ABSTRACT
@#ObjectiveTo screen a high alkaline xylanase-producing strain,subject to molecular identification and characterization of enzymatic property,and optimize its fermentation condition.MethodsThe farmland soil samples from Shanxi,Henan and Zhejiang Provinces were collected aseptically,from which the high xylanase-producing strains were screened by enrichment culture,isolation and identification of 16S r DNA,and determined for enzymatic properties.The carbon source(xylan,lactose,soluble starch,sucrose,bran and glucose),nitrogen source[ammonium oxalate,peptone,yeast powder,(NH_4)_2SO_4,NH_4Cl,Na NO_3,urea,beef extract,bean powder,KNO_3,(NH_4)_2HPO_4or random mixture of two of peptone,yeast powder,beef extract,(NH_4)_2HPO_4and bean powder],metal ion[CuCl_2,MgCl_2,ZnCl_2,Al_2(SO_4)_3,CoCl_2,MnCl_2,AgNO_3,NaCl,CaCl_2,FeCl_2,BaCl_2 and FeCl_3],pH value(3.0～10.0)in medium and the fermentation temperature(25,28,30,33,35,37 and 40℃)were optimized by single factor test,while the contents of components[xylan,(NH_4)_2HPO_4and bean powder]by response surface method.The high alkaline xylanase-producing strain was fermented by using the optimized medium under the optimized condition,and determined for xylanase activity,and the result was compared with that predicated in model.ResultsA high alkaline xylanase-producing strain named as SX6-18 was screened and identified as Paemibacillus based on 16s rDNA sequence analysis.The xylanase produced by the strain maintained more than 70%of relative activity at temperatures of 25～55℃and pH 6.0～10.0.Mg(2+)promoted while Fe(2+)promoted while Fe(3+),Mn(3+),Mn(2+)and Cu(2+)and Cu(2+)inhibited the activity of xylanase.The optimal carbon source,nitrogen source and metal ion of the medium were xylan,bean powder+(NH_4)_2HPO_4 and Fe(2+)inhibited the activity of xylanase.The optimal carbon source,nitrogen source and metal ion of the medium were xylan,bean powder+(NH_4)_2HPO_4 and Fe(3+),while the optimal p H value and temperature for fermentation were 8.0 and 30℃，respectively.However,the optimal component contents were 15.00 g/L xylan,3.03 g/L(NH_4)_2HPO_4and 3.28 g/L bean powder.The activity of xylanase cultured in optimized medium under opti-mized condition for fermentation reached 701.08 U/m L,which was closed to the expected value(693.96 U/m L).ConclusionA high alkaline xylanase-producing strain SX6-18 was successfully screened,which maintained relatively high enzyme activity in alkaline condition.This study laid a foundation of application of xylanase in papermaking and washing fields.
ABSTRACT
There is no consensus on the content, accumulation, transformation and content determination methods of phenolic acids in fresh Salvia miltiorrhiza. In order to find out the true content of phenolic acids in fresh S. miltiorrhiza, a variety of treatment me-thods were used in this study to prepare sample solution. The content changes of phenolic acids in S. miltiorrhiza samples with different dehydration rates were investigated during drying and shade drying processes. Polyphenol oxidase(PPO) of S. miltiorrhiza was extracted and purified by ammonium sulfate precipitation and dialysis to investigate the enzymatic properties. The content of rosmarinic acid, lithosperic acid and S. nolic acid B in S. miltiorrhiza was determined by UPLC. The results showed that the content of phenolic acids in fresh S. miltiorrhiza was highest when it was homogenized with 1 mol·L~(-1) HCl solution or 1 mol·L~(-1) HCl methanol solution. There was no significant difference in the content of phenolic acids in S. miltiorrhiza with different dehydration rates, indicating that there was no correlation between phenolic acid content and dehydration rate. The optimum pH of S. miltiorrhiza PPO was 7.6 and the optimum temperature was 40 ℃. With catechol as substrate, S. miltiorrhiza PPO had the enzymatic browning reaction which was in compliance with Michaelis equation, with Michaelis constant K_m of 0.12 mol·L~(-1) and V_(max) of 588.23 U·min~(-1). The inhibitory effect of citric acid, disodium ethylenediamine tetraacetate, ascorbic acid and sodium sulfite on S. miltiorrhiza PPO increased with the increase of inhibitor concentration, and sodium sulfite showed the strongest inhibitory effect. The present study proved that there were a large number of phenolic acids in fresh S. miltiorrhiza, which were the secondary metabolite of primitive accumulation during the growth of S. miltiorrhiza, rather than the induced product of postharvest drying and dehydration stress. This study has reference value and significance for the cultivation, harvest and processing of S. miltiorrhiza.
