Effects of site-directed mutagenesis of A79Y and T81Q at Loop structure on thermal stability of xylanase XynASP / 中国生物制品学杂志

@#ObjectiveTo identify the sites of thermal stability at Loop structure of xylanase Xyn ASP in Aspergillus saccharolyticus JOP 1030-1 and improve the thermal stability.MethodsThe amino acid sites related to thermal stability of xylanase were predicted,and beneficial mutation sites at Loop structure were screened by Fireprot online server.Singlepoint mutants Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and double-point mutants Xyn(T81Q)and double-point mutants Xyn(LQ)(A79Y/T81Q)were constructed by site-directed mutagenesis.The recombinant mutant plasmid was transformed to E.coli BL21(DE3)and induced by IPTG.The recombinant xylanase was purified by Ni-NTA protein purification kit,and determined for the optimum temperature,thermal stability,optimum p H and p H stability.ResultsBeneficial mutation sites A79Y and T81Q were screened at Loop structure,and the purified protein samples showed high purity.Compared with that of wild type Xyn ASP,the optimum temperature of mutants Xyn(LQ)(A79Y/T81Q)were constructed by site-directed mutagenesis.The recombinant mutant plasmid was transformed to E.coli BL21(DE3)and induced by IPTG.The recombinant xylanase was purified by Ni-NTA protein purification kit,and determined for the optimum temperature,thermal stability,optimum p H and p H stability.ResultsBeneficial mutation sites A79Y and T81Q were screened at Loop structure,and the purified protein samples showed high purity.Compared with that of wild type Xyn ASP,the optimum temperature of mutants Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)increased by 10℃，5℃and 5℃，respectively.After heat treatment at 40℃for 30 min,wild type Xyn ASP retained only 30.2%residual relative activity,while the mutant Xyn(LQ)increased by 10℃，5℃and 5℃，respectively.After heat treatment at 40℃for 30 min,wild type Xyn ASP retained only 30.2%residual relative activity,while the mutant Xyn(T81Q)retained 37.7%,Xyn(T81Q)retained 37.7%,Xyn(A79Y)and Xyn(A79Y)and Xyn(LQ )still retained more than 65%.After heat treatment at 60℃for 30 min,the enzyme activity of wild type was 7.1%,while that of Xyn(LQ )still retained more than 65%.After heat treatment at 60℃for 30 min,the enzyme activity of wild type was 7.1%,while that of Xyn(LQ)remained 26.4%.The thermal stability of mutants was improved compared with that of wild type Xyn ASP.The optimum p H of Xyn(LQ)remained 26.4%.The thermal stability of mutants was improved compared with that of wild type Xyn ASP.The optimum p H of Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)was 5.0,which was lower than that of wild type Xyn ASP(p H 6.0).The p H stability of Xyn(LQ)was 5.0,which was lower than that of wild type Xyn ASP(p H 6.0).The p H stability of Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)at pH 3.0～8.0 showed no significant change compared with the wild type.ConclusionSite-directed mutagenesis(A79Y and T81Q)was carried out at Loop structure,and xylanase mutants with obviously improved thermal stability were obtained,which laid a foundation of the later research on the structure,function and relationship of the enzyme and its industrial application.

Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum / 生物工程学报

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Engineering the plastic degradation enzyme Ple629 from marine consortium to improve its thermal stability / 生物工程学报

Petrochemical-derived polyester plastics such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT) have been widely used. However, the difficulty to be degraded in nature (PET) or the long biodegradation cycle (PBAT) resulted in serious environmental pollution. In this connection, treating these plastic wastes properly becomes one of the challenges of environment protection. From the perspective of circular economy, biologically depolymerizing the waste of polyester plastics and reusing the depolymerized products is one of the most promising directions. Recent years have seen many reports on polyester plastics degrading organisms and enzymes. Highly efficient degrading enzymes, especially those with better thermal stability, will be conducive to their application. The mesophilic plastic-degrading enzyme Ple629 from the marine microbial metagenome is capable of degrading PET and PBAT at room temperature, but it cannot tolerate high temperature, which hampers its potential application. On the basis of the three-dimensional structure of Ple629 obtained from our previous study, we identified some sites which might be important for its thermal stability by structural comparison and mutation energy analysis. We carried out transformation design, and performed expression, purification and thermal stability determination of the mutants. The melting temperature (Tm) values of mutants V80C and D226C/S281C were increased by 5.2 ℃ and 6.9 ℃, respectively, and the activity of mutant D226C/S281C was also increased by 1.5 times compared with that of the wild-type enzyme. These results provide useful information for future engineering and application of Ple629 in polyester plastic degradation.

