Breeding of D (–)-Lactic Acid High Producing Strain by Low-energy Ion Implantation and Preliminary Analysis of Related Metabolism
Ting-Ting Xu &Zhong-Zhong Bai &Li-Juan Wang &Bing-Fang He
Received:21January 2008/Accepted:5May 2008/Published online:24June 2008#Humana Press 2008
Abstract The low-energy nitrogen ion beam implantation technique was used in the breeding of mutant D (–)-lactic-acid-producing strains.The wild strain Sporolactobacillus sp.DX12was mutated by an N +ion beam with energy of 10keV and doses ranging from 0.4×1015to 6.60×1015ions/cm https://www.doczj.com/doc/6316707953.html,bined with an efficient screening method,an efficient mutant Y2-8was selected after two times N +ion beam implantation.By using the mutant Y2-8,121.6g/l of D -lactic acid was produced with the molar yields of 162.1%to the glucose.The yield of D -lactic acid by strain Y2-8was 198.8%higher than the wild strain.Determination of anaerobic metabolism by Biolog MT2was used to analyze the activities of the concerned enzymes in the lactic acid metabolic pathway.The results showed that the activities of the key enzymes responded on the substrates such as 6-phosphofructokinase,pyruvate kinase,and D -lactate dehydrogenase were considerably higher in the mutants than the wild strain.These might be affected by ion beam implantation.
Keywords Nitrogen ion beam implantation .D (–)-Lactic-acid-producing strain .Mutation .Breeding .Metabolic influence
Introduction
For centuries,lactic acid has traditionally been used in the food,textile,chemical,and pharmaceutical industries.In recent years,poly-L -lactic acid (PLLA)has attracted much interest as a renewable alternative to conventional petroleum-based plastics.Its properties make it useful for many applications such as biodegradable packaging and agricultural mulch film [1].However,thermal stability of PLA is not sufficiently high to some
Appl Biochem Biotechnol (2010)160:314–321DOI 10.1007/s12010-008-8274-4
T.-T.Xu :Z.-Z.Bai :L.-J.Wang :B.-F.He (*)
College of Life Science and Pharmaceutical Engineering,Nanjing University of Technology,Xinmofan Road 5,Nanjing,210009Jiangsu,China e-mail:bingfanghe@https://www.doczj.com/doc/6316707953.html,
applications.A polymer blend of PLLA and poly-D-lactic acid(PDLA)gives a racemic crystal called a stereo-complex;this blended PLLA/PDLA polymer has a melting temperature(T m)of225°C,50°C higher than that of pure PLLA(177°C)due to the unusually strong interaction between PLLA and PDLA chains[2,3].The production of D-lactic acid through fermentation has been reported in several articles[4–6],but their yields were quite low compared with L-lactic acid.Therefore,the efficient production of D-lactic acid would be a breakthrough,allowing the large-scale application of poly lactic acid with a high melting point.
Low-energy ion implantation,a new mutagenesis technique,was used in the breeding of rice seeds for the first time by Yu et al.[7].From then on,this method has been widely applied in the breeding of crops,plants,and microbes[8–10].When low-energy ions are implanted into organic cells,some important biomolecules such as amino acids and nucleotides are decomposed,and DNA can be destroyed[11].This means that ion beam mutation has a wide range of biological effects.Experimental results showed that low-energy ion implantation has many advantages,such as a low injury rate,higher mutation rate,and wider spectrum of mutation than traditional mutation methods[12].The mechanism of low-energy ion implantation acting on microbes has not yet been clarified.
In this work,we screened an efficient mutant for D-lactic acid production through ion beam implantation and conducted a pilot study on the influence of N+ion implantation on the metabolism concerned with the lactic acid fermentation pathway of the cells. Materials and Methods
Microorganism and Media
Sporolactobacillus sp.DX12was isolated from soil and stored in our lab[13].The agar medium contained(per liter):glucose20g,yeast extract2g,peptone2g,KH2PO41g,corn steep liquor2ml,sodium acetate2g,MgSO40.2g,MnSO410mg,and FeSO410mg.The selection agar plates had the same compositions except that100g/l(150g/l for second screen)of glucose and0.1g/l bromocresol green(pH3.6yellow?pH5.2blue)were added [14].The seed culture medium was composed of(g/l):glucose20,yeast extract2,peptone 2,corn steep liquor5ml,MgSO40.2,MnSO40.01,FeSO40.01,and CaCO320,and the fermentation medium was composed of(g/l):glucose100,(150g/l for second screen),yeast extract3,corn steep liquor20ml,MgSO40.5,MnSO40.01,FeSO40.01,and CaCO3100.
Ion Implantation Equipment
The implantation sources were produced by an ion beam implanting instrument LZD-900 designed by Chinese Southwestern Institute of Physics.
Procedure of Ion Implantation on the Cells of Sporolactobacillus sp.DX12
Active cells were harvested from the culture after48h anaerobic incubation at37°C in agar medium.The fresh cultured cells of DX12were diluted in sterilized physiological salt solution,and80μl suspension was spread on an empty sterilized Petri dish(75mm)and desiccated by filtrated air to make a dry membrane of cells.Then,the dishes were put into the sample holder and implanted by nitrogen ion beam with energy of10keV.The dose for implantation ranged from0.40×1015to6.60×1015ions/cm2.
Selection of Efficient D-Lactic-Acid-Producing Mutants
After ion implantation,the DX12cells were washed out from the plates with sterilized physiological salt solution.The suspension was spread over three to five selection agar plates to isolate high-yield D-lactic-acid-producing mutants.The colonies showing large halos(yellow)on the agar medium were selected.For the flask test,the mutants selected after48h culture on selection agar plates(without bromocresol)were inoculated to a150-ml flask containing60ml seed culture medium and were shaken anaerobically at170rpm at 37°C with liquid sealing by liquid paraffin for24h.A250-ml flask containing120ml of the fermentation medium was inoculated with10%of the above inoculums and cultured in a rotary shaker at150rpm at37°C for7days.
Analysis of Concentration and Optical Purity of Produced Lactic Acid
After the cells and CaCO3had been removed by centrifugation,fermentation fluid was put through cation exchange resin to change calcium lactate into lactic acid eluted with distilled water for further detection.The concentration of lactic acid in the fermentation supernatant was measured by high-performance liquid chromatography(HPLC)using a C18column (Allteck)under the following conditions:mobile phase,2.5mM NH4H2PO4;flow rate, 0.5ml/min;temperature,30.0°C.The detection wavelength was UV230mm.The optical purity of the produced lactic acid was measured by HPLC with a UV detector(254nm) under the following conditions:ligand exchange column,Sumichiral CA5000(150×4.6mm,Sumika Chemical Analysis Service,Osaka,Japan);mobile phase,1mM CuSO4; flow rate,1.0ml/min and temperature,35.0°C.
Cell Growth
Growth was determined by measurement of optical density(OD)at660nm.As it is difficult to measure growth by OD in the presence of calcium carbonate,the sample of broth was settled down for a steady few minutes.The most of calcium carbonate precipitated with few cells because the sediment speed of calcium carbonate is faster than the cells’.Then,the upper layer of the sample broth was diluted to measure the optical densities at660nm as the rough cell growth.It was confirmed that the OD660nm of cell growth showed an approximate direct proportional relation to the protein concentration of the broth.
Metabolic Activity Assay Using Biolog Microstation System
Biolog Inc.(Biolog,Inc.,Hayward,CA,USA)provides a rapid convenient technique to conduct simultaneous testing of different bacterial phenotypes using cell metabolism as the reporter system[15,16].The commercially available Biolog microplates contain a certain amount of tetrazolium violet,different substrates,and the necessary buffer.Tetrazolium violet is used as a redox dye to colorimetrically detect(at590nm)the metabolic activity of cells for the substrates.The more metabolic activity the cells have,the more purple color will accumulate due to a reduction of the dye[17,18].In this research,the Biolog MT2MicroPlate[19] containing a tetrazolium redox dye and a necessary buffer without substrates was used to analyze the metabolic activities of the mutant and wild strain.The substrates related to the metabolic pathway of lactic acid such as glucose,fructose-6-phosphate,glycerophosphate disodium,and sodium pyruvate were selected and added to the different wells of MT2plate to assay the relevant metabolic activity of the D-lactic acid fermentation pathway.
