Production of inulooligosaccharides by endoinulinases from Aspergillus ficuum

Inulooligosaccharide (IOS) production from inulin was studied using a partially purified endoinulinase and a purified endoinulinase, which originated from Aspergillus ficuum. At the optimal conditions, including 50 g/L inulin, an enzyme concentration of 10 U/g substrate, 45 °C, and pH 6.0, the inulin-degrading degree by partially purified endoinulinase was 74% and an IOS yield over 50% were observed after 72 h. The major products were identified as DP2 to DP4. The purified Endo-I was used for inulo-oligosaccharide production at the optimal conditions obtained with orthogonal experiments, including pH 5.0, 45 °C, 50 g/L inulin, and an enzyme concentration of 10 U/g substrate. With pure inulin as substrate, the maximum inulin-hydrolyzing degree was 75% and the total IOS yield was 70% after 72 h. The hydrolysis products consisted of DP2 to DP8 with HPLC, and DP3 and DP4 were relatively high. With Jerusalem artichoke juice as substrate, the inulin-hydrolyzing extent reached 89% and the maximum IOS production was up to 80% after 72 h. Various IOS with different DP (mainly DP2, DP3, DP4, DP5, DP7, DP8) were evenly distributed in the final reaction products.     

Inulin Potential for Enzymatic Obtaining of Prebiotic Oligosaccharides

Oligosaccharides have been marketed since the 80s as low-calorie agents and recently have gained interest in the pharmaceutical and food industry as functional sweeteners and prebiotic enriching population of Bifidobacteria. Currently, they have an approximated value of $200 per kg and recently, inulin has been proposed as a feedstock for production of oligosaccharides through selective hydrolysis by action of endoinulinase. High optimum temperature (60°C) and thermostability are two important criteria that determine suitability of this enzyme for industrial applications as well as enzyme cost, a major limiting factor. Significant reduction in cost can be achieved by employing low-value and abundant inulin-rich plants as Jerusalem artichoke, dahlia, yacon, garlic, and onion, among others. In general, the early harvested tubers of these plants contain a greater amount of highly polymerized sugar fractions, which offer more industrial value than late-harvested tubers or those after storage. Also, development of recombinant microorganisms could be useful to reduce the cost of enzyme technology for large-scale production of oligosaccharides. In the case of fungal inulinases, several studies of cloning and modification have been made to achieve greater efficiency. The present paper reviews inulin from vegetable sources as feedstock for oligosaccharides production through the action of inulinases, the impact of polymerization degree of inulin and its availability, and some strategies to increase oligosaccharide production.     

Aspergillus ficuum 内切菊粉酶的酶解条件优化及酶解动力学研究

采用Aspergillus ficuum 内切型菊粉酶Endo-Ⅰ 酶解商品菊粉制备低聚果糖,经正交试验确定其最适酶解条件为：pH5.0、温度45℃、底物浓度50g/L、加酶量10U/g,在此酶解条件下,低聚果糖得率可达70.37%,酶解产物以DP3 和DP4 为主,同时含有一定量的DP2、DP5、DP6、DP7、DP8;在此条件下酶解自制菊芋干粉时,低聚果糖得率为41.72%,酶解产物以DP3～DP6 的低聚果糖为主,DP2 含量很少,同时酶解液中还含有大量的果糖;在此条件下酶解自制菊芋提取液时,低聚果糖得率高达79.80%,酶解产物中除DP6 含量较低外,其他各聚合度的低聚糖分布比较平均。在此相同酶解条件下酶解72h 时,3 种底物中,以菊芋提取液酶解后的低聚果糖得率最高。因此,应用Aspergillus ficuum 内切型菊粉酶Endo-Ⅰ酶解菊芋制备低聚果糖宜选择菊芋提取液作底物。     

Production of thermotolerant N-carbamyl-D-amino acid amidohydrolase by recombinant Escherichia coli

