Directed evolution improves the enzymatic synthesis of L-5-hydroxytryptophan by an engineered tryptophan synthase

L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) was 4.36 mM− 1∙s− 1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

Biochemical Characterization of a Novel Bacterial Laccase and Improvement of Its Efficiency by Directed Evolution on Dye Degradation

Laccase is a copper-containing polyphenol oxidase with a wide range of substrates, possessing a good application prospect in wastewater treatment and dye degradation. The purpose of this research is to study the degradation of various industrial dyes by recombinant laccase rlac1338 and the mutant enzyme lac2-9 with the highest enzyme activity after modification by error-prone PCR. Four enzyme activities improved mutant enzymes were obtained through preliminary screening and rescreening, of which lac2-9 has the highest enzyme activity. There are four mutation sites, including V281A, V281A, P309L, S318G, and D232V. The results showed that the expression of the optimized mutant enzyme also increased by 22 ± 2% compared to the unoptimized enzyme and the optimal reaction temperature of the mutant enzyme lac2-9 was 5°C higher than that of the rlac1338, and the optimal pH increased by 0.5 units. The thermal stability and pH stability of mutant enzyme lac2-9 were also improved. With ABTS as the substrate, the kcat/Km of rlac1338 and mutant strain lac2-9 are the largest than other substrates, 0.1638 and 0.618 s–1M–1, respectively, indicating that ABTS is the most suitable substrate for the recombinant enzyme and mutant enzyme. In addition, the Km of the mutant strain lac2-9 (76 μM) was significantly lower, but the kcat/Km (0.618 s–1M–1) was significantly higher, and the specific enzyme activity (79.8 U/mg) increased by 3.5 times compared with the recombinant laccase (22.8 U/mg). The dye degradation results showed that the use of rlac1338 and lac2-9 alone had no degradation effect on the industrial dyes [indigo, amaranth, bromophenol blue, acid violet 7, Congo red, coomassie brilliant blue (G250)], however, adding small molecular mediators Ca2+ and ABTS at the same time can significantly improve the degradation ability. Compared to the rlac1338, the degradation rates with the simultaneous addition of Ca2+ and ABTS of mutant enzyme lac2-9 for acid violet 7, bromophenol blue and coomassie brilliant blue significantly improved by 8.3; 3.4 and 3.4 times. Therefore, the results indicated that the error-prone PCR was a feasible method to improve the degradation activity of laccase for environmental pollutants, which provided a basis for the application of laccase on dye degradation and other environmental pollutants.

MO061IS THE PM290I MUTATION RELATED TO ORGAN INVOLVEMENT OF FABRY DISEASE?

Background and Aims Fabry disease (FD) is an X-linked hereditary disease. It results from mutations in the GLA gene, leading to deficient activity of the enzyme alpha-galactosidase A and progressive accumulation of undegraded glycosphingolipids in cell lysosomes. Enzyme replacement therapy improved the natural course of this disease, but an early diagnosis is crucial for a successful treatment. Method A screening study for GLA gene mutations was conducted for all patients under dialysis, from a single centre. All the probands with a detectable mutation were analysed individually. Data on the patient's family and personal pathological history were retrospectively collected, by consulting the clinical file. Results 35 years-old female diagnosed with chronic proteinuric kidney disease in the postpartum period. Despite optimal medical treatment the disease progressed, and she started renal replacement therapy with peritoneal dialysis. Five years later she was enrolled in a pilot screening study for FD and the heterozygous mutation c.870G>C (p.Met290Ile; M290I) in exon 6 of the GLA gene was found. The proband didn’t meet the criteria for a definitive FD diagnosis, but she remained under follow-up at our nephrology metabolic diseases consultation, as the mutation was described as pathogenic and associated with a classic FD phenotype. Later that same year, reassessment exams revealed a worsening left ventricle mass index, a new ischemic cerebral lesion and a substantial increase in serum globotriaosylsphingosine (LysoGb3) levels. These clinical changes led to the decision to initiate enzyme replacement therapy. Until now there are only a few descriptions of this genetic variant in the scientific literature. A Portuguese study analysed a total of 11 FD patients and described 2 patients with p.M290I mutation, without detectable Gb3 accumulation. Another study was designed to evaluate the genotype-phenotype relationship in 73 Chinese FD patients. Contrary to other reports, the p.M290I mutation was not associated to the classic FD phenotype. A Swiss investigation with a similar design analysed 69 FD patients during their routine annual examinations. M290I mutant enzyme was found in a 48-year-old heterozygous female with a classic FD phenotype but with a low serum LysoGb3. A Spanish newborn screening identified one male patient with FD and the p.M290I genetic variant but was unable to provide any information about the clinical expression of this mutation, since the diagnosis was made between the third and fifth days of life. The study describing the most patients carrying the M290I mutant enzyme is Brazilian and screened a total of 25,223 dialysis patients. Among 89 FD-positive patients, the p.M290I mutation was present in 22. However, the authors did not provide detailed information about the clinical manifestations or α-Gal A activity and LysoGb3 levels of these patients. Finally, a recent Portuguese screening of 150 hypertrophic cardiomyopathy patients found 25 patients with FD. Of these, one female carried the GLA gene variant p.M290I, with a non-detectable LysoGb3 plasma level. Conclusion We describe a case of FD due to a previously known but still poorly described GLA mutation, which offers strong evidence of its pathogenicity. To our knowledge, this is the first report of p.M290I mutation-associated disease activity evidenced by elevated levels of serum LysoGb3. Despite the absence of classic FD symptoms such as neuropathic pain, cornea verticillata and angiokeratoma, the presence of severe multiple organ evolvement, characterized by renal failure, cardiac disease and ischaemic stroke, strongly suggests a classic phenotype. Consequently, it is our opinion that the presence of a p.M290I GLA mutation should require a strict ongoing patient follow-up, as it may cause clinically significant disease.