Following the established Biolog procedures [15],suspensions of the wild strain and mutant were adjusted to give 60%transmittance with the Biolog turbidimeter.Subsequent-ly,15μl of different substrates and 135μl of the cell suspension were added into the MT2MicroPlates.The microplates were incubated in an anaerobic culture box (Bug box dual gas,Ruskinn,UK)at 37°C,and the reduction of tetrazolium violet was determined at 6-h intervals.
Results and Discussion
Survival Rate Curve and Choice of Mutant Dose
The ion source can be varied depending in the specific purpose,and different ion species such as N +,C 2+,and Ti 2+can be produced.In this study,N +was chosen as the ion source,and the energy was fixed at 10keV .Figure 1shows the effect of the dose of nitrogen ion beam irradiation to DX12cells on the survival rate.The survival rate was related to the dose of N +implantation and showed a characteristic curve shaped like a saddle.The survival rate decreased as the dose was increased (0.6×1015to 2.6×1015ions/cm 2),but when the dose was increased within a short range (3.2×1015to 4.8×1015ions/cm 2),the survival rate showed a temporary increase and then decreased as the dose continued to increase.The falling,rising,and then falling survival pattern was called “saddle curve ”[20].
The mechanisms of low-energy ion irradiation are not totally understood.Low dosage ions only incur damage and etching of the cell surface;thus,the survival rate is higher.With the increase in dosage,the superficial injury to the cell becomes serious,and massive free OH radicals and radials cause the survival rate to drop.When they drop to a certain extent (3.2×1015–4.8×1015ions/cm 2),some kind of internal repair mechanism of the cell such as SOS (error-prone repair)is activated,and the survival rate have a temporary rise.But when the dose is further increased,the cell damage becomes irreparable.According to some reports,the “saddle ”region of the survival curve,in which the survival rate ranges from 10%to 30%,is the region prone to mutate [9,20],and doses ranging from 2.4×1015to 5.2×1015ions/cm 2were decided as the appropriate dose for further mutation.
1
2
4
67
20406080
100S u r v i v a l r a t e (%)
Dose of nitrogen ions (×1015ions/cm 2)
5
3
Fig.1The survival rate curve of nitrogen ion beam irradiated Sporolactobacillus sp.DX12cells.The survival rate can be calculated using the number of clones scored in control plates without ion implantation as 100%.The averages for
two independent experiments are presented
Screening Using Agar Plates Containing High Concentrations of Glucose and Bromocresol Green
Combining the selection on selection agar plates,the mutants were isolated after DX12cells had been implanted by N +ions (2.49×1015to 4.82×1015ions/cm 2).Twenty colonies showing comparatively large yellow halos were selected out.They were inoculated and fermented in flasks as described above.It was found that the D -lactic acid yields of all mutants were higher than that of the wild strain.Table 1shows the growth and lactic acid yield of eight mutants,which produced more than 80g/l of D -lactic acid (D -LA)from 100g/l glucose.The yields were almost two times greater than the wild strain,which could only produce 40.7g/l D -lactic acid from 60g/l glucose (the wild strain could not resist more than 60g/l glucose [13]).Mutant Y1-14produced 89g/l D -lactic acid and was used in a second mutation also by N +ion implantation.
Mutant Y1-14was further mutated by N +ion beam implantation with the dose of 3.80×1015–5.40×1015ions/cm 2according to the survival rate curve.The glucose concentration of the selection agar plates and fermentation medium were increased to 150g/l to screen more efficient mutants.Ten colonies were selected out and detected through flask batch fermentation.These mutants showed higher producibility than strain Y1-14(Table 2),and the mutant Y2-8was confirmed to produce 143.6g/l D -lactic acid.This was equivalent to a 191.5%molar yield to the glucose.
Mutant Y2-8was generated and inoculated to flask fermentations to test their stability in producing lactic acid (Table 3).It can be seen from the results that the D -lactic acid producibility of the high-yield mutant fell during the initial generation and then remained relatively stable in subsequent generations.Average yield of D -lactic acid during two to four generations was 121.6g/l.
Detection of Enantiomeric Purity of Produced Lactic Acid
The enantiomeric purity of the product by mutant Y2-8was 99.08%of D -type,higher than the parent strain (96%).This means that a better quality of product was obtained using Sporolactobacillus sp.mutant Y2-8.
In recent studies,a high productivity (120g/l)of D -lactic acid was obtained by genetically engineered Corynebacterium glutamicum ,but it was fermented using nutrient-Table 2The D -lactic acid yield and growth of mutants after second N +implantation.Strain
Y2-1Y2-3Y2-4Y2-5Y2-6Y2-7Y2-8Y2-9Growth (OD 660)7.779.719.41 6.118.31 6.2011.247.84D -LA (g/l)
115.2
120.6
124.2
96.3
111.7
106.2
143.6
100.3
The averages for three independent experiments are presented.
Table 1The D -lactic acid yield and growth of mutants after first N +implantation.Strain
Wild Y1-2Y1-3Y1-7Y1-9Y1-13Y1-14Y1-16Y1-18Growth (OD 660) 4.83 4.77 4.09 5.27 5.19 5.87 5.30 5.36 5.90D -LA (g/l)
40.7a
86.8
81.8
88.4
80.4
88.2
89.2
87.3
87.3
The averages for three independent experiments are presented.a
The wild strain could not resist more than 60g/l glucose.
rich medium with the supplement of biotin,thiamine,and some antibiotics [21].Lactobacillus delbrueckii produced D -lactic acid from sugarcane juice or sugar beet juice,but the optical purity is relatively low [22].In this work,high productivity (121.6g/l)of D -lactic acid was obtained by mutant Y2-8with related longer fermentation times.Future study will focus on breeding more efficient mutants through further ion beam implantation to short fermentation period.
Analysis of Metabolic Activity Related with D -Lactic Acid Fermentation for Mutant and Wild Strain
Typical homolactic metabolic pathway including glucose becomes pyruvate through the glycolytic flux and pyruvate toward D -lactic acid production through D -lactate dehydroge-nase (LDHD)under anaerobic conditions (see Fig.2).Within the pathway,glucokinase (GK),6-phosphofructokinase (6-PFK),pyruvate kinase (PYK),and LDHD are the key enzymes [23].In this research,the Biolog approach was used to analyze the metabolic activities of the concerned substrates in mutant Y2-8and the wild strain.
As Fig.3shows,the amounts of reduced tetrazolium increased rapidly in the metabolic analysis of mutant Y2-8using four kinds of substrates related with the key enzymes of
glucokinase
glucosephosphate isomerase
Dihydroxy-acetone-P
pyruvate kinase
Lactate
Pi
+
+
NADH+H NAD Fig.2The homolactic metabolic pathway of lactic acid fermenta-tion.Within the pathway,glucokinase (GK ),
6-phosphofructokinase (6-PFK ),pyruvate kinase (PYK ),and
D -lactate dehydrogenase (LDHD )are the key enzymes
Table 3Stability of mutant Y2-8during generation.Regeneration times 1234
Growth (OD 660)11.249.028.889.67D -LA (g/l)
143.6
121.3
119.8
123.6
lactate fermentation,while the amounts of reduced tetrazolium stayed at low levels
according to the metabolic analysis of the wild strain.The metabolic activity of mutant Y2-8using glycerophosphate and pyruvate as substrate were 50%higher than the wild strain.These results suggest that the activities of the related enzymes,especially lactate dehydrogenase,in mutant Y2-8was obviously higher than in the wild strain DX12.Glucokinase responding on glucose was a key enzyme that turns glucose into an Embden –Meyerhof –Parnas pathway.Another key enzyme responding on fructose-6-phosphate was 6-phosphofructokinase.The activity levels of mutant Y2-8metabolizing D -glucose and fructose-6-phosphate were 40%and 100%higher than that of the wild strain.All of the metabolic speeds of the tested substrates concerned with the key enzyme of lactic acid formation were significantly faster in the mutant than in the wild strain.These results suggested that the enzymes concerned with the lactic acid fermentation pathway were activated through the mutation by N +ion beam implantation.