The use of micronucleus assay in exfoliated oral cells in patients undergoing fixed orthodontic therapy: a systematic review with meta-analysis

Abstract The aim of this systematic review was to evaluate published papers regarding the micronucleus assay in oral mucosal cells of patients undergoing orthodontic therapy (OT). A search of the scientific literature was made in the PubMed, Scopus, and Web of Science databases for all data published until November, 2021 using the combination of the following keywords: "fixed orthodontic therapy," "genetic damage", "DNA damage," "genotoxicity", "mutagenicity", "buccal cells", "oral mucosa cells," and "micronucleus assay". The systematic review was designed according to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. Nine studies were retrieved. Some authors demonstrated that OT induces cytogenetic damage in oral mucosal cells. Out of the nine studies included, two were classified as strong, five as moderate, and two as weak, according to the quality assessment components of the Effective Public Health Practice Project (EPHPP). Meta-analysis data revealed no relationship between mutagenicity in oral cells and OT in different months of treatment. At one month, the SMD = 0.65 and p = 0.08; after three months of OT, the SMD = 1.21 and p = 0.07; and after six months of OT, the SMD = 0.56 and p = 0.11. In the analyzed months of OT, I2 values were >75%, indicating high heterogeneity. In summary, this review was not able to demonstrate that OT induces genetic damage in oral cells. The study is important for the protection of patients undergoing fixed OT, given that mutagenesis participates in the multi-step process of carcinogenesis.

Mutagenic damage among bronchiectasis patients attending in the pulmonology sector of a hospital in southern Brazil

SUMMARY OBJECTIVE: Bronchiectasis is a chronic respiratory disease characterized by inflammation, irreversible dilation of the bronchi, and recurrent pulmonary infections, with a high morbidity and mortality rate, but is less studied from the point of view of its prevalence and associated factors not directly related to respiratory prognosis. As it is a disease related to the exacerbation of the inflammatory process and oxidative stress, this study searched to investigate the micronucleus frequency in patients with and without bronchiectasis treated at a specialized pulmonology service in a hospital in the extreme south of Brazil. METHODS: Patients with a confirmed tomographic diagnosis of bronchiectasis were defined as cases. Mutagenicity was evaluated by the micronucleus test in patients' oral mucosa cells. Data collection was performed through a questionnaire containing socioeconomic, demographic, lifestyle, and health condition information. RESULTS: Of the 95 patients involved in this study, 21 (22.1%) were diagnosed with bronchiectasis aged between 12 and 89 years. There was no significant difference in the frequency of micronucleus between patients with and without bronchiectasis. There was a significant positive association between age and frequency of micronucleus among patients with bronchiectasis, but this association does not occur among patients without the disease. CONCLUSION: This is the first study to investigate data on the prevalence and clinical and epidemiological aspects of this chronic disease in Brazil, especially those related to the genotoxicity outcome.

Mutagénesis inducida en plantas de Solanum betaceum Cav. mediante el uso de dietil sulfato (DES) / Induced mutagenesis in Solanum betaceum Cav. plants by the use of diethyl sulphate (DES)

RESUMEN La materia prima del fitomejoramiento es la variabilidad genética, que se presenta baja, en especies en proceso de domesticación, que no han sido sometidas a selección, como en Solanum betaceum. Una de las tecnologías para incrementar la variabilidad genética es la inducción de mutagénesis. El objetivo del estudio fue evaluar, a través de marcadores RAMs, las variaciones moleculares presentes en plántulas de S. betaceum, provenientes de semillas sometidas a diferentes concentraciones del agente mutante dietil sulfato (DES). Los loci polimórficos oscilaron entre 87,5 y 100 % y el número de alelos efectivos (Ne), entre 1,0 y 1,99. Los loci más polimórficos se observaron en TG, AG, ACA y CGA, que mostraron una heterosis media insesgada entre 0,34 y 0,51, que permite establecer que estos marcadores sean útiles para obtener mayor discriminación entre mutantes en S. betaceum. Las distancias genéticas oscilaron entre 0,30 y 1,0. El 81,28 % de estos registros se dieron entre 0,60 y 0,90; esto revela bajo nivel de cambios, debido al DES. Estos pequeños cambios contribuyeron a enriquecer la variabilidad genética de la muestra tratada con DES. Los marcadores RAMs fueron útiles para detectar cambios entre plantas provenientes de semillas tratadas con DES y plantas normales. La variabilidad genética entre tratamientos con DES fueron más altos que tratamientos sin DES. Las similitudes genéticas fueron bajas entre plantas tratadas y no tratadas y fueron altas, entre no tratadas. Los cambios producidos por DES fueron de baja magnitud; sin embargo, produjeron cambios en los niveles de variabilidad genética.