A plasmid, pNT4553, was constructed for high level production of N-carbamyl-d-amino acid amidohydrolase (DCase), the thermostability of which has been improved by amino acid substitution. The DCase activity and the stability of the plasmid in the host cells were dependent on the Escherichia coli strains used. E. coli HB101 was the most suitable host strain among the 13 types of E. coli tested. E. coli HB101 exhibited the highest activity, i.e. 6.36 units/ml of culture broth in 2YT medium (1.6% tryptone, 1.0% yeast extract, and 0.5% NaCl, pH 7.0), and the plasmid was stably maintained by cultivation in 5 types of E. coli including HB101. Casamino acids, NZ-amine, peptone, and protein extract (a mixture of hydrolyzates of corn gluten, wheat gluten and soybean), were found to be suitable as natural nitrogen sources for both enzyme activity and growth. When cultivation was carried out in the presence of high concentrations of glycerol (6.5%) as the carbon source, and protein extract (3.0%) as the nitrogen source, in a small volume of the medium (20 ml of medium in a 500-ml shaking flask), in which the aeration level was estimated to be high, growth and activity reached OD550=63.8 (17.1 mg of dry cell weight/ml of culture broth) and 22.9 units/ml of culture broth, respectively. The economical hyperproduction of DCase using only inexpensive constituents for the medium was achieved.     

Establishment of a mass-production system for NADP using bacterial inorganic polyphosphate/ATP-NAD kinase

The inorganic polyphosphate/ATP-dependent NAD kinase (Ppnk) of Mycobacterium tuberculosis was applied to the mass-production of NADP from NAD and inorganic polyphosphate (metaphosphate). When Ppnk purified from recombinant Escherichia coli cells overexpressing the M. tuberculosis Ppnk was used, 30 mM (27 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The recombinant E. coli cells were immobilized in polyacrylamide gel, and treated with acetone to render the cells permeable to substrates and products. When acetone-treated immobilized cells were used, 16 mM (14 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The isolation of NADP formed in the reaction mixture was easy because of the absence of by-products (ATP degradation compounds), and this NADP production system using purified Ppnk or immobilized recombinant E. coli cells expressing Ppnk is thought to be feasible in the production of NADP on an industrial scale.     

牛蒡菊糖酶法提取

采用固定化酶法提取牛蒡菊糖。结果表明酶水解提取牛蒡菊糖的最佳工艺为：13.5g/100mL 中性蛋白酶、pH 7、固液比1:15、50℃、酶水解6h,菊糖提取率为14.57%;固定化酶制备最佳工艺为：以甲醛(40%):NaOH(2mol/L)=2:3 为凝结液、pH7.5、壳聚糖2.5g/100mL、60℃、加酶量7.5mg/mL,固定8h,酶活力回收率可达到39.13%;固定化酶提取牛蒡菊糖最适条件为：pH7、固液比1:15、60℃、固定化酶加入量13. 5 g/100mL、酶解5h,在此条件下菊糖提取率达到12.89%。固定化酶的稳定性与游离酶相比有显著的提高,连续反应10 次后,固定化酶仍然具有良好的使用性能,此时牛蒡菊糖的提取率为9.42%。     

STABILITY AND PROPERTIES OF A THERMOSTABLE β-GALACTOSIDASE IMMOBILIZED ON CHTTIN

Thermostable β‐galactosidase from an E. coli transformant containing the enzyme gene from P. woesei was immobilized at pH 4.0 and a glutaraldehyde concentration of 10 mM on chitin isolated from shrimp Crangon crangon shells. These preparations had a specific activity of 43,000 U/g of chitin at 85C using ONPG as substrate. The optimum pH and temperature for immobilized β‐galactosidase activity were 5.2 and 93C. Immobilization shifts the optimum pH for the activity of the enzyme by 0.2 units towards the acid side. The immobilized enzyme is stable at temperatures close to the optimal value, and the residual activity for ONPG hydrolysis of the preparations incubated 5 h in 0.1 M phosphate citrate buffer (pH 5.4) at 90C and 100C was 70% and 40% of the initial value, respectively.     

棉织物对甲苯磺酰氯活化法固定化菊粉酶工艺的研究

以4-甲苯磺酰氯活化的棉纤维为载体,对固定化菊粉酶的条件进行探讨。通过对不同纤维成分布料的比较,绒布为载体时得到了较高的固定化酶活。分别采用单因素试验和正交试验,得到优化的工艺参数:每克干基棉布加入10ml干吡啶,对甲苯磺酰氯与干吡啶的比例为1g:1ml,偶联pH值4.5,离子强度0.2mol/L,加酶量80U/g,菊粉酶酶活回收率达到83.60%。在优化的工艺条件下,对糖分组分进行HPLC分析,固定化酶与250g/L菊糖(5U/g菊糖)作用1.5h,低聚果糖含量达到28.21%。     

Nonthermal death of Escherichia coli.