There are only a few of research on mechanisms of mutation by ion beam implantation.One important biological effect is the mutation of DNA.DNA mutation can affect the activities of enzymes and cause the increase or decrease of some metabolites.The decomposition of amino acids and nucleotides induced the change of DNA structure or even the break of DNA strand [24,25].The effects of ion beam implantation on metabolic ability related to the main key enzymes of D -lactic acid fermentation were elucidated in this
0.1
0.20.30.40.50.60.70.80.91.0Incubation Time(h)
A b s o r b a n c e V a l u e (O D 590)
0.10.20.30.40.50.60.70.80.91.0 Incubation Time(h)
A b s o r b a n c e V a l u e (O D 590)
a
b
Fig.3The metabolic activities of mutant Y2-8(a )and the wild strain (b )on the substrates related to the key enzymes of lactate fermentation.The MT2microplate with the addition of the substrates was incubated anaerobically.The averages for two independent experiments are presented.The reduction of
tetrazolium violet is expressed by the change of the absorbance value at 590nm.D -Glucose
(filled square ),6-p-fructose (filled circle ),glycerophosphate (filled triangle ),pyruvate (filled inverted triangle )
study;continuing studies will yield further insight into the differences of transcription regulation in the key enzyme of lactic acid formation between the mutants with the wild strain.
Acknowledgments We thank Dr.Long Yu of the Nanjing University of Technology for technical support. This work was supported by the973program(grant no.2003CB716004)and the863program(grant no. 2006AA020102)of China.
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生物化学学号： 淀粉酶酶学性质的研究 学生姓名：#### 指导教师：##### 所在院系：生命科学学院 所学专业：####### 学号：##### #### 大学 中国·哈尔滨 2011 年12 月
摘要： 酶是酶是一种生物催化剂，它具有催化剂属性，同是也具有一些无机催化剂所不具有的特性。催化特定化学反应的蛋白质、RNA或其复合体。是生物催化剂，能通过降低反应的活化能加快反应速度，但不改变反应的平衡点。本实验通过利用淀粉酶水解还原糖，还原糖能使3，5-二硝基水杨酸还原，生成棕色的3-氨基-5硝基水杨酸。淀粉酶活力与还原糖的量成正比，用比色法测定淀粉酶作用于淀粉后生成的还原糖的量，以单位质量样品在一定时间内生成还原糖的量表示酶活力。酶的活性又同时受到温度、PH、激活剂抑制剂等的影响。 关键词： 淀粉酶活力温度 PH 激活剂和抑制剂 前言： 淀粉酶是水解淀粉和糖原的酶类总称，通常通过淀粉酶催化水解织物上的淀粉浆料，由于淀粉酶的高效性及专一性，酶退浆的退浆率高，退浆快，污染少，产品比酸法、碱法更柔软，且不损伤纤维。淀粉酶的种类很多，根据织物不同，设备组合不同，工艺流程也不同，目前所用的退浆方法有浸渍法、堆置法、卷染法、连续洗等，由于淀粉酶退浆机械作用小，水的用量少，可以在低温条件下达到退浆效果，具有鲜明的环保特色。 此酶以Ca2+为必需因子并作为稳定因子和激活因子，也有部分淀粉酶为非Ca2+依赖型。淀粉酶既作用于直链淀粉，亦作用于支链淀粉，无差别地随机切断糖链内部的α－1，4-链。因此，其特征是引起底物溶液粘度的急剧下降和碘反应的消失，最终产物在分解直链淀粉时以葡萄糖为主，此外，还有少量麦芽三糖及麦芽糖，其中真菌a-淀粉酶水解淀粉的终产物主要以麦芽糖为主且不含大分子极限糊精，在烘焙业和麦芽糖制造业具有广泛的应用。另一方面在分解支链淀粉时，除麦芽糖、葡萄糖、麦芽三糖外，还生成分支部分具有α-1，6-键的α-极限糊精（又称α-糊精）。一般分解限度以葡萄糖为准是35-50%，但在细菌的淀粉酶中，亦有呈现高达70%分解限度的（最终游离出葡萄糖）。 通过研究淀粉酶的性质，能使我们更好的了解它，并充分利用于工业生产、食品加工、医疗等产业。
３结 论 植物乳杆菌素Ｌ－１经硫酸铵沉淀，透析除盐后效价达１２８０ＡＵ／ｍｌ，作用方式为杀菌。在７、１５、３０、３７℃下，添加植物乳杆菌素Ｌ－１对单增李斯特菌都有一定的抑制作用。７℃下该细菌素在１４４ｈ内控制住初始菌数，温度较高的情况下则可以在短时间内迅速降低活菌数。在选用的六种ｐＨ下，ｐＨ７．０时植物乳杆菌素Ｌ－１的抑菌效果最好。不论在培养基中还是ｐＨ７．０，５ｍｍｏｌ／Ｌ的磷酸缓冲液中，盐对该细菌素具有一定的拮抗作用，各盐分之间和同种盐不同浓度之间差异不显著。有关吸附作用的研究发现：低ｐＨ（５．０～５．５）下，植物乳杆菌素Ｌ－１不能吸附在单核细胞增生李斯特氏菌上，而ｐＨ６．０～７．５下有５０％吸附在指示菌上。盐对该细菌素吸附单核细胞增生李斯特氏菌没有显著影响。 参考文献： ［１］ 吕燕妮， 李平兰， 江志杰． 乳酸菌３１－１菌株产细菌素的初步研究［Ｊ］． 中国食品学报， ２００３， 增刊： １３０－１３３． ［２］郁庆福， 蔡宏道， 何晓青， 等． 现代卫生微生物学［Ｍ］． 北京： 人民卫生出版社， １９９５． １１６－１１７． ［３］ Ｓｏｐｈｉｅ Ｍ Ｐ， Ｅｍｉｌｉａ Ｆ， Ｒｉｃｈａｒｄ Ｊ． Ｐｕｒｉｆｉｃａｔｉｏｎ， Ｐａｒｔｉａｌ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎａｎｄ ｍｏｄｅ ｏｆ ａｃｔｉｏｎ ｏｆ ｅｎｔｅｒｏｃｏｃｃｉｎ ＥＦＳ２， ａｎ ａｎｔｉｌｉｓｔｅｒｉａｌ ｂａｃｔｅｒｉｏｃｉｎｐｒｏｄｕｃｅｄ ｂｙ ａ ｓｔｒａｉｎ ｏｆ Ｅｎｔｅｒｏｃｏｃｃｕｓ ｆａｅｃａｌｉｓ ｉｓｏｌａｔｉｏｎ ｆｒｏｍ ａ ｃｈｅｅｓｅ［Ｊ］．Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｊｏｕｒｎａｌ ｏｆ Ｆｏｏｄ Ｍｉｃｒｏｂｉｏｌｏｇｙ， １９９６， ３０： ２５５－２７０． ［４］ Ａｔｒｉｈ Ａ， Ｒｅｋｈｉｆ Ｎ， Ｍｏｉｒ Ａ Ｊ Ｇ， ｅｔ ａｌ． Ｄｅｔｅｃｔｉｏｎ ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａｂａｃｔｅｒｉｏｃｉｎ ｐｒｏｄｕｃｅｄ ｂｙ Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ Ｃ１９［Ｊ］． ＣａｎａｄａｎＪｏｕｒｎａｌ ｏｆ Ｍｉｃｒｏｂｉｏｌｏｇｙ， １９９３， ３９： １１７３－１１７９． ［５］ Ａｔｒｉｈ Ａ， Ｒｅｋｈｉｆ Ｎ， Ｍｏｉｒ Ａ Ｊ Ｇ， ｅｔ ａｌ． Ｍｏｄｅ ｏｆ ａｃｔｉｏｎ，ｐｕｒｉｆｉｃａｔｉｏｎ ａｎｄａｍｉｎｏ ａｃｉｄ ｓｅｑｕｅｎｃｅ ｏｆ ｐｌａｎｔａｒｉｃｉｎ Ｃ１９， ａｎ ａｎｔｉ－Ｌｉｓｔｅｒａ ｂａｃｔｅｒｉｏｃｉｎ ｐｒｏ－ｄｕｃｅｄ ｂｙ Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ Ｃ１９［Ｊ］． Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｊｏｕｒｎａｌ ｏｆ ＦｏｏｄＭｉｃｒｏｂｉｏｌｏｇｙ， ２００１， ６８： ９３－１０４． ［６］ Ｒｏｎｇｇｕａｎｇ Ｙ， Ｍｏｎｔｙ Ｃ Ｊ， Ｂｉｂｅｋ Ｒ． Ｎｏｖｅｌ ｍｅｔｈｏｄ ｔｏ ｅｘｔｒａｃｔ ｌａｒｇｅａｍｏｕｎｔｓ ｏｆ ｂａｃｔｅｒｉｏｃｉｎｓ ｆｒｏｍ ｌａｃｔｉｃ ａｃｉｄ ｂａｃｔｅｒｉａ［Ｊ］． Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉ－ｒｏｎｍｅｎｔ Ｍｉｃｒｏｂｉｏｌｏｇｙ， １９９２， ５８： ３３５５－３３５９． ［７］还连栋， 贾士芳， 庄增辉， 等． 乳链菌肽（ＮＩＳＩＮ）的杀菌作用机制［Ｊ］．中国食品添加剂， １９９７， （４）： ２０－２３． ［８］ Ｓ Ｔｏｄｏｒｏｖ， Ｂ Ｏｎｎｏ， Ｏ Ｓｏｒｏｋｉｎｅ， ｅｔ ａｌ． Ｄｅｔｅｃｔｉｏｎ ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆａ ｎｏｖｅｌ ａｎｔｉｂａｃｔｅｒｉａｌ ｓｕｂｓｔａｎｃｅ ｐｒｏｄｕｃｅｄ ｂｙ Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍＳＴ ３１ ｉｓｏｌａｔｅｄ ｆｒｏｍ ｓｏｕｒｄｏｕｇｈ［Ｊ］． Ｉｎｔ Ｊ Ｆｏｏｄ Ｍｉｃｒｏｂｉｏｌ， １９９９， ４８： １６７－１７７． 收稿日期：２００５－０１－２１ 作者简介：毕金峰（１９７０－），男，副教授，博士后，主要从事食品化学与生物技术研究。 耐高温α－淀粉酶的酶学性质研究 毕金峰１，董福奎２ （１．中国农业科学院农产品加工研究所 农业部农业核技术与农产品加工重点实验室，北京 １０００９４； ２．内蒙古呼和浩特市赛罕区蔬菜技术推广站，内蒙古 呼和浩特 ０１００２０） 摘 要：耐高温α－淀粉酶是淀粉生产麦芽糖的关键酶。本文对两种耐高温α－淀粉酶的酶学性质进行了对比研究。结果表明：两种酶的耐高温能力差别较大，酶活差别明显；最适ｐＨ值均为７．０，耐酸性较差；当Ｃａ２＋浓度在７～９ｍｍｏｌ／Ｌ时，酶活提高明显。关键词：耐高温α－淀粉酶；性质 Ｓｔｕｄｉｅｓ ｏｎ Ｅｎｚｙｍｅ Ｐｒｏｐｅｒｔｉｅｓ ｏｆ Ｈｅａｔ－ｒｅｓｉｓｔｉｎｇ α－ａｍｙｌａｓｅ ＢＩ Ｊｉｎ－ｆｅｎｇ１，ＤＯＮＧ Ｆｕ－ｋｕｉ２ （１．Ｉｎｓｔｉｔｕｔｅ ｏｆ Ａｇｒｏ－Ｆｏｏｄ Ｓｃｉｅｎｃｅ ａｎｄ Ｔｅｃｈｎｏｌｏｇｙ， Ｃｈｉｎｅｓｅ Ａｃａｄｅｍｙ ｏｆ Ａｇｒｉｃｕｌｔｕｒａｌ Ｓｃｉｅｎｃｅｓ， Ｋｅｙ Ｌａｂｏｒａｔｏｒｙ ｏｆ Ａｇｒｉｃｕｌｔｕｒａｌ Ｎｕｃｌｅａｒ Ｔｅｃｈｎｏｌｏｇｙ ａｎｄ Ａｇｒｏ－Ｆｏｏｄ Ｐｒｏｃｅｓｓｉｎｇ， ＭＯＡ， Ｂｅｉｊｉｎｇ １０００９４， Ｃｈｉｎａ；２．Ｖｅｇｅｔａｂｌｅ Ｔｅｃｈｎｏｌｏｇｙ Ｐｏｐｕｌａｒｉｚｅ Ｓｔａｔｉｏｎ ｏｆ Ｓａｉｈａｎ Ｄｉｓｔｒｉｃｔ ｉｎ Ｈｕｈｅｈａｏｔｅ Ｃｉｔｙ， Ｈｕｈｅｈａｏｔｅ ０１００２０， Ｃｈｉｎａ） Ａｂｓｔｒａｃｔ ：Ｈｅａｔ－ｒｅｓｉｓｔｉｎｇ α－ａｍｙｌａｓｅ ｉｓ ａ ｃｒｉｔｉｃａｌ ｅｎｚｙｍｅ ｆｏｒ ｐｒｏｄｕｃｉｎｇ ｍａｌｔｏｓｅ． Ｅｎｚｙｍｅ ｐｒｏｐｅｒｔｉｅｓ ｏｆ ｔｗｏ ｓｐｅｃｉｅｓ ｏｆ ｈｅａｔ－ｒｅｓｉｓｔｉｎｇα－ａｍｙｌａｓｅｓ ｗｅｒｅ ｓｔｕｄｉｅｄ． Ｔｈｅ ｒｅｓｕｌｔｓ ｗｅｒｅ ａｓ ｆｏｌｌｏｗｓ： ｔｈｅ ｈｅａｔ－ｒｅｓｉｓｔｉｎｇ ａｂｉｌｉｔｙ ｆｏｒ ｔｗｏ ｓｐｅｃｉｅｓ ｏｆ ｅｎｚｙｍｅｓ ｗａｓ ｄｉｆｆｅｒｅｎｔ，ａｎｄ ｔｈｅｒｅ ｗａｓ ａｎ ｅｖｉｄｅｎｔ ｄｉｆｆｅｒｅｎｃｅ ｉｎ ｅｎｚｙｍｅ ａｃｔｉｖｉｔｙ． Ｔｈｅ ｏｐｔｉｍｕｍ ｐＨ ｗａｓ ７．０， ａｎｄ ｔｈｅ ａｃｉｄ－ｒｅｓｉｓｔｉｎｇ ａｂｉｌｉｔｙ ｗａｓ ｐｏｏｒ． Ｔｈｅ
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球孢白僵菌Bb174固态发酵产几丁质酶产酶及酶学特征研究 作者：张洁， 蔡敬民， 吴克， 金胜先， 潘仁瑞， 樊美珍 作者单位：张洁,蔡敬民,吴克,金胜先,潘仁瑞(合肥学院生物科学与技术系,合肥,23002)， 樊美珍(安徽农业大学虫生真菌研究所,合肥,230036) 刊名： 应用生态学报 英文刊名：CHINESE JOURNAL OF APPLIED ECOLOGY 年，卷(期)：2004,15(5) 被引用次数：6次 参考文献(19条) 1.Bradford MM Rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding[外文期刊] 1976 2.Cai JM;Wu K;Zhang J Production,Properties and Application of Xylanase from Aspergillus Niger A3,Volume 864 1998 3.蔡荟梅;刘斌;蔡敬民Study on production conditions and properties of chitinase by Metarhizium anisopliae Ma83[期刊论文]-食品与发酵工业 2003(02) 4.陈三凤;李季伦The study about prospectand history on chittinase 1993(03) 5.Felse PA;Panda T Regulation and clonining of microbial chitinase gene[外文期刊] 1999(2) 6.Fumio N;Mariko I;Kycouke K Purification,thansglu cosylation reaction of β -N-acetylglucosaminidase from Nocardia orientalis 1990(04) 7.Izume M;Ohtakara A Preparation of D-glucoesmine oligosaccharides by the enzymatic hydrolysis of chitosan 1987 8.Jeunianx C Chitinase[外文期刊] 1966 9.Lowry DH Protein messurement with the folin phenol reagent 1951 https://www.doczj.com/doc/6316707953.html,ler GL Use of dinitrosalicylic acid reagent for determination of reducing sugar[外文期刊] 1959(03) 11.Ohtakara A;Mitsutomi M;Uchida YJ Purification and some properties of chitinase from ribrio species 1979(03) 12.Quakfaoui SEL;Potvin C;Brzesinski R A Streptomyces chitosanase is active in transgenic tobacco[外文期刊] 1995 13.