γ-Glutamyltranspeptidase essential for the metabolism of γ-glutamyl compounds in bacteria and its application

ABSTRACT The enzymatic characteristics of γ-glutamyltranspeptidase were elucidated. The catalytic nucleophile of the enzymatic reaction of Escherichia coli γ-glutamyltranspeptidase was identified as the Oγ of the N-terminal Thr-residue of the small subunit. It was demonstrated that the inactive precursor of γ-glutamyltranspeptidase is processed autocatalytically and intramolecularly into the active heterodimeric mature enzyme via an ester intermediate. The catalytic nucleophile of this processing reaction was identified as the same Oγ atom of the N-terminal Thr-residue of the small subunit. These results were also supported by the three-dimensional structures of the γ-glutamyl enzyme intermediate and of the precursor-mimicked T391A nonprocessable mutant enzyme. Applications of transpeptidation and hydrolysis activities of bacterial γ-glutamyltranspeptidases were developed. Using transpeptidation activity, efficient enzymatic production of useful γ-glutamyl compounds, such as prodrug for Parkinson's disease, theanine and kokumi compound, was enabled. Hydrolysis activity was used as glutaminase and the mutant enzymes gaining glutaryl-7-aminocephalosporanic acid acylase activity were isolated.

Probing the Role of the Conserved Arg174 in Formate Dehydrogenase by Chemical Modification and Site-Directed Mutagenesis

The reactive adenosine derivative, adenosine 5′-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD+. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (KD = 0.66 ± 0.15 mM, first order maximum rate constant k3 = 0.198 ± 0.06 min−1). NAD+ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD+ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH.

Directed evolution and immobilization of new lipase Lip 906

In this experimental study, a new lipase named Lip 906 was screened out from a metagenomic library in the laboratory. To improve the stability of the enzyme and develop and apply it as soon as possible, we adopted directed evolution and immobilization methods. A random mutation library was constructed by error-prone PCR and finally, a mutant lipase Lip 5-D with increased enzyme activity was screened out and immobilized. The activity of the mutant enzyme Lip 5-D was improved by 4 times compared with the wild-type lipase Lip 906. The optimal reaction temperature rose by 4 °C, and by 3 °C after immobilization. The optimal reaction pH increased from 7.8 to 7.5. Both temperature stability and pH stability were improved. The mutant enzyme Lip 5-D can maintain about 70% of the relative activity after incubation at 65 °C for 2 h, and it can keep 60% at pH 3-10. Error-prone PCR and immobilization improve the catalytic activity and stability of the enzyme, and promote its development and application in many industries.

Thioglycoligase derived from fungal GH3 β-xylosidase is a multi-glycoligase with broad acceptor tolerance

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal β-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.

Directed Evolution and Immobilization of a Novel Lipase LIP 906

Background: The object of this experimental study is a new lipase screened from metagenomic libraries in the early stage of the laboratory and named it LIP 906. In order to improve the stability of the enzyme and develop and apply it as soon as possible, experiments use directed evolution and immobilization.Results: A random mutation library was constructed by error-prone PCR technology, and finally a mutant lipase LIP 5-D with improved enzyme activity was screened out and then immobilized. Compared with the wild-type lipase LIP 906, the enzyme activity of the mutant enzyme LIP 5-D increased 4 times; the optimum reaction temperature was increased by 4 °C by mutation and 3 ℃ by immobilization; and the optimum reaction pH is changed from 7.8 to 7.5; temperature stability and pH stability has been improved. The mutant enzyme LIP 5-D can maintain a relative enzyme activity of about 70% at a temperature below 65 °C for 2 hours, and can also maintain a relative enzyme activity of about 60% at different pH 3 -10.Conclusions: Error-prone PCR and immobilization improved the catalytic activity and stability of the enzyme, and promoted its development and application in many industries. The research on the properties and modification of the new lipase LIP906 provides a solid foundation for my next innovative research in application and environmental protection.