Optimization of Enzymatic Extraction Process of Polysaccharides from Pseudostellaria heterophylla Fibrous Roots by Response Surface Methodology and Its Pilot Application

[Objectives]To study and optimize the process conditions of enzymatic hydrolysis technology for extracting polysaccharides from Pseudostellaria heterophylla fibrous roots and its application in workshop pilot tests.[Methods]P.heterophylla fibrous roots were taken as the matrix material,and Box Behnken design was used to analyze the extraction time,composite enzyme addition amount,and liquid-solid ratio for response surface optimization experiments,and then applied to the pilot extraction of P.heterophylla fibrous roots.[Results]Response surface analysis showed that all factors had a significant impact on the experimental indicators.The optimal extraction process conditions for polysaccharides from P.heterophylla fibrous roots were extraction time of 2.7 h,compound enzyme addition of 2.5%,and liquid-solid ratio of 32.The yield of polysaccharides from P.heterophylla fibrous roots was 4.83%.The water extraction process of P.heterophylla fibrous roots extraction pilot was used as the control group for response surface optimization of the pilot experiment.The optimization results showed that the extraction time was 3 h,the amount of composite enzyme added was 2.5%,and the liquid-solid ratio was 28.The polysaccharide yield was 4.75%,an increase of 4.63%compared to the control group.[Conclusions]This paper could provide feasibility for the innovation of enzy-matic hydrolysis technology for P.heterophylla fibrous roots and its workshop pilot practice application,as well as a reference for the industrial application of its medicinal resources.

Enzymatic Transformation of Notoginsenoside Fe by β-Glucanase

Aim An industrial enzyme β-glucanase was used to transfortn notoginsenoside Fe for the first time. Methods Notoginsenoside Fe was isolated from the leave saponin of Panax notoginseng （Burk.） Chen FH. The enzymatically transformed compounds were detected by HPLC and two transformed compounds were identified as 20 （S） -protopanaxadiol-20- O- α-L-arabinofuranosyl （ 1→6 ） - β-gluco- pyranoside, ginsenoside-Mc） and 20（S）-protopanaxadiol-20-O-β-D-glucopyranoside compound-K （C-K） respectively on the basis of their ^1H NMR and ^13 C NMR spectral data. Results Based on the enzymolytic kinetic curve, the transformation rate of notoginsenoside Fe reached 95% after 24 h. Conclusion The enzymatic transformation pathway of notoginsenoside Fe by β-glucanase has been proposed as notoginsenoside Fe→ginsenoside Mc→C-K.

Enzymatic Degradation of Parvifloside

Parvifloside (1), a new furostanol pentaglycoside, was isolated from the fresh rhizomes of Dioscorea parviflora C. T. Ting. On the basis of spectroscopic and chemical methods, its structure was elucidated as (25R)-26-O-β-glucopyranosyl-furost-5-en-3β,22ξ,26-triol 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosyl (1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside. Six prosapogenins (2-7)were obtained from the enzymatic degradation of 1by cellulase, but only 3 and 4 were obtained by β-glucosidase. The structures of all compounds were determined by spectroscopic data. The activity of the isolated compounds on deformation of mycelia germinated from Pyricularia oaryzae P-2b conidia was evaluated.

Isolating and Screening of Effective Decomposing Strain of Silkworm Chrysalis Protein and Study on Parts of Its Enzymatic Properties

[Objective] The research aimed to obtain an effectively-decomposing strain of silkworm chrysalis protein and discuss its enzymatic properties.[Method] The effectively-decomposing bacteria of protein was isolated from the decayed silkworm chrysalis by using dilution plate and its enzymatic properties were tested after primary screening and second screening.The enzyme activity was determined and the intermediate and small molecule protein content in silkworm chrysalis was measured after solid-state fermentati...

Studies on the Isolation and Screening of α-amylase Producing Strain and Their Enzymatic Properties

[Objective] The aim was to obtain α-amylase producing strains with some excellent properties like high temperature resistance,strong acid resistance,strong alkali resistance,etc..[Method] α-amylase producing strains were isolated and screened; furthermore their enzymatic properties were studied.[Result] 10 strains with an obvious starch hydrolysis cycle were screened out from starch screening plate coated by diluted sample,from which 3 strains with higher α-amylase activity were screened out,that was X6,X8 and X10.As for X6,X8 and X10,their optimum pH values all belonged to neutral,and their optimum temperatures were all 60 ℃.Meanwhile,Ca^2＋ could increase their enzyme thermal stability.When the concentration of Ca^2＋ was 0.02-0.04 mol/L,the enzyme thermal stability of X6 and X8 reached the highest; When the concentration of Ca^2＋ was 0.03-0.04 mol/L,that of X10 reached the highest; When the concentration of Ca^2＋ was increased continuously,those of the 3 strains all decreased.[Conclusion] The research provides theoretical basis for satisfying the demands of different industries for α-amylase with different characteristics.