彭仁旺;管考;黄秀梨Induction and purification of two extra cellular chitinases from Beauveria bassiana 1996(02) 14.邱立友;汪世山;余功德Screening of chitinase formingstrains and conditions for enzyme production [期刊论文]-微生物学通报 2000(04) 15.邱立友Microbe chitinase and its application topest control 1995(02) 16.夏国庆;马应伦;贾新成Production conditions of chitinase from Aspergillus sp.F-817 1996(04) 17.夏国庆;董志扬;苗雪霞Some properties of the chitinase from Aspergillus sp.F817 1997 18.徐同;柳良好Chitinases from Trichoderma spp.and their antagonism against phytopathogenic fungi 2002 19.郑爱萍;李平Advances of researches on bacterial chitinase[期刊论文]-中国生物工程杂志 2002(04)
1.6 酶学性质研究 （1）pH 的影响：分别测定粗酶液在pH3.0、4.0、5.0、6.0、7.0、8.0下的酶活力，确定其最适反应pH 值；将粗酶液用上述pH 缓冲液稀释后，45℃水浴保温4小时后，测定其剩余酶活力。 （2）温度的影响：分别在40~95℃下测定酶活力，确定其最适反应温度；将酶液在40～90℃范围内的不同温度下保温60 min 后，测定其剩余酶活力。 （3）金属离子的影响：在酶液中分别添加各种金属离子，使其浓度为4 mmol ／L ，然后测定酶活力。 2.5 纤维素酶粗酶液酶学性质 2.5.1酶反应的最适pH 值和酶的pH 稳定性 粗酶液在不同pH 值下测得的酶活及在不同pH 值下处理4小时后测得的相对酶活示于图11。结果表明，CMCase 在pH 3.5～4.5有较高的酶活力，最适反应pH 值为4.0；β-Gluase 在pH 4.5～5.5酶活力较高，最适反应pH 值为5.0，同样方法测得FPA 最适反应pH 为5.0。可见，该菌株所产的各组分纤维素酶是酸性酶。 图11表明，该菌产CMCase 在pH3.0～6.0的范围内，β-Gluase 在pH3.5～5.5的范围内，酶活力均可保持在80％以上，说明该菌株所产酸性纤维素酶可在较宽的pH 值范围内保持其酶活力的稳定性。2.5.2 酶反应的最适温度和酶的热稳定性 在不同温度下直接进行酶促反应测得的酶活及在不同温度下热处理60 min 后于最适反应温度和最适pH 下测得的相对酶活(以4℃保存的酶液活力为100%)示于图12。结果表明，CMCase 、β-Gluase 及FPA 最适反应温度均为65℃。 c e l l u l a s e a c t i v i t y ( U .m l -1) pH r e l a t i v e y a c t i v i t y （%） c e l l u l a s e a c t i v i t y ( U .m l -1) temperature ( o C ) r e l a t i v e y a c t i v i t y （%） 图11 pH 值对酶活力及酶稳定性的影响 Fig.10 Effects of pH value on Cellulase activity and stability 图12 温度对酶活力及酶稳定性的影响 Fig.11 Effects of temperature on activity and stability of cellulase
实验四酶学性质研究 一、实验目的 1、了解pH、温度、金属离子对酶的活性的影响机理； 2、掌握如何选择酶催化反应的最适pH、温度和获得最适pH条件的确定、以及Km常数的测定。 二、实验原理 酶促反应速度受介质pH的影响，一种酶在几种pH介质中测其活力，可看到在某一pH时酶促效率最高，这个pH称为该酶的最适pH。pH影响酶分子的活性部位的解离，另外，也影响底物的解离状态，从而影响酶活性中心的结合与底物或催化。其次，有关基团解离状态的改变影响酶的空间构象，甚至会使酶变性。酶的最适pH不是酶的特征性常数，如缓冲液的种类与浓度，底物浓度等均可改变酶作用的最适pH。 在一定温度范围内，酶促反应速率随温度的升高而加快；但当温度高到一定限度时，酶促反应速率不仅不再加快反而随着温度的升高而下降，最终，酶因高温变性失去活性，失去了催化能力。在一定条件下，每一种酶在某一温度时活力最大，这个温度称为这种酶的最适温度 在进行酶学研究时一般都要制作一条pH与酶活性的关系曲线，即保持其他条件恒定，在不同pH条件下测定酶促反应速度，以pH值为横坐标，反应速度为纵坐标作图。由此曲线，不仅可以了解反应速度随pH值变化的情况，而且可以求得酶的最适pH。最适温度的实验方法和pH类似。 酶促动力学研究酶促反应的速度及影响速度的各种因素，而米氏常数K m值等于酶促反应速度为最大速度一般时所对应的底物浓度，其值大小与酶的浓度无关，是酶促反应的特征常数。不同酶的K m值不同，同一种酶与不同的底物反应
时，其Km值也不同，Km值反映了酶和底物亲和力的强弱程度，Km值越大，表明酶和底物的亲和力越弱；Km值越小，表明酶与底物的亲和力越强。 酶的活力就是酶所催活的反应速度，通常用单位时间内底物的减少或产物的增加来表示。酶反应过程中产物的生成和时间的关系可以用进程曲线来说明，曲线的斜率就是酶反应过程中的反应速度。从进程曲线来看，在一定时间内反应速度维持恒定，但随着时间的延长，反应速度逐渐降低，这是由多种因素造成的。所以，为了准确表示酶的反应速度必须采用初速度，即保持恒定时的速度。同样，不同酶浓度下的反应进程曲线也可以说明这个问题。V=Vmax[S]/Km+[S]，Vmax 指该酶促反应的最大速度，[S]为底物浓度，Km是米氏常数，V是在某一底物浓度时相应的反应速度。双倒数作图（将米氏方程两边取倒数，可转化为下列形式：1／V=Km／Vmax.1／[S]+1／Vmax，可知，1/V对1/[S]的作图得一直线，其斜率是Km/V，在纵轴上的截距为1/Vmax，横轴上的截距为-1/Km。此作图除用来求Km和Vmax值外，在研究酶的抑制作用方面还有重要价值 三、实验器材与试剂 1、试剂：磷酸二氢钠、柠檬酸、ABTS、酸性靛蓝。 2、器材：可见分光光度计、恒温水浴锅、试管、酸度计 四、操作步骤 1、配置缓冲溶液 按下表配置缓冲溶液，其溶液pH值以酸度计测定值为准。
实验四多酚氧化酶的活性的测定及酶学性质 This model paper was revised by the Standardization Office on December 10, 2020