Subject(s)
Catechol Oxidase , Desiccation , Hydroxybenzoates , Plant Roots , Salvia miltiorrhizaABSTRACT
Wheat quiescin sulfhydryl oxidase was expressed in Escherichia coli for developing a new biological flour improver. The synthesized wqsox gene was constructed into the vector pMAL-c5x and expressed in E. coli, then the expression conditions of recombinant protein was optimized. The MBP fusion label in recombinant protein was removed by protease digestion after affinity purification. Moreover, enzymatic properties of the purified wQSOX and its effect on bread quality were investigated. The synthesized wqsox gene contained 1 359 bp and encoded 453 amino acids with a deduced molecular weight of 51 kDa. The constructed recombinant vector pMAL-c5x-wqsox could successfully express soluble recombinant protein MBP-wQSOX in E. coli Rosetta gamiB(DE3), and the optimal induced expression conditions for recombinant protein were 25 °C, 0.3 mmol/L IPTG and 6 h. MBP fusion tag was cut out by factor Xa protease and wQSOX was prepared after affinity purification. wQSOX could catalyze the oxidation of DTT, GSH and Cys, accompanying the production of H2O2, and exhibited the highest substrate specificity for DTT. Furthermore, enzymatic properties results demonstrated that the optimal temperature and pH for wQSOX catalyzing oxidation of DTT was 50 °C and 10.0, respectively, and wQSOX presented a good stability under high temperature and alkaline environment. The addition of wQSOX with 1.1 U/g flour significantly (P<0.05) increased 26.4% specific volume of the bread, and reduced 20.5% hardness and 24.8% chewiness of bread crumb compared to the control, indicating a remarkable ability to improve the quality of bread.
Subject(s)
Bread , Escherichia coli/genetics , Hydrogen Peroxide , Oxidoreductases , TriticumABSTRACT
The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols.
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β-xylosidase (EC 3.2.1.37) is an important part of the xylanolytic enzymes system. In the present research, β-xylosidase gene Sxa derived from Selenomonas ruminantium was expressed in Pichia pastoris GS115. According to the codon bias and rare codons of P. pastoris, mRNA secondary structure and GC content, Sxa gene was optimized. The optimized full-length gene mSxa was obtained by gene synthesis technique and the recombinant yeast expression vector pPIC9K-mSxa was constructed. After being digested by restriction enzyme BglⅡ, the mSxa gene was transformed into P. pastoris GS115. Then, phenotype and geneticin G418 resistance screening, and PCR were adopted to identify the positive transformants. Finally, the recombinant P. pastoris GS115-pPIC9K-mSxa was obtained. Based on enzymatic activity assay, a high-level expression clone was picked up and then the enzymatic characteristics of the recombinant β-xylosidase were studied. The results showed that the molecular weight of the mSxa expressed in P. pastoris G115 was about 66 kDa. The maximum activity was achieved 287.61 IU/mL at fermenter level. Enzymatic characterization showed the β-xylosidase was stable between 40 ℃ and 60 ℃, and pH between 5.0 and 7.0. The optimal reaction temperature and pH were 55 ℃ and 6.0, and preferentially degrading the β-xylose glycosidic bond. The enzymatic activity was activated by Mn²⁺ and Ca²⁺, and inhibited by Fe³⁺, Cu²⁺, Co²⁺, Mg²⁺, EDTA and SDS. The study indicates that the modified β-xylosidase gene mSxa from Selenomonas ruminantium can express successfully with high activity in P. pastoris. The study lays a foundation for further industrial application of the β-xylosidase.
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A new wood-degrading fungus Monodictys asperospera(Cooke & Massee) Ellis with a high level of laccase production was chosen to study.This laccase was purified by ammonium sulfate precipitation,DEAE-cellulose and sephacryl S-300.Purification of about 8.1 fold was achieved with an overall yield of 5.7%.Its molecular weight was estimated to be about 77 kD.The optimum temperature and pH of the lac-case activity were 55?C and 6.0,respectively.Kinetic studies of the laccase showed that the Km and the Vmax for using syringaldazine as substrate was 0.163 mmol/L and 0.194 mmol/(L.min),respectively.The carbo-hydrate content was 18.14%.In addition,it was found that laccase activity was significantly inhibited by Cu2+.