The destruction of Escherichia coli M23 OR.H- using lethal water activity levels and nonlethal temperatures was investigated. Death rates were measured for a combination of four growth-permissible temperatures (15 degrees C, 25 degrees C, 40 degrees C and 45 degrees C) and four distinctly lethal water activities (0.75, 0.83, 0.88 and 0.90). In addition, death rates were measured at two temperatures (4 degrees C and 50 degrees C) outside the growth range of E. coli. Death rate increased both at higher temperature or lower water activity. Inactivation curves resulting from exposure of E. coli to osmotic stress were biphasic. The initial rate of death was followed by a slower second phase decline, or "tailing" effect. Addition of chloramphenicol to the suspending medium reduced the tailing effect and suggested that tailing was caused by de novo protein synthesis.     

Properties of Rhodotorula gracilis D-amino acid oxidase immobilized on magnetic beads through his-tag

D-amino acid oxidase catalyzes one of the key steps in the production of semisynthetic cephalosporins. We expressed and purified recombinant Rhodotorula gracilis D-amino acid oxidase with C-terminal his-tags. This engineered enzyme was immobilized onto Ni(2+)-chelated nitrilotriacetic acid magnetic beads through the interaction between his-tag and Ni(2+). The kinetic constants, storage properties, and the reusability of the immobilized d-amino acid oxidase were determined. The effects of temperature, pH, and hydrogen peroxide on the activity of immobilized d-amino acid oxidase were also studied. The highest activity recovery was 75%. Thermal stability was improved after immobilization; the relative activity of the immobilized enzyme was 56% whereas the free enzyme was completely inactivated after incubation at 50 degrees C for 1 h. In the presence of 10 mM hydrogen peroxide, the immobilized enzyme did not show a rapid loss of activity during the first 2 h of incubation, which was observed in the case of the free enzyme; the residual activity of the immobilized enzyme after 9 h was 72% compared with 22% of the free form. The long-term storage stability was improved; the residual activity of the immobilized enzyme was 74% compared with 20% of the free enzyme when stored at room temperature for 10 d. The immobilized form retained 37% of its initial activity after 20 consecutive reaction cycles.     

Inulinase from Rhodotorula mucilaginosa: immobilization and application in the production of fructooligosaccharides

The crude extract containing inulinase from Rhodotorula mucilaginosa was obtained by submerged fermentation. Inulinase was immobilized on chicken eggshell by physical adsorption and covalent crosslinking, using glutaraldehyde as a crosslinking reagent, and Celite by adsorption. Fructooligosaccharides production was performed using immobilized inulinase (5%, w/v) and inulin substrate solution under experimental conditions evaluated through Doehlert experimental design. The production of inulinase was optimized for concentrations of D-glucose and yeast extract at 12.5 and 0.5 g/L, respectively, resulting in an optimal activity of 0.62 U. The optimal pH and temperature for enzyme activity were 8.0 and 75 °C, respectively, leading to an optimal activity of 3.54 U. The highest immobilization efficiency (46.27%) was obtained upon immobilization on Celite. Immobilization by adsorption to eggshell allowed for specific activity of 4.15 U/g, and adsorption to Celite resulted in specific activity of 3.70 U/g. The highest titer in fructooligosaccharides was obtained with an initial inulin concentration of 250 g/L (25%, w/v), and a reaction time of 16 h. Hence, immobilized inulinase proved to be a promising catalyst for fructooligosaccharides production since the formulation is performed through a simple, low-cost, and large-scale applicable methodology.