实验四、马铃薯块茎多酚氧化酶（PPO）活性测定及酶学性质一、实验目的 1掌握分光光度法测定多酚氧化酶活性的一般原理及操作技术方法。 2了解酶的活性与植物组织褐变以及生理活动之间的关系。 二、实验原理 马铃薯不耐储藏，在加工过程中去皮切分后非常容易发生酶促褐变，使外观品质和营养价值大为降低，制约着马铃薯的开发利用。酶促褐变是马铃薯加工产业必须解决的难题。其中多酚氧化酶是导致马铃薯等果蔬发生酶促褐变的重要酶类。多酚氧化酶活性大小直接影响酶促褐变程度。 多酚氧化酶(polyphenol oxidase，PPO)又称酪氨酸酶、儿茶酚酶、酚酶等．是自然界中分布极广的一种含铜氧化酶．普遍存在于植物、真菌、昆虫的质体中。植物受到机械损伤和病菌侵染后，PPO催化酚与O2氧化形成醌，使组织形成褐变．以便损伤恢复，防止或减少感染，提高抗病能力。研究多酚氧化酶的特性对食品的加工与保藏工艺有非常重要的意义。因此，检测食品中多酚氧化酶具有重要意义。 多酚氧化酶是一种含铜的氧化酶，在一定的温度、pH条件下，有氧存在时，能使催化邻苯二酚氧化生成有色物质，单位时间内有色物质在410nm处的吸光度与酶活性强弱成正相关，在分光光度计410nm处使反应体系的OD值产生变化，通过OD值的变化确定PPO的酶活大小。 多酚氧化酶 邻苯二酚（儿茶酚）＋1∕2O 2——————→邻醌＋H 2 O
三、试验材料、试剂及试验用品 1．材料：马铃薯块茎。 2．仪器：分光光度计；离心机；恒温水浴；研钵；试管；移液管；容量瓶 3．试剂：L 磷酸缓冲液（pH=）；L邻苯二酚；L磷酸氢二钠；L磷酸二氢钠；10mmol/L 柠檬酸；10mmol/L抗坏血酸；10mmol/L乙二胺四乙酸二钠（EDTA）；10mmol/L亚硫酸钠 四、实验方法： 1.多酚氧化酶的提取 取马铃薯块茎样品，加入预冷的磷酸缓冲液（）3ml，研磨匀浆，转移到离心管中，再用7mL磷酸缓冲液冲洗研钵，合并提取液，在４℃下离心（8000r/min）5min，取上清液为多酚氧化酶提取液，并量取粗酶液体积。 2.多酚氧化酶活性测定 采用比色法测定。将ｍl邻苯二酚加入2ｍl磷酸缓冲液（ｐＨ）中，加入ｍl酶提取液，立即于波长410nm下测定吸光值，2min后再计吸光值，以不加酶提取液的反应液做对照(注意空白为： ml缓冲液和 mL邻苯二酚溶液)。以每分钟吸光度变化为1个多酚氧化酶活性单位。 表1 多酚氧化酶活性测定
萌发小麦种子中淀粉酶酶学性质研究（东北农业大学，生命科学学院，黑龙江省哈尔滨市150030） 摘要： 酶是酶是一种生物催化剂，它具有催化剂属性，同是也具有一些无机催化剂所不具有的特性。催化特定化学反应的蛋白质、RNA或其复合体。是生物催化剂，能通过降低反应的活化能加快反应速度，但不改变反应的平衡点。本实验通过利用淀粉酶水解还原糖，还原糖能使3，5-二硝基水杨酸还原，生成棕色的3-氨基-5硝基水杨酸。淀粉酶活力与还原糖的量成正比，用比色法测定淀粉酶作用于淀粉后生成的还原糖的量，以单位质量样品在一定时间内生成还原糖的量表示酶活力。以淀粉在碘液中显蓝色性质，探究酶活性影响因素，常见的影响因素有：温度 pH 活性剂和抑制剂等。 Abstract:Enzyme is a biological catalyst is an enzyme, the catalyst having the property, the same also has some inorganic catalysts do not have the characteristics. Proteins catalyze specific chemical reactions,RNA or a composite thereof. Are biological catalysts,by reducing the activation energy of the reaction to accelerate the reaction rate, but does not change the equilibrium reaction. In this study, the use of enzymatic hydrolysis of starch sugar, sugar makes 3,5-dinitrosalicylic acid reduction ,a brown 3-amino-nitro-salicylic acid.Proportional to the amount of amylase activity and reducing sugars,measuring the amount of amylase in starch sugar produced by colorimetry ,a unit mass of the sample at the certain time. 关键词： 淀粉酶活性温度 PH 激活剂和抑制剂 引言： 新陈代谢是生命活动的基础，是生命活动最重要的特征。而构成新陈代谢的许多复杂而有规律的物质变化与能量变化，都是在酶催化下进行的。生物的生长发育、繁殖、遗传、运动、神经传导等生命活动都与酶的催化过程紧密相关，可以说，没有酶的参与，生命活动一刻也不能进行。酶是细胞产生的，受多种因素调节控制的具有催化能力的生物催化剂，与一般催化剂比较有以下不同点：酶易失活、酶具有很高的催化效率、酶具有高度专一性、酶活性受到调节和控制。而调节和控制又包括调节酶浓度、抑制剂和激活剂的调节等。[1] 按照淀粉酶水解淀粉的作用方式，可以分为α-淀粉酶、β-淀粉酶、异淀粉酶和麦芽糖酶四种类型。实验证明，当谷类种子萌发时，两类淀粉酶（α，β型）都存在，淀粉酶总酶
小麦种子淀粉酶酶学性质的研究 小麦种子淀粉酶酶学性质的研究Enzymatic properties of amylases from wheat 摘要：在小麦种子中提取淀粉酶，研究相关酶学性质，了解温度、PH值以及激活剂和抑制剂对淀粉酶活性的影响，并且对酶的活力进行测定。不同的温度、PH条件下和加激活剂或抑制剂情况下淀粉酶将淀粉水解的程度不同，产物遇碘呈现不同的颜色，由此可知道酶活性的最适温度和最适PH，也可知道激活剂能使酶的性增加，抑制剂能使酶的活性降低。对酶的活力进行测定时，是测定产物麦芽糖的量，来表示酶的活力。 麦芽糖能将3、5—二硝基水杨酸还原成棕红色的氨基化合物（520nm处有最大吸收峰），其颜色深浅与麦芽糖浓度成正比，利用分光光度法测定棕红色的氨基化合物吸光值，从而得到产物麦芽糖的量，来表示酶的活力[1]。 关键词：小麦种子；淀粉酶；温度；PH值；激活剂；抑制剂；酶的活力;分光光度计 研究背景：二十一世纪是生物信息时代，各种生物学领域研究层出不穷 ,对酶的研究是其中一个重要方面,目前对酶的研究已转入了后期，各种酶的生化性质也相继被研究出，酶是一种具有催化活性的蛋白质，由氨基酸通过肽链连接而成,只有在适当的温度、pH和离子强度下才具有生物活性,有些酶还需要辅酶或者辅因子[2]。通过此次实验研究，让我们进一步加深对淀粉酶的认识和学习，同时培养我们设计实验的基本思路，学会科学的实验组合，提出合理的实验方案,为以后研究其他种类的酶提供了研究方法和实验依据,也为我们以后更多的设计型实验作好铺垫。 目的：通过此次实验研究让我们进一步的加深对淀粉酶的学习和认识。同时，培养我们设计
