Environmental biocatalysis: from remediation with enzymes to novel green processes

Modern biocatalysis is developing new and precise tools to improve a wide range of production processes, which reduce energy and raw material consumption and generate less waste and toxic side-products. Biocatalysis is also achieving new advances in environmental fields, from enzymatic bioremediation to the synthesis of renewable and clean energies and biochemical cleaning of 'dirty' fossil fuels. Despite the obvious benefits of biocatalysis, the major hurdles hindering the exploitation of the repertoire of enzymatic processes are, in many cases, the high production costs and the low yields obtained. This article will discuss these issues, pinpointing specific new advances in recombinant DNA techniques amenable to future biocatalyst development, in addition to drawing the attention of the biotechnology community to the active pursuit and development of environmental biocatalysis, from remediation with enzymes to novel green processes.     

Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) > phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 degrees C, pH 4.5, yielded values of 26, 0.43, and 0.45 microm and 18, 660, and 1175 s(-1), respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.     

Development of chimeric laccases by directed evolution

DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high‐redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α‐factor prepro‐leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high‐throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability. Biotechnol. Bioeng. 2012; 109: 2978–2986. © 2012 Wiley Periodicals, Inc.     

Human Papillomavirus Infection in HIV-1 Infected Women in Catalonia (Spain): Implications for Prevention of Cervical Cancer

Background

High-risk human Papillomavirus infection is a necessary factor for cervical squamous intraepithelial lesions and invasive cervical cancer. In HIV-1-infected women, HPV infection is more prevalent and a higher risk of cervical cancer has been identified. We aimed to calculate the prevalence of infection by HR-HPV, determine the factors associated with this infection and abnormal cytology findings and to describe the history of cervical cancer screening in HIV-1-infected women.

Methods

We enrolled 479 HIV-1–infected women from the PISCIS cohort. Each patient underwent a gynecological check-up, PAP smear, HPV AND Hybrid capture, HPV genotyping, and colposcopy and biopsy, if necessary. We applied questionnaires to obtain information on sociodemographic, behavioral, clinical, and cervical screening variables. We present a cross-sectional analysis.

Results

Median age was 42 years. The prevalence of HR-HPV infection was 33.2% and that of high-grade squamous intraepithelial lesions (HSIL) was 3.8%. The most common genotypes were 16(23%), 53(20.3%), and 52(16.2%). The factor associated with HR-HPV infection was age <30 years (odds ratio[OR],2.5; 95%confidence interval[CI],1.1–5.6). The factors associated with the presence of HSIL or low-grade squamous intraepithelial lesions (LSIL) were CD4T-lymphocyte count <200cells/mm³ versus >500cells/mm³ (OR,8.4; 95%CI,3.7–19.2), HIV-1 viral load >10,000copies/mL versus <400copies/mL (OR,2.1; 95%CI,1.0–4.4), and use of oral contraceptives (OR,2.0; 95%CI,1.0–3.9). Sixty percent of HIV-1–infected women had had one Pap smear within the last 2 years.

Conclusions

The high prevalence of HPV infection and cervical lesions in the HIV-1–infected population in Catalonia, as well as the low coverage and frequency of screening in this group, means that better preventive efforts are necessary and should include vaccination against HPV, better accessibility to screening programs, training of health care professionals, and specific health education for HIV-1–infected women.     

Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra

The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a nonmutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S. cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with approximately 3-fold better kinetics than parent type showed two consecutive amino acid changes (G614D -GGC/GAC- and E615K -GAG/AAG-) because of the in vivo shuffling of the mutant libraries. Both mutations are located in the C-terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C-terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C-terminal tail for the functionality of the ascomycete laccases.     

Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.     

Immobilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F on Eupergit C supports

Dextransucrase from Leuconostoc mesenteroides B-512F was immobilized on epoxy-activated acrylic polymers with different textural properties (Eupergit C and Eupergit C 250L). Prior to immobilization, dextransucrase was treated with dextranase to remove the dextran layer covering the enzyme surface, thus increasing the accessibility of its reactive groups to the epoxide centers of the support. Elimination of 99% of the initial carbohydrate content was determined by the anthrone method. To prevent enzyme inactivation, the immobilization was carried out at pH 5.4, at which the coupling to the support took place through the carboxylic groups of the enzyme. The effects of the amount (mg) of dextransucrase added per gram of support (from 0.2:1 to 30:1), temperature and contact time were studied. Maximum activity recovery of 22% was achieved using Eupergit C 250L. Using this macroporous support, the maximum specific activity (710 U/g biocatalyst) was significantly higher than that obtained with the less porous Eupergit C (226 U/g biocatalyst). The dextransucrase immobilized on Eupergit C 250L showed similar optimal temperature (30 degrees C) and pH (5-6) compared with the native enzyme. In contrast, a notable stabilization effect at 30 degrees C was observed as a consequence of immobilization. After a fast partial inactivation, the dextransucrase immobilized on Eupergit C 250L maintained more than 40% of the initial activity over the following 2 days. The features of this immobilized system are very attractive for its application in batch and fixed-bed bioreactors.     

Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death

We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.