Advances in Application of Non-aqueous Phase Enzymatic Catalysis in Food Additive Production

Non-aqueous phase enzymatic catalysis technology has been widely ap- plied in the area of food additives production. This paper reviewed the types of re- action medium of non-aqueous phase enzymatic catalysis reaction, introduced the application of non-aqueous phase enzymatic catalysis technology in catalysis of L-ascorbic （isoascorbic） acid esters, short-chain acid esters, sugar esters, vitamin A esters, vi- tamin E esters, and other food additives, and finally predicted the prospects of non- aqueous phase enzymatic catalysis technology.

产β-mannanase内生菌的诱变育种和酶学特性研究

以实验室筛选的产伊甘露聚糖酶的黄豆内生菌Bacillussubtilis HD-1（54．6U／mL）为出发菌株，分别采用紫外线，硫酸二乙酯，紫外线-光复活和紫外线．硫酸二乙酯对其进行诱变，结果表明，以紫外线-硫酸二乙酯复合诱变效果最好，获得-株高产伊甘露聚糖酶的突变菌株BacillussubtilisLID．20，酶活达89．5u／mL，比出发菌株提高了63．9D／01遗传性能稳定。该伊甘露聚糖酶的最适反应温度和pH分别为50℃和7．0，该酶在50℃以下，pH5．0-8．0之间稳定性较好，属于中性酶。亚铁离子和钴离子对酶有较强的激活作用，相对酶活分别提高了13．2％和31．7％，但锰离子对酶有强烈的抑制作用，相对酶活仅有对照组的55．6％；金属离子钾、钙、钴和钡都能增强酶热稳定性，其中钴离子的增强效果最为明显，残余酶活约是对照组的1．5倍。

Effects of Chemical Pretreatment on Enzymatic Saccharification and Fermentation of Forages

[ Objective] To study the effect of pretreatment with chemical substances on enzymatic saccharification of affalfa （ Medic, ago sativa L. ）, sorghum hybrid sudan grass [ Sorghum bicolor （ L. ） Moench x Sorghum sudanese （Piper） Stapf], erect milkvetch （Astraga/us adsurgens Pall. ） and pearl millet （ Pennisetum americanum （ L. ） Leeke）. [ Method ] The forages were pretreated with sulfuric acid at different concentration, and then the content of cellulose, hemicellulose, and lignin were detected and compared with that before pretreatment. The concentration of glucose and ethanol after different fermentation time was also determined. [ Result] After pretreatment, the content of cellulose increased, while that of hemicel- lulose and lignin decreased. After treatment with 1.0% （W/V） sulfuric acid, the four kinds of forages all had the highest concentration of ethanol in the citric acid-sodium citrate buffer system （pH 4.8）. Dudng fermentation process, the concentration of glucose and ethanol first increased and then declined, peaking respectively at 24 h and 48 h post fermentation. [Condusion] Pretreatment promotes the enzymatic saccharification and fermen- tation of alfalfa, sorehum hvbrid sudan orass. Dead millet, and erect milkvetch, and their enerov performance decreases in order.

Effect of Chitosan-Cysteine Conjugate on Enzymatic Degradation and Hypoglycemic effect of Insulin