碱性蛋白酶的分离纯化及其酶学特性的研究碱性蛋白酶(alkaline protease)是一类最适pH为碱性的蛋白酶,是一种非常重要的水解酶类,在医药、饲料、食品等领域中都有广泛应用。本论文以Cellulomonas bogoriensis sp.nov为出发菌株,对其产酶条件、碱性蛋白酶的分离纯化及酶学性质等方面进行了研究,并得到以下结论:利用单因素法和响应面Box-Behnken组合设计方法对菌株产碱性蛋白酶的发酵条件进行了优化,得出菌株最佳产酶条件为:培养温度30℃,培养基初始pH 10.5,装液量75/250 mL,1.5%(w/v)蔗糖为碳源、1.5%(w/v)的牛肉膏酵母浸粉(1:1)为混合氮源、NaCl 含量为2.67%(w/v),培养时间120 h。 经硫酸铵分级沉淀和CM Sepharose Fast Flow柱层析纯化得到一个相对分子质量约为18.3 kDa的碱性蛋白酶,纯化倍数为10.83,回收率为25.79%。酶的最适反应温度70℃,最适pH为11.0。 在4-60℃、pH3.0-12.0范围内酶活力稳定;1 mM的Mg2+、Fe2+会增加蛋白酶活力;而1 mM的Ba2+、Mn2+、Ca2+对蛋白酶活力几乎没有影响,但在10mM时会明显抑制蛋白酶的活力。EDTA浓度为10m M时,碱性蛋白酶的酶活力仍能达到77%。 抑制剂PMSF,TPCK对酶有强烈的抑制作用,表明该酶属于丝氨酸蛋白酶家族中的胰凝乳蛋白酶。酶对甲醇、乙醇、甘油等有机溶剂具有较强的耐受能 力,Triton X-100强烈抑制酶活力。 该蛋白酶最佳作用底物为酪蛋白,可以水解血红蛋白和牛血清白蛋白,不能水解明胶和胶原蛋白,米氏常数Km为19.2μg/mL,Vmax为2000μg/min。该蛋白酶与市售的洗涤剂均有良好的相容性,具有良好的脱毛效果、处理血污的能力和
Breeding of D (–)-Lactic Acid High Producing Strain by Low-energy Ion Implantation and Preliminary Analysis of Related Metabolism Ting-Ting Xu &Zhong-Zhong Bai &Li-Juan Wang &Bing-Fang He Received:21January 2008/Accepted:5May 2008/Published online:24June 2008#Humana Press 2008 Abstract The low-energy nitrogen ion beam implantation technique was used in the breeding of mutant D (–)-lactic-acid-producing strains.The wild strain Sporolactobacillus sp.DX12was mutated by an N +ion beam with energy of 10keV and doses ranging from 0.4×1015to 6.60×1015ions/cm https://www.doczj.com/doc/6316707953.html,bined with an efficient screening method,an efficient mutant Y2-8was selected after two times N +ion beam implantation.By using the mutant Y2-8,121.6g/l of D -lactic acid was produced with the molar yields of 162.1%to the glucose.The yield of D -lactic acid by strain Y2-8was 198.8%higher than the wild strain.Determination of anaerobic metabolism by Biolog MT2was used to analyze the activities of the concerned enzymes in the lactic acid metabolic pathway.The results showed that the activities of the key enzymes responded on the substrates such as 6-phosphofructokinase,pyruvate kinase,and D -lactate dehydrogenase were considerably higher in the mutants than the wild strain.These might be affected by ion beam implantation. Keywords Nitrogen ion beam implantation .D (–)-Lactic-acid-producing strain .Mutation .Breeding .Metabolic influence Introduction For centuries,lactic acid has traditionally been used in the food,textile,chemical,and pharmaceutical industries.In recent years,poly-L -lactic acid (PLLA)has attracted much interest as a renewable alternative to conventional petroleum-based plastics.Its properties make it useful for many applications such as biodegradable packaging and agricultural mulch film [1].However,thermal stability of PLA is not sufficiently high to some Appl Biochem Biotechnol (2010)160:314–321DOI 10.1007/s12010-008-8274-4 T.-T.Xu :Z.-Z.Bai :L.-J.Wang :B.-F.He (*) College of Life Science and Pharmaceutical Engineering,Nanjing University of Technology,Xinmofan Road 5,Nanjing,210009Jiangsu,China e-mail:bingfanghe@https://www.doczj.com/doc/6316707953.html,
碱性磷酸酶的分离纯化与酶学性质研究 董静 201131301031 摘要：以猪肝为原料，采用低浓度醋酸钠(低渗破膜作用)制备肝匀浆，醋酸镁则有保护和稳定AKP 的作用，匀浆中加入正丁醇可使部分杂蛋白变性，释出膜中酶，过滤后，以去除杂蛋白。含有AKP 的滤液用冷丙酮和冷乙酸进行重复分离纯化。根据AKP 在33％的丙酮或30％的乙醇中溶解，而在50％的丙酮或60％的乙酸中不溶解的性质，用冷丙酮和冷乙醇重复分离提取，可从含有AKP 的滤液中获得较为纯净的碱性磷酸酶。酶学性质研究表明该酶催化对磷酸苯二钠水解反应的最适PH值为10.0，最适温度为37℃,米氏常数值Km为3.154mmol/L，酶的热稳定性研究表明该酶在pH在8-9区间和温度在25℃-37℃区间下稳定。 关键字：碱性磷酸酶分离纯化热稳定性酸碱稳定性 碱性磷酸酶(Alkalinephosphatase，EC3．1．3．1，简称AKP)广泛存在于微生物界和动物界，它在动物机体的骨化过程中及在磷化物和其他一些营养物质的消化、吸收和转运过程中起着重要作用。此外，通过催化磷蛋白的水解，碱性磷酸酶在细胞调节过程中也具有一定的作用。它可专一性的水解磷酸单酯化合物而释放无机磷，主要用于核酸研究，分析、测定核苷酸顺序及其基团的重组、分离，也是酶标免疫测定技术的常用工具酶之一。药用化妆品中添加碱性磷酸酶有益于皮肤细胞的再生和新陈代谢，还可用于核苷酸脱磷产生核苷等。它是一种膜结合蛋白，在生物体内直接参与磷酸基团的转移和代谢等生理过程[1]。临床上通过测定AKP的活力，可作为诊断骨骼及肝脏疾病的重要生化指标。因此纯化和研究AKP的性质、调控等有重要意义。本实验尝试以猪肝为材料，分离纯化猪肝碱性磷酸酶，研究其酶学性质，旨在探讨以猪肝研制科研用碱性磷酸酶生化试剂的方法，同时也为进一步的深入研究该酶提供理论上的参考。此外，猪肝来源广泛，价格便宜，可以作为生产碱性磷酸酶生化试剂的原料[2-3]。 研究酶的生物学特性，其之一是它对酸碱度的敏感性， pH对酶活性的影响极为显著，通常各种酶只有在一定的范围内才表现出活性，同一种酶在不同的pH条件下所表现的活性不同，其表现活性最高时的pH值称为酶的最适pH。pH 不仅对酶活性有很大影响，而且对酶的稳定性也有很大的影响，而且对酶的稳定