     

Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae β-galactosidase

Aspergillus oryzae β-galactosidase was immobilized by three different techniques, namely adsorption on celite, covalent coupling to chitosan and aggregation by cross-linking. These techniques were compared in terms of the yield of immobilized preparation, enzymatic characteristics, stability and efficiency in oligosaccharide synthesis. Immobilization led to increase in K_m in each case. Immobilization on chitosan gave maximum enzyme yield and oligosaccharide synthesis. At 60 °C, the chitosan-immobilized enzyme was stabilized (by 1.6-fold) due to protection effect of the matrix. However, at 65 °C, the t_1/2 of cross-linked enzyme aggregates (CLEA) of β-galactosidase was 1.07 h as compared to 0.79 h in the case of free enzyme. Both chitosan-immobilized enzyme and CLEA were used for oligosaccharide synthesis. Using 20% (w/v) lactose, the chitosan-immobilized enzyme gave maximum oligosaccharide yield (17.3% of the total sugar) as compared to free enzyme (10.0%) in 2 h at 40 °C. CLEA were instead found effective in lactose hydrolysis yielding 78% monosaccharide in 12 h.     

Sources, properties and suitability of new thermostable enzymes in food processing

Investigations concerning recombinant a-amylases from Pyrococcus woesei and thermostable a-glucosidase from Thermus thermophilus indicate their suitability for starch processing. Furthermore, the study of recombinant ss-galactosidase from Pyrococcus woesei suitable for purpose of low lactose milk and whey production are also presented. The activity of this enzyme in a wide pH range of 4.3-6.6 and high thermostability suggests that it can be used for processing of dairy products at temperatures which restrict microbial growth during a long operating time of continuous-flow reactor with an immobilized enzyme system. Preparation of recombinant a-amylase and ss-galactosidase was facilitated by cloning and expression of genes from Pyrococcus woesei in Escherichia coli host. Satisfactory level of recombinant enzymes purification was achieved by thermal precipitation of native proteins originated from Escherichia coli. The obtained a-amylase has maximal activity at pH 5.6 and 93 degrees C. The half-life of this preparation (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. An advantageous attribute of recombinant a -amylase is independence of its activity and stability on calcium salt. a-Glucosidase from Thermus thermophilus also not require metal ions for stability and retained about 80% of maximal activity at pH range 5.8-6.9. Thus, this enzyme can be used together with recombinant a-amylase.     

大孔树脂D101固定中性脂肪酶及其生物催化应用

以大孔树脂D101固定化中性脂肪酶,研究了固定化条件对酶催化活性的影响,得到了最佳固定化条件:给酶量为90IU/g,固定化温度为35℃,时间为12h,此时固定化酶的活力回收约为60%。固定化酶的半失活温度由游离酶的53℃提高到57℃,最适反应温度和最适pH分别由42℃上升至44℃和由7.5下降到7.3。对固定化中性脂肪酶在生物柴油合成中应用也进行了初步研究。     

Novel quantitative assays for estimating the antimicrobial activity of fresh garlic juice

Novel agar diffusion and broth dilution assays were developed for quantitatively estimating the antimicrobial activity of fresh garlic juice. Bacteria found to be inhibited by garlic juice in agar diffusion assay included two gram-positive and five gram-negative species. Leuconostoc mesenteroides was not inhibited. Escherichia coli B-103 (HB101, with pJH101, ampicillin resistant, 100 microg ml(-1)) was inhibited and chosen as the standard culture for quantitative assays. The agar diffusion assay was based on the slope ratio method, where the slope of dose response for garlic juice was divided by the slope of dose response for methylmethane thiosulfonate (MMTSO2). Juice from fresh garlic varied in activity between 1.76 and 2.31 microg of MMTSO2 per mg of garlic juice. The activity of juice decreased during 11 months of storage of garlic cloves at 5 degrees C from 2.31 to less than 0.1 microg of MMTSO2 per mg of juice. The broth dilution assay also used the E. coli B-103 culture, which permitted selective enumeration of this bacterium when 100 microg ml(-1) of ampicillin was incorporated into the enumerating agar. Selective enumeration was essential since the garlic juice was not sterile and, thus, contained natural flora. Growth of E. coli was unaffected by 0.1%, delayed by 0.25%, and completely inhibited at 0.5 and 2% garlic juice in broth during 24 h of incubation at 37 micro C. The minimum inhibition concentration of garlic juice by broth dilution assay was, thus, estimated to be 0.5%, which is equivalent to 3.46 microg of MMTSO2 per mg of garlic juice by the agar diffusion assay.     