Aim To evaluate the inhibitory effect of chitosan-cysteine conjugate onenzymatic degradation and hypogly-cemic enhancement effect of insulin. Methods Chitosan-cysteineconjugate was synthesized. The protective effect of the conjugate against degradation of insulin byα-chymotrypsin and trypsin was evaluated in vitro. Insulin enteric- microspheres were prepared byusing O_1 /Q_2 emulsion solvent evaporation method. The hypoglycemic enhancement effect of theconjugate was studied by oral administration of insulin solution or enteric-microspheres to rats.Results The thiol group content of the synthesized conjugate was about 200 μmol·g^(-1) polymer,which showed a strong protective effect on insulin from enzymatic degradation in vitro. Almost allthe insulin incubated in a-chymotrypsin solution or trypsin solution without chitosan-cysteineconjugate was degraded entirely within 1 h and 5 h respectively, whereas above 75% of insulinremained in the same content of the enzymatic solution containing 4 mg·mL^(-1) conjugate. The drugloading of insulin enteric-microspheres was about 7% . In vivo experiment, chitosan-cysteineconjugate (85 μg·kg^(-1)) prolonged the hypoglycemic time of insulin solution orenteric-microspheres when administered simultaneously with the absorption enhancer SNAC. ConclusionChitosan-cysteine conjugate has a marked inhibitory effect on the enzymatic degradation of insulinin vitro, and it displays a significant hypoglycemic enhancement effect on insulin oral formulationin vivo.

Enhanced Enzymatic Hydrolysis of Miscanthus sinensis Following Synergic Pretreatment with ^(60)Co γ-ray Irradiation and Alkaline Hydrogen Peroxide

[Objective] This study aimed to investigate the effects of different pretreat- ments on enzymatic saccharification of Miscanthus sinensis and improve reducing sugar yield in the enzymolysis process. [Method] M. sinensis was pretreated with 60Co y-ray irradiation and alkaline hydrogen peroxide, to analyze their effects on re- ducing sugar yield of enzymatic hydrolysis. [Result] After pretreatment with 400 kGy 60Co y-ray irradiation, reducing sugar yield in the enzymolysis process of M sinensis was 76.24 mg/g; after synergic pretreatment with 400 kGy 60Co y-ray irradiation and alkaline hydrogen peroxide, reducing sugar yield in the enzymolysis process of M. sinensis was 505.08 mg/g, which was improved by 5.6 times compared to that in pretreatment with 400 kGy 60Co y-ray irradiation. Based on process optimization, the optimal hydrolysis conditions were obtained： pretreatment temperature 30 ℃, NaOH concentration 1.2%, hydrogen peroxide concentration 2%, pretreatment time 6 h. [Conclusion] Synergic pretreatment with 60Co y-ray irradiation and alkaline hydrogen peroxide could significantly improve reducing sugar yield in the enzymolysis process of M. sinensis, which provided a new theoretical basis for preparing fuel ethanol with M. sinensis.

Soil enzymatic activities and microbial community structure with different application rates of Cd and Pb

This study focused on the changes of soil microbial diversity and potential inhibitory effects of heavy metals on soil enzymatic activities at different application rates of Cd and/or Pb. The soil used for experiments was collected from Beijing and classified as endoaquepts. Pots containing 500 g of the soil with different Cd and/or Pb application rates were incubated for a period of 0, 2, 9, 12 weeks in a glasshouse and the soil samples were analyzed for individual enzymes, including catalase, alkaline phosphatase and dehydrogenase, and the changes of microbial community structure. Results showed that heavy metals slightly inhibited the enzymatic activities in all the samples spiked with heavy metals. The extent of inhibition increased significantly with increasing level of heavy metals, and varied with the incubation periods. The soil bacterial community structure, as determined by polymerase chain reaction- denaturing gradient gel electrophoresis techniques, was different in the contaminated samples as compared to the control. The highest community change was observed in the samples amended with high level of Cd. Positive correlations were observed among the three enzymatic activities, but negative correlations were found between the amounts of the heavy metals and the enzymatic activities.

Enzymatic vitrectomy for diabetic retinopathy and diabetic macular edema

The aim of this paper is to determine the role of enzymatic vitrectomy performed by intravitreal injection of autologous plasmin enzyme(APE)in the management of diabetic retinopathy and diabetic macular edema(DME).Diabetic patients with proliferative diabetic retinopathy or DME and evident posterior hyaloid adherence to the retinal surface were included.All cases were treated with an initial intravitreal injection of APE and reevaluated one month later,measuring changes in best-corrected visual acuity(BCVA),macular thickness and the status of the posterior hyaloid.A second APE injection was performed in cases with no evident posterior vitreous detachment(PVD)after the initial treatment.Sixty-three eyes were included in the present review.A complete PVD appeared in 38%of cases(24 eyes)after one injection of plasmin and the total increased to 51%(32 eyes)after the second injection,separated at least by one month.The central macular thickness improved in all cases(100%)and BCVA in89%.Finally,in 50%of eyes with proliferative diabetic retinopathy,a high reduction of new vessels regression was observed.Enzymatic vitrectomy could be considered a good therapeutic alternative in diabetic retinopathy and macular edema.