２００６年第１０期第１０期（总第７９期）农产品加工!学刊 Ｎｏ．１０２００６年１０月 ＡｃａｄｅｍｉｃＰｅｒｉｏｄｉｃａｌｏｆＦａｒｍＰｒｏｄｕｃｔｓＰｒｏｃｅｓｓｉｎｇ Ｏｃｔ． 文章编号"１６７１－９６４６（２００６）１０－００６７－０４ 收稿日期"２００６－０８－２１ 作者简介"于宏伟#１９７８－!!$%男%河北人%硕士%研究方向"酶工程技术& !为通讯作者"贾英民%教授%博士生导师%研究方向"酶工程技术研究&Ｅｍａｉｌ"ｙｍｊｉａ＠ｈｅｂａｕ．ｅｄｕ．ｃｎ& 蛋白酶系催化分解蛋白质肽键的一群酶的总称%它作用于蛋白质%将其分解为蛋白胨’多肽及游离氨基酸&此酶种类繁多%广泛存在于所有生物体内%按其来源可分为植物蛋白酶’动物蛋白酶’微生物蛋白酶(按其作用形式可分为肽链内切酶’肽链外切酶(按所产蛋白酶性能分为酸性蛋白酶’霉菌蛋白酶’中性蛋白酶和碱性蛋白酶&目前%蛋白酶制剂主要被用于提高风味’生产抽提物’改善营养价值%以及改变物理性质等方面&如皮革工业的脱毛和软化已大量利用蛋白酶%既节省时间%又改善劳动卫生条件&蛋白酶还可用于蚕丝脱胶’肉类嫩化和酒类澄清&临床上蛋白酶可作药用%如用胃蛋白酶治疗消化不良%用酸性蛋白酶治疗支气管炎%用弹性蛋白酶治疗脉管炎%用胰蛋白酶’胰凝乳蛋白酶对外科化脓性创口的净化及胸腔间浆膜粘连的治疗&加酶洗衣粉是洗涤剂中的主要产品%含碱性蛋白酶%能去除衣物上的血渍和蛋 白污物［１］&本实验筛选得到了一株产中性蛋白酶的菌株%对其产酶条件与酶学性质作了初步研究& １材料与方法 １．１．１ 菌株 从肉联厂’污水沟等地附近的土壤中分离得到&１．１．２主要试剂和仪器 酪蛋白’ＳｉｇｍａＣ－５８９０酪氨酸%第二军医大学政翔化学试剂研究所提供(脱脂奶粉%内蒙古伊利实业集团股份有限公司产品(ＳＰ－７５６Ｐ型紫外可见分光光度计%上海光谱仪器有限公司提供(ＧＬ－２０Ｇ－!型高速冷冻离心机%上海安定科学仪器厂生产(ＰＨＳ－３Ｃ型酸度计%上海康仪仪器有限公司提供(ＨＺＱ－Ｑ型旋转式恒温摇床%哈东联电子技术开发有限公司提供& 于宏伟%栗志丹%郝珊珊% ＊ 贾英民 #河北农业大学食品科技学院%河北保定 ０７１００１ $蛋白酶产生菌的筛选及酶学性质研究 摘要"从养牛厂附近的土壤中分离得到一株产蛋白酶性质优良的菌株%初步鉴定为芽孢杆菌%对其最适产酶条件进行了研究&试验表明%最适碳源为玉米粉%最适氮源为玉米浆%３００ｍＬ摇瓶最适装液量为６０ｍＬ%最适接种量为 ２％%起始ｐＨ值为４．５和６．５时有２个产酶高峰%并对其进行酶学性质的研究&该酶最适温度为６５"%最适ｐＨ值为 ７．５%该酶在５０#以下保温３０ｍｉｎ酶活仍较高%在７０$以上酶活全部丧失&在ｐＨ值７．０￣９．０时比较稳定%而在ｐＨ值５．０以下时酶活下降很快&金属离子Ｐｂ２＋%鳌合剂ＥＤＴＡ能抑制蛋白酶的活性%而Ｆｅ２＋能激活蛋白酶活性& 关键词"蛋白酶(芽孢杆菌(酶学性质中图分类号"ＴＱ９２０ 文献标志码"Ａ #ＣｏｌｌｅｇｅｏｆＦｏｏｄＳｃｉｅｎｃｅａｎｄＴｅｃｈｎｏｌｏｇｙ%ＨｅｂｅｉＡｇｒｉｃｕｌｔｕｒｅＵｎｉｖｅｒｓｉｔｙ%Ｂａｏｄｉｎｇ%Ｈｅｂｅｉ０７１００１%Ｃｈｉｎａ$ Ｆｒｏｍｔｈｅｓｏｉｌｏｆｔｈｅｒａｉｓｉｎｇｃｏｗｆａｃｔｏｒｉｅｓ%ｗｅｇａｔｈｅｒｅｄａｓｔｒａｉｎｐｒｏｄｕｃｅｄｐｒｏｔｅａｓｅ．Ｔｈｅｓｔｒａｉｎｗｉｔｈｔｈｅｈｉｇｈｅｓｔ－ｙｉｅｌｄ ｐｒｏｔｅａｓｅｗａｓｉｄｅｎｔｉｆｉｅｄａｓＢａｃｉｌｌｕｓ．Ｉｎａｄｄｉｔｉｏｎ%ｔｈｅｅｘｐｅｒｉｍｅｎｔａｌｒｅｓｕｌｔｓｒｅｖｅａｌｅｄｔｈａｔｔｈｅｍｏｓｔｓｕｉｔａｂｌｅｆｅｒｍｅｎｔｉｎｇｃｏｎｄｉｔｉｏｎｓｆｏｒｅｘｃｒｅｔｉｎｇｐｒｏｔｅａｓｅｗｅｒｅ"ｔｈｅｃａｒｂｏｎａｎｄｎｉｔｒｏｇｅｎｓｏｕｒｃｅｓ%ｃｏｒｎｆｌｏｕｒ%ｃｏｒｎｓｔｅｅｐｌｉｑｕｏｒ；ｔｈｅｖｏｌｕｍｅｏｆｆｅｒｍｅｎｔｉｎｇｌｉｑｕｏｒｏｆ３００ｍＬｆｌａｓｋ%６０ｍＬ(ｉｎｏｃｕｌｕｍｓｉｚｅ%２％(ｒｅｓｐｅｃｔｉｖｅｌｙ．ＡｎｄｗｅｃｏｎｃｌｕｄｅｄｔｈｅｒｅｗｅｒｅｔｗｏｐｅａｋｓｔｈａｔｐｒｏｄｕｃｅｄｐｒｏｔｅｉｎａｓｅｉｎｔｈｅｏｒｉｇｉｎａｌｐＨ４．５ａｎｄｐＨ６．５．ＴｈｅａｃｔｉｖｉｔｙｏｆｔｈｉｓｅｎｚｙｍｅｗａｓｔｈｅｈｉｇｈｅｓｔａｔｐＨ７．５ａｎｄ６５%．ＴｈｉｓｅｎｚｙｍｅｗａｓｓｔａｂｌｅｏｖｅｒｔｈｅｒａｎｇｅｏｆｐＨ７．０￣９．０ａｎｄｂｅｌｏｗ５０&%ａｎｄｉｔｓｅｎｚｙｍｅａｃｔｉｖｉｔｙｗａｓｉｎｈｉｂｉｔｅｄｂｙＰｂ２＋ａｎｄＥＤＴＡ．Ｆｅ２＋ｃａｎｓｔｒｏｎｇｌｙａｃ－ｔｉｖａｔｅｔｈｅｅｎｚｙｍｅ． ｐｒｏｔｅｏｌｙｔｉｃｅｎｚｙｍｅ(ｂａｃｉｌｌｕｓ(ｃｒｕｄｅｅｎｚｙｍｅｐｒｏｐｅｒｔｉｅｓ
酶学分析方法及酶活性测定复习与练习 一、有关酶的基本理论知识 1、酶浓度与酶促反应速度的关系是？ 2、底物浓度与酶促反应速度的定量关系式是？ 3、影响酶促反应速度的因素主要有哪些？ 4、Km及Vm代表的含义及它们在临床酶活性测定中应用及意义。 二、酶活性测定的基本知识 1、酶活性的概念及酶活性单位如何表示表示 2、目前酶活性单位有-------、----------、-------------三种表示方式。？ 3、解释酶活性国际单位、催量（Katal）的概念，并比较两者的关系？ 4、酶活性单位的计算：几类计算公式要求理解并熟练应用。 1）通用公式 2）惯用单位的计算公式（测定产物生成类的计算公式；测定底物消耗类的计算公式）3）**连续监测法根据摩尔吸光系数进行酶活性浓度的计算； **连续监测法因素F值（全自动化生化仪多用K表示）的计算和应用？ 三、酶活性的测定方法 1、酶活性测定的基本原则是什么？ 2、酶反应时间进程曲线有————、—————、————三个时期 酶活性测定是在————期测定，此时期反应速度不受------------的影响也称------期3、根据反应时间，酶活性测定分为两类方法是---------和--------------两种 从概念、结果计算、优缺点叙述并比较两类方法？其中连续监测法 4、按检测对象的酶活性测定方法分为----------和------------法；其直接法的方法类型有哪些？ 5、何谓酶偶联反应？写出酶偶联反应的基本模式？什么是待测酶、辅助酶、指示酶？ 6、何谓工具酶？酶作为工具应用于临床生化检验可进行酶活性测定或进行代谢物浓度（底物）的测定时一般有哪两大指示系统？何谓-Trinder反应？Trinder反应的主要缺点或影响因素有哪些？ 7、何谓同工酶？同工酶分析的常用原理有哪些？检测同工酶有何临床意义？并举例说明？ 8、酶活性测定条件及影响因素有哪些？ 9、从几个酶类测定（如AMY、ALT、LD、ALP|等）的实验操作中，讨论酶活性测定应注意的问题？ 10、试述影响血清酶的生理变异有哪些？ 11、酶活性测定，标本的采集、处理、贮存及样品试剂比有何要求？ 四、体液酶测定： 1、常用于临床协助诊断的血浆酶有哪些？用于协助诊断心肌损伤、肝脏疾病、胰腺疾病、骨骼骨骼肌、前列腺疾病等的常用的血清酶有哪些？常用的临床诊断酶谱有哪些？ 2、将各血清酶活性测定的方法学原理进行分析归纳，请总结出有哪几大反应基础。 3、书写出AMS、ALP两种（化学比色法、（连续监测法）方法及ALT、AST、GGT、LDH、CK酶测定（连续监测法）反应原理、主要试剂成分和作用？影响结果准确性因素有哪些？评价LD-L反应和 LD-P反应试剂盒的优缺点？ 4、血清ALT、AST、ALP、GGT、AMS、CK、LD等酶测定的临床意义？