Immobilization and characterization of C‐S lyase from onion (Allium cepa) bulbs

Cysteine sulfoxide lyase (C‐S lyase; EC 4.4.1.4), the enzyme responsible for flavor potentiation in minced Allium tissues, was immobilized by entrapment within an alginate gel. Both the free and immobilized C‐S lyase had an optimum pH of 7.5 for activity. Also, similar Km values were observed for both forms of the enzyme using (±)‐S‐methyl‐L‐cysteine sulfoxide (± MCSO; 14.2–19.2 mM) and an alk(en)yl‐L‐cysteine sulfoxide extract (0.9–1.8 mM) prepared from onion bulbs. Both forms of the enzyme were stable for 6–8 weeks at 4°C. At 25°C, the immobilized enzyme was fully stable for 5 weeks whereas the free enzyme lost 50% of its activity within 2 weeks. As the alginate content in the gel was increased from 0.25 to 3%, the % yield of activity of the immobilized enzyme system decreased from 65 to 44%. However, as % alginate content was increased, the yield of active enzyme immobilized in the gel increased from 62 to 97%. Reaction of both the free and alginate‐entrapped C‐S lyase with (±) MCSO resulted in the production of a distinctive cabbage‐like aroma. Onion aroma was liberated when either form of the enzyme was recombined with an odorless alk(en)yl‐L‐cysteine sulfoxide extract from onion.     

内切菊糖酶的分离纯化及性质研究

采用DEAE-Sepharose CL-6B离子交换层析从Fructozyme (Aspergillus niger) 中分离得到三个菊糖酶组份EⅠ、EⅡ和EⅢ,其中EⅠ主要表现为内切菊糖酶,其分子量为65,000,以菊糖为底物其最适pH为4.6,最适温度为60℃,Km值为16mmol/L,对pH及温度有较高的稳定性,并利用液相色谱对菊糖酶EI催化水解菊糖的产物进行分析研究。     

Construction of membrane-anchoring fusion protein of Thermococcus kodakaraensis glycerol kinase and its application to repetitive batchwise reactions

We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Thermus thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70°C. In this study, TkGK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70°C for 2h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. coli cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities.     

菊芋酶解制备低聚果糖糖浆的工艺优化及功能评价

菊芋块茎富含菊糖,是制备低聚果糖（Fructooligosaccharides,FOS）的主要原料之一,新鲜菊芋块茎直接酶法加工用于功能性糖浆的制备可以丰富菊芋综合加工的应用。本研究以新鲜菊芋块茎为原料,通过系统研究菊芋内源酶、菊粉内切酶、葡聚糖内切酶、木聚糖酶、聚半乳糖醛酸酶和单宁酶在鲜菊芋酶法加工制备低聚果糖糖浆中的作用规律和酶解效果,建立并优化了酶法制备低聚果糖糖浆的工艺。结果表明,最优酶解工艺如下：菊芋浆在50℃和pH5.0条件下,加入0.08 U/g单宁酶酶解4 h,再加入0.08 U/g葡聚糖内切酶、0.08 U/g木聚糖酶、0.07 U/g聚半乳糖醛酸酶和12 U/g菊糖内切酶组合酶解8 h,酶解液浓缩2倍后获得低聚果糖糖浆成品。成品中低聚果糖和单宁的含量分别为53.72和3.11 g/L,DPPH自由基清除率、羟基自由基清除率和总抗氧化能力分别为82.23%、30.47%和2.78μmol/mL。以制备的低聚果糖糖浆为唯一碳源替代MRS培养基中的葡萄糖,植物乳杆菌、嗜热链球菌和副干酪乳杆菌的生长速率较未经酶解的菊芋原浆作为MRS培养基的唯一碳源时,分别提高了33.33%...     

Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol

The synthesis of galacto-oligosaccharides (GOS) by the action of Aspergillus oryzae β-galactosidase free and immobilized on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) was studied. A maximum GOS concentration of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50%, w/v lactose solution at pH 4.5 and 40 °C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield. GOS formation was not affected by enzyme immobilization in the mPOS-PVA matrix, indicating the absence of diffusional limitations in the enzyme carrier. Furthermore, this water insoluble magnetic derivative was reutilized 10-times and retained about 84% of the initial activity. In addition, the kinetic parameters for various initial lactose concentrations were determined and compared for the free and immobilized enzyme.