Cloning,expression in Escherichia coli,and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology （more than 60%） with bacterial laccases in the genome and protein databases and the highest degree of similarity （61% identity） was observed with the multicopper oxidase of KlebsieUa sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS （2,2＇-azino-bis（3-ethylbenzthiazolinesulfonic acid） and DMP （2,6-dimethoxyphenol）, respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Thinning intensity affects microbial functional diversity and enzymatic activities associated with litter decomposition in a Chinese fir plantation

Microbial functional diversity and enzymatic activities are critical to maintaining material circulation during litter decomposition in forests.Thinning,an important and widely used silvicultural treatment,changes the microclimate and promotes forest renewal.However,how thinning affects microbial functional diversity and enzymatic activities during litter decomposition remains poorly understood.We conducted thinning treatments in a Chinese fir plantation in a subtropical region of China with four levels of tree stem removal（0,30,50,and 70%）,each with three replicates,and the effects of thinning on microbial functional diversity and enzymatic activities were studied 7 years after treatment by collecting litter samples four times over a 1-year period.Microbial functional diversity and enzymatic activities were analyzed using Biolog Ecoplates（Biolog Inc.,Hayward,CA,USA）based on the utilization of 31 carbon substrates.Total microbial abundance during litter decomposition was lower after the thinning treatments than without thinning.Microbial functional diversity did not differ significantly during litter decomposition,but the types of microbial carbon-source utilization did differ significantly with the thinning treatments.Microbial cellulase and invertase activities during litter decomposition were significantly higher under the thinning treatments due to changes in the litter carbon concentration and the ratios of carbon and lignin to nitrogen.The present study demonstrated the important influence of thinning on microbial activities during litter decomposition.Moderate-intensity thinning may maximize vegetation diversity and,in turn,increase the available substrate sources for microbial organisms in litter and promote nutrient cycling in forest ecosystems.

Structure, composition and enzymatic hydrolysis of steam-exploded lespedeza stalks

Pretreatment of lespedeza stalks by steam explosion has been studied. The results indicate that steam-exploded pretreatment has strong effects on physical features, morphology, crystallinity, and composition of lespedeza stalks as shown by scanning electron microscopy （SEM）, infrared （IR）, and X-ray diffraction spectrometry methods. After steam explosion, the cellulose and lignin contents of lespedeza stalks varied only slightly, but the hemicellulose content had decreased from 29.34% to 7.48%. The cellulose obtained by steam-exploded pretreatment had a higher degree of crystallinity than that of the raw material. At the explosion condition of 2.25 MPa and 4 min, lignocellulose is easier to hydrolyze by enzyme than the original lignocellulose. The concentration of reduced sugar in the hydrolyzate liquid increased from 71.77 to 162.84 g·L^-1.

Regioselective enzymatic acylation of troxerutin in nonaqueous medium

A series of monosubstituted troxerutin esters have been synthesized by enzyme-catalyzed regioselective acylation of troxerutin in nonaqueous medium.Using divinyl dicarboxylates(CH_2=CH-OOC-(CH_2)_n-COO-CH=CH_2,n = 2,3,4,7,8,11) featuring different chain length as acyl donors and alkaline protease from Bacillus subtilis as catalyst,troxerutin was regioselective acylated at B ethoxyl group.The results indicated that the regioselectivity of the enzyme-catalyzed acylation was not affected by the chain length o...

Effect of Soil Drought Stress on Leaf Water Status, Membrane Permeability and Enzymatic Antioxidant System of Maize

A simulation experiment on the responses of maize (Zea mays L.) from the third leaf stage to maturity for different soil water levels (well-watered, moderately stressed, and severely stressed) was conducted by controlling irrigation and using a mobile rain shelter in a neutral loam, meadow soil to determine the effects on leaf water status, membrane permeability and enzymatic antioxidant system for different growth stages. The results indicated that drought stress relied on drought intensity and duration, with more severe drought stress creating more serious effects on maize. Compared with well-watered conditions, during the silking and blister stages moderate stress did not significantly change the relative water content (RWC) and did change significantly the relative conductivity (RC) (P < 0.05) of the leaves; however, severe stress did significantly decrease (P < 0.01) the leaf RWC and increase (P < 0.01) membrane permeability (leaf relative conductivity). Furthermore, under severe drought stress antioxidant enzyme activities declined significantly (P < 0.01) in later stages, namely for superoxide dismutase (SOD) the tasseling and blister stages, for peroxidase (POD) the milk stage, and for catalase (CAT) during the tasseling, blister, and milk stages. Meanwhile, membrane lipid peroxidation (measured as malondialdehyde content) significantly increased (P < 0.01) in all stages.

