coenzyme NADPH. Three Gox2181 mutants displaying high structure stability and structural compatibility to NADH/NADPH were chosen for experimental confirmation. Among the three Gox2181 mutants, Gox2181‐Q20R&D43S showed the highest enzymatic activity by utilizing NADPH as its coenzyme, which was even better than the wild‐type enzyme. In addition, isothermal titration calorimetry analysis further verified that Gox2181‐Q20R&D43S was able to interact with NADPH but the wild‐type enzyme could not. This novel computational strategy represents an insightful approach for altering the cofactor preference of target enzymes.

Purification of Xylitol Dehydrogenase and Improved Production of Xylitol by Increasing XDH Activity and NADH Supply in Gluconobacter oxydans

Gluconobacter oxydans is known to be a suitable candidate for producing xylitol from d-arabitol. In this study, the enzyme responsible for reducing d-xylulose to xylitol was purified from G. oxydans NH-10 and characterized as xylitol dehydrogenase. It has been reported that XDH depends exclusively on NAD(+)/NADH as cofactors with a relatively low activity, which was proposed to be the direct reason for its limiting the overall conversion process. To better produce xylitol, an engineered G. oxydans PXPG was constructed to coexpress the XDH gene and a cofactor regeneration enzyme (glucose dehydrogenase) gene from Bacillus subtilis. Activities for both enzymes were more than twofold higher in the G. oxydans PXPG than in the wild strain. Approximately 12.23 g/L xylitol was obtained from 30 g/L d-arabitol by resting cells of the engineered strain with a conversion yield of 40.8%, whereas only 7.56 g/L xylitol was produced by the wild strain with a yield of 25.2%. These results demonstrated that increasing the XDH activity and the cofactor NADH supply could improve the xylitol productivity notably.

Template-based modeling of a psychrophilic lipase: conformational changes, novel structural features and its application in predicting the enantioselectivity of lipase catalyzed transesterification of secondary alcohols.

In order to fully explore the structure-function relationship of a Proteus lipase (LipK107) that was screened from the soil in our previous study, we have modeled the three-dimensional (3-D) structures of the enzyme in its active and inactive conformations on the basis of crystal structures of Burkholderia glumae and Pseudomonas aeruginosa lipases in the present study. Both homology models suggested that LipK107 possessed a catalytic triad (Ser79-Asp232-H254), an oxyanion hole (Leu13 and Gln80) which was used to stabilize the reaction tetrahedral intermediates, and a lid substructure that controlled the access of the substrate to the active site. The existence of the lid was further verified by carrying out the interfacial activation experiment. The conformational change of LipK107 which was caused by lid opening action was predicted by superimposing the two theoretical models for the first time. Finally, both 3-D structures were used to predict the enantioselectivity of LipK107 when the enzyme was used to catalyze the resolution of racemic 1-phenylethanol. Lid-open model of LipK107 identified the R-enantiomer as the preferred enantiomer, while lid-closed mode showed that the S-enantiomer was more favored. However, only the lid-open conformational model could led to predictions that agreed with the following the experimental result of real biocatalysis reaction of 1-phenylethanol.

Novel α-l-Arabinofuranosidase from Cellulomonas fimi ATCC 484 and Its Substrate-Specificity Analysis with the Aid of Computer

In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.

Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of (R)-o-Chloromandelic Acid

ABSTRACT The nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic α-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzing o-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained from Burkholderia cenocepacia strain J2315 (BCJ2315) in hydrolyzing o-chloromandelonitrile. Four hot spots (T49, I113, Y199, and T310) responsible for the enantioselectivity and activity of BCJ2315 were identified by random mutagenesis. An effective double mutant (I113M/Y199G [encoding the replacement of I with M at position 113 and Y with G at position 199]), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess [ee] compared to 89.2% ee for the wild type) and relative activity (360% of the wild type), was created by two rounds of site saturation mutagenesis, first at each of the four hot spots and subsequently at position 199 for combination with the selected beneficial mutation I113M. Notably, this mutant also demonstrated dramatically enhanced enantioselectivity and activity toward other mandelonitrile derivatives and, thus, broadened the substrate scope of this nitrilase. Using an ethyl acetate-water (1:9) biphasic system, o-chloromandelonitrile (500 mM) was completely hydrolyzed in 3 h by this mutant with a small amount of biocatalyst (10 g/liter wet cells), resulting in a high concentration of (R)-o-chloromandelic acid with 98.7% ee, to our knowledge the highest ever reported. This result highlights a promising method for industrial production of optically pure (R)-o-chloromandelic acid. Insight into the source of enantioselectivity and activity was gained by homology modeling and molecular docking experiments.

Cloning of a novel thermostable glucoamylase from thermophilic fungus Rhizomucor pusillus and high-level co-expression with α-amylase in Pichia pastoris.

BackgroundFungal amylase, mainly constitute of fungal α-amylase and glucoamylase, are utilized in a broad range of industries, such as starch hydrolysis, food and brewing. Although various amylases have been found in fungi, the amylases from Aspergillus dominate the commercial application. One of main problems exist with regard to these commercial use of amylases is relatively low thermal and acid stability. In order to maximize the efficiency of starch process, developing fungal amylases with increased thermostability and acid stability has been attracting researchers’ interest continually. Besides, synergetic action of glucoamylase and α-amylase could facilitate the degradation of starch. And co-expressing glucoamylase with α-amylase in one host could avoid the need to ferment repeatedly and improves cost-effectiveness of the process.ResultsA novel fungal glucoamylase (RpGla) gene encoding a putative protein of 512 amino acid residues was cloned from Rhizomucor pusillus. BLAST analysis revealed that RpGla shared highest identity of 51% with the Rhizopus oryzae glucoamylase (ABB77799.1). The fungal glucoamylase RpGla was expressed in Pichia pastoris (KM71/9KGla) with maximum activity of 1237 U ml-1. The optimum pH and temperature of RpGla were pH 4.0 and 70°C, respectively. Fungal α-amylase (RpAmy) gene was also cloned from R. pusillus and transformed into KM71/9KGla, resulted in recombinant yeast KM71/9KGla-ZαAmy harboring the RpGla and RpAmy genes simultaneously. The maximum saccharogenic activity of KM71/9KGla-ZαAmy was 2218 U ml-1, which improved 79% compared to KM71/9KGla. Soluble starch hydrolyzed by purified RpGla achieved 43% glucose and 34% maltose. Higher productivity was achieved with a final yield of 48% glucose and 47% maltose catalyzed by purified enzyme preparation produced by KM71/9KGla-ZαAmy.ConclusionsA novel fungal glucoamylase and fungal α-amylase genes were cloned from Rhizomucor pusillus. The two enzymes showed good thermostability and acid stability, and similar biochemical properties facilitated synergetic action of the two enzymes. A dramatic improvement was seen in amylase activity through co-expressing RpGla with RpAmy in Pichia pastoris. This is the first report of improving activity through co-expression glucoamylase with α-amylase in P. pastoris. Besides, fungal glucoamylase and α-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.