ABSTRACT The raw material for plant breeding is genetic variability, which is low in species in the process of domestication that have not been subjected to selection, as is the case with Solanum betaceum. One of the technologies to increase genetic variability is mutagenesis induction. The objective was to evaluate, through RAMs markers, the molecular variations present in S. betaceum seedlings from seeds previously subjected to different concentrations of the mutant agent diethyl sulfate (DES). The polymorphic loci ranged from 87.5 to 100%, number of effective alleles (Ne) between 1.0 and 1.99. The most polymorphic loci were observed in TG, AG, ACA, and CGA, which showed a mean unbiased heterosis between 0.34 and 0.51 with an average of 0.44, which allows establishing that these markers are useful to obtain greater discrimination between mutants in S. betaceum. Genetic distances ranged from 0.30 to 1.0. The 81.28% of these records were between 0.60 and 0.90. This reveals a low level of changes due to DES. These small changes contribute to enriching the genetic variability of the DES-treated sample. The RAMs markers were useful for detecting changes between plants from DES treated seeds and normal plants. Genetic variability between DES treatments was higher than non-DES treatments. Genetic similarities were low between treated and untreated plants and were high among untreated plants. The changes produced by DES were of low magnitude, however, they produced changes in the levels of genetic variability.

Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design / 生物工程学报

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

Identification and verification of key salt-tolerant amino acid sites of banana MaNHX5 / 生物工程学报

In order to improve the salt tolerance of banana NHX genes, we cloned a MaNHX5 gene from Musa acuminata L. AAA group and predicted the key salt-tolerant amino acid sites and mutant protein structure changes of MaNHX5 by using bioinformatics tools. The 276-position serine (S) of MaNHX5 protein was successfully mutated to aspartic acid (D) by site-directed mutagenesis, and the AXT3 salt-sensitive mutant yeast was used for a functional complementation test. The results showed that after the mutated MaNHX5 gene was transferred to AXT3 salt-sensitive mutant yeast, the salt tolerance of the mutant yeast was significantly improved under 200 mmol/L NaCl treatment. It is hypothesized that Ser276 of MaNHX5 protein plays an important role in the transport of Na+ across the tonoplast.

Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations / 生物工程学报

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

Advances in the microbial production of the compatible solute ectoine: a review / 生物工程学报

Ectoine is an amino acid derivative and an important natural product in halophilic microorganisms. It plays an important role in protecting cells and stabilizing biological macromolecules, and can be widely used in biomedical fields such as drug preparation adjuvants, organ transplantation and preservation, skin wound repair and cosmetics. Due to the medical value and commercial market demand of ectoine, this article summarized the recent advances in the microbial production of ectoine, including the mutation and breeding of hyper-producing strains, construction of genetically and metabolically engineered strains, optimization of fermentation processes, and extraction and purification processes. The application of multi-omics technologies and computational biology to develop an ectoine producing cell factory was prospected, with the aim to provide a reference for ectoine overproduction.

Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis / 生物工程学报

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower Km value and a higher kcat/Km value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.

Panorama mundial de las diferentes plataformas de vacunas contra el COVID-19: revisión y reflexión de la literatura actual / Worldwide Overview of the Different COVID-19 Vaccine Platforms: A Review and Reflection on the Current Evidence

RESUMEN Las vacunas son productos biológicos que contienen antígenos que buscan generar protección contra a la exposición real de un agente patógeno. En cuanto a su importancia, hacen parte de las intervenciones más costo-efectivas en salud pública, siendo superadas únicamente por el agua potable. A grandes rasgos, podemos dividir las vacunas en vivas atenuadas e inactivadas; no obstante, el nuevo coronavirus ha producido la emergencia de plataformas innovadoras que utilizan mecanismos intracelulares y moleculares con el mismo objetivo de generar inmunidad. Se realizó una búsqueda sistemática de la literatura utilizando las bases de datos electrónicas Pubmed, Scopus y Web of Science. Todos los tipos de diseño de estudio fueron considerados, sin embargo, se prefierieron aquellos redactados en idioma inglés o español. Se hace una revisión de la literatura presente sobre las plataformas existentes para generar inmunidad frente al coronavirus SARS-CoV-2 y se desarrolla cada una según su ruta y forma de acción en aquellas basadas en subunidades proteicas, vector viral recombinante, ácidos nucleicos, virus inactivados, partículas virales y virus vivos atenuados. Los mecanismos por los cuales dichas vacunas generan inmunogenicidad son diferentes, no obstante, la constante inserción de mutaciones por parte del virus sigue siendo un objeto de interés y preocupación por los investigadores.

ABSTRACT Vaccines are biological products containing antigens that aim to generate protection against real exposure to an infectious pathogen. They constitute the most cost-effective interventions in public health, being surpassed only by drinking water. Generally speaking, we can divide the vaccines into live attenuated and inactive; However, the new coronavirus has produced innovative platforms that use intracellular and molecular mechanisms with the same objective of generating immunity. A systematic literature search was carried out using the PUBMED, SCOPUS, and Web of Science electronic databases. All types of study design were selected, those written in English or Spanish were prioritized. We reviewed the existing platforms to generate immunity against the SARS-CoV-2 coronavirus. Each one is developed according to its route and form of action, and can be classified as protein subunits, recombinant viral vector, nucleic acids, inactivated viruses, viral particles, and live attenuated viruses. The mechanisms by which these vaccines generate immunogenicity are different; however, the constant insertion of mutations by the virus remains an object of interest and concern for researchers.

Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene / 生物工程学报

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.

Evaluation of the production of antifungal metabolites against Colletotrichum gloeosporioides in Streptomyces 5.1 by random mutagenesis

Streptomyces 5.1 is a bacterium isolated from rice soils in the south of the Tolima department (Colombia). This microorganism is characterized by its antagonistic activity against rubber tree phytopathogens like Colletotrichum gloeosporioides, the causal agent of leaf anthracnose. The antifungal activity of this Streptomyces isolate has been associated with secondary metabolites production. However, the identity of those metabolites is unknown because its purification and identification have not been possible through classic chemical studies. Therefore, aiming to contribute in the study of the secondary metabolites produced by 5.1 from a molecular approach, this research seeks to identify -preliminarily- the genomic fingerprint changes associated with the production of antifungal secondary metabolites produced by Streptomyces 5.1 through the evaluation of a mutant library of 5.1 obtained by random mutagenesis using controlled ultraviolet light exposure. The antifungal activity of obtained mutants was evaluated using Colletotrichum gloeosporioides (C1) fungus as a biosensor, isolated by the Biotechnology Institute of Universidad Nacional de Colombia. In this way, the library of mutants of 5.1, initially formed by 300 isolations, was classified into two phenotypic groups of interest: enhanced mutants (1 isolate) and null mutants (11 isolates) of secondary metabolites. The genomic changes in both groups were analyzed by obtaining the genomic profile of the isolates using Repetitive Extragenic Palindromic (Rep-PCR). The obtained profiles evidenced the presence of one additional band in the enhanced mutant, and the absence of a specific band in the non-producing mutants, both in comparison with the original strain. These bands are proposed for a future sequencing study which will define their role in the production process of metabolites with antifungal activity in Streptomyces 5.1.

Vitamin D-dependent rickets (VDDR) type 1: case series of two siblings with a CYP27B1 mutation and review of the literature / Raquitismo dependente de vitamina D: tipo 1: série de casos de dois irmãos com mutação CYP27B1 e revisão da literatura

Abstract Two siblings presented with clinical and biochemical features of rickets, initially suspected as hypophosphatemic rickets. There was no improvement initially, hence the siblings were reinvestigated and later diagnosed as having vitamin D-dependent rickets (VDDR) type 1 due to a rare mutation in the CYP27B1 gene encoding the 1α-hydroxylase enzyme. Both siblings improved with calcitriol supplementation. The initial presentation of VDDR is often confusing and algorithmic evaluation helps in diagnosis. We also present a brief review of the literature, including genetics.

Resumo Dois irmãos apresentaram características clínicas e bioquímicas do raquitismo, com suspeita clínica inicial de raquitismo hipofosfatêmico. Não houve melhora no início, portanto os irmãos foram reavaliados e, posteriormente, diagnosticados com raquitismo dependente de vitamina D (VDDR) tipo 1 devido a uma rara mutação no gene CYP27B1, que codifica a enzima 1a-hidroxilase. Ambos os irmãos melhoraram com a suplementação de calcitriol. A apresentação inicial do VDDR geralmente é confusa e a avaliação algorítmica ajuda no diagnóstico. Também apresentamos uma breve revisão da literatura, incluindo genética.

Response surface methodology-artificial neural network based optimization and strain improvement of cellulase production by Streptomyces sp / Metodologia de superfície de resposta - otimização baseada em redes neurais artificiais e melhoria de estirpes na produção de celulases por Streptomyces sp

Thirty-seven different colonies were isolated from decomposing logs of textile industries. From among these, a thermotolerant, gram-positive, filamentous soil bacteria Streptomyces durhamensis vs15 was selected and screened for cellulase production. The strain showed clear zone formation on the CMC agar plate after Gram's iodine staining. Streptomyces durhamensis vs15 was further confirmed for cellulase production by estimating the reducing sugars through the dinitrosalicylic acid (DNS) method. The activity was enhanced by sequential mutagenesis using three mutagens of ultraviolet irradiation (UV), N methyl-N'-nitro-N-nitrosoguanidine (NTG), and Ethyl methanesulfonate (EMS). After mutagenesis, the cellulase activity of GC23 (mutant) was improved to 1.86-fold compared to the wild strain (vs15). Optimal conditions for the production of cellulase by the GC 23 strain were evaluated using Response Surface Methodology (RSM) and Artificial Neural Network (ANN). The effects of pH, temperature, duration of incubation, and substrate concentration on cellulase production were evaluated. Optimal conditions for the production of cellulase enzyme using Carboxymethyl cellulose as a substrate are 55 ºC of temperature, pH of 5.0, and incubation for 40 h. The cellulase activity of the mutant Streptomyces durhamensis GC23 was further optimized to 2-fold of the activity of the wild type by RSM and ANN

Trinta e sete colônias diferentes foram isoladas de toras em decomposição das indústrias têxteis. Dentre estes, uma bactéria do solo filamentosa termotolerante, Gram-positiva, Streptomyces durhamensis vs15, foi selecionada e rastreada quanto à produção de celulase. A cepa mostrou uma formação de zona clara na placa de ágar CMC após a coloração com iodo Gram. Streptomyces durhamensis vs15 foi ainda confirmado para a produção de celulase, estimando os açúcares redutores pelo método do ácido dinitrosalicílico (DNS). A atividade foi aprimorada por mutagênese sequencial usando três mutagênicos de irradiação ultravioleta (UV), N metil-N'-nitro-N-nitrosoguanidina (NTG) e metanossulfonato de etil (EMS). Após a mutagênese, a atividade celulase do GC23 (mutante) foi melhorada para 1,86 vezes em comparação com a cepa selvagem (vs15). As condições ideais para a produção de celulase pela cepa GC 23 foram avaliadas usando a Metodologia de Superfície de Resposta (RSM) e a Rede Neural Artificial (RNA). Os efeitos do pH, temperatura, duração da incubação e concentração de substrato na produção de celulase foram avaliados. As condições ideais para a produção da enzima celulase usando Carboximetilcelulose como substrato são 55 ° C de temperatura, pH de 5,0 e incubação por 40 h. A atividade da celulase do mutante Streptomyces durhamensis GC23 foi ainda otimizada para 2 vezes a atividade do tipo selvagem por RSM e RNA.

Enhanced pyruvic acid yield in an osmotic stress-resistant mutant of Yarrowia lipolytica

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.

Optimized inverse PCR strategy for constructing multilocus mutants efficiently / 生物工程学报

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.

Site-directed mutagenesis of long gene by partial amplification combining with double fragments ligation / 生物工程学报

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

Identifying protein epitopes recognized by monoclonal antibodies / 生物工程学报

To establish a method for identifying protein epitopes recognized by therapeutic monoclonal antibodies, the programmed death receptor-1 (PD-1) was selected as the target protein. Based on the alanine scanning strategy, a rapid expression method of antigen mutants combining site-directed mutagenesis with mammalian cell expression system was established, the conditions for eukaryotic expression element amplification and cell transfection expression were established. 150 PD-1 protein mutants were co-expressed, and the binding ability of these mutants to anti-PD-1 antibody Pembrolizumab was identified. The epitopes of Pembrolizumab were determined based on the binding ability of protein mutants to antibodies and combined with protein structure analysis, which was highly consistent with the reported crystal structure-based epitopes, indicating that this method is simple and accurate and can be used for epitope mapping of therapeutic monoclonal antibodies.