Pretreatment of agricultural residues by supercritical CO_2 at 50–80 °C to enhance enzymatic hydrolysis

Various agricultural crop residues including corn stover,corn cob,and sorghum stalk with a moisture content of 75 wt%were subjected to a long pretreatment(12–60 h)with supercritical CO_2(scCO_2),at low temperature(50–80°C)and a pressure of 17.5–25.0 MPa.The sugar yields from the enzymatic hydrolysis(EH)of the pretreated samples were as much as three-to fourfold greater than those afforded by the raw materials.However,when pretreatment was conducted within a short time(e.g.0.5 h),as previously reported in the literature,only a slight increase in the EH sugar yields was observed.The proposed sc CO_2pretreatment mechanism demonstrated the role of moisture in the system.Wetting,softening,and swelling were observed to mainly affect the lignocellulose when a suitable amount of water was added.Finally,the samples were analysed by X-ray diffraction and scanning electron microscopy,before and after pretreatment,to investigate the changes in the microscopic structure of the biomass.

Enzymatic Synthesis of Agmatine by Immobilized Escherichia coli Cells with Arginine Decarboxylase Activity

A new method for the enzymatic synthesis of agmatine by immobilized Escherichia coli cells with arginine decarboxylase（ADC） activity was established and a series of optimal reaction conditions was set down. The arginine decarboxylase showed the maximum activity when the pyridoxal phosphate（PLP） concentration was 50 mmol/L, pH=7 and 45 °C. The arginine decarboxylase exhibited the maximum production efficiency when the substrate concentration was 100 mmol/L and the reaction time was 15 h. It was also observed that the appropriate concentration of Mg2＋, especially at 0.5 mmol/L promoted the arginine decarboxylase activity; Mn2＋ had little effect on the arginine decarboxylase activity. The inhibition of Cu2＋ and Zn2＋ to the arginine decarboxylase activity was significant. The immobilized cells were continuously used 6 times and the average conversion rate during the six-time usage was 55.6%. The immobilized cells exhibited favourable operational stability. After optimization, the maximally cumulative amount of agmatine could be up to 20 g/L. In addition, this method can also catalyze D,L-arginine to agmatine, leaving the pure optically D-arginine simultaneously. The method has a very important guiding significance to the enzymatic preparation of agmatine.

Identification of Odor Volatile Compounds and Deodorization ofPaphia undulata Enzymatic Hydrolysate

Unfavorable fishy odour is an inevitable problem in aquatic products. In the present study, headspace solid-phase mi- croextraction gas chromatography mass spectrometry （HS-SPME-GC-MS） analysis of volatiles from untreated samples and three deodorized samples （under the optimal conditions） ofPaphia undulata enzymatic hydrolysate revealed that the compounds contrib- uting to the distinctive odor were 1-octen-3-ol, n-hexanal, n-heptanal, 2,4-heptadienal, and 2,4-decadienal, whereas n-pentanal, n-octanal, n-octanol, benzaldehyde, 2-ethylfuran and 2-pentylfuran were the main contributors to the aromatic flavor. The deodoriz- ing effects of activated carbon （AC） adsorption, yeast extract （YE） masking and tea polyphenol （TP） treatment on a P. undulata en- zymatic hydrolysate were investigated using orthogonal experiments with sensory evaluation as the index. The following optimized deodorization conditions were obtained： AC adsorption （35 mg mL-1, 80℃, 40 rain）, YE masking （7 mgmL l, 45 ℃, 30 min） and TP treatment （0.4mgmL-l, 40℃, 50min）. AC adsorption effectively removed off-flavor volatile aldehydes and ketones. YE masking modified the odor profile by increasing the relative contents of aromatic compounds and decreasing the relative contents of aldehydes and ketones. The TP treatment was not effective in reducing the odor score, but it significantly reduced the relative content of alde- hydes while increasing that of alkanes. It is also notable that TP effectively suppressed trimethylamine （TMA） formation in a P. un- dulate hydrolysate solution for a